iv Cryopreservation of Tropical Plant Germplasm

Foreword Cryopreservation, i.e. the storage of biological material at ultra-low temperature, usually that of liquid nitrogen (–196°C), is the only method currently available to ensure the safe and cost-effective long-term conservation of genetic resources of species that have recalcitrant seeds or are vegetatively propagated. Dramatic progress has been made over the last 10 years in this area with the development of cryopreservation techniques for well over 100 plant species. Cryopreservation protocols are increasingly becoming available for routine application in genebanks. However, much of the work to date has been done on temperate species, with research on tropical and subtropical species lagging behind. This is of particular concern given the large number of tropical species that are either vegetatively propagated or that produce recalcitrant seeds. Both JIRCAS and IPGRI are heavily involved in cryopreservation research. In the framework of its Visiting Fellowship Programme, JIRCAS has carried out a project specifically to develop techniques for the long-term preservation of vegetatively propagated crop germplasm. During the project, visiting scientists from developing countries have developed cryopreservation protocols for selected tropical crops. For more than 15 years, IPGRI and its predecessor IBPGR has supported cryopreservation research in collaboration with partners in Asia, the Pacific and Oceania, Africa, the Americas and Europe. As a result of their experience in the field of cryopreservation, JIRCAS and IPGRI, in October 1998, jointly organized an international workshop to assess the current state of the science, to explore cryopreservation applications and to examine outstanding problems. The focus of the workshop was on the use of cryopreservation to conserve the germplasm of tropical plant species. An additional objective was to identify priority areas for collaborative research, technology development, transfer and application. The workshop was attended by a large number of cryopreservation experts from both developing and developed countries who presented their latest research results and contributed to the discussions. This publication of the proceedings of the workshop thus presents a comprehensive overview of current knowledge concerning the biological and physical mechanisms involved in cryopreservation, and the status of the development of protocols for new species and their application in genebanks. We trust that it will help to stimulate further collaborative research and thus contribute to the wider application of cryopreservation for the safe long-term and cost-effective conservation of genetic resources of tropical species.

Nobuyoshi Maeno, Director General Geoff Hawtin, Director General Japan International Research Center for International Plant Genetic Agricultural Sciences (JIRCAS) Resources Institute (IPGRI) Tsukuba, Japan Rome, Italy v

Acknowledgements The editors hereby acknowledge the invaluable contributions made by Profs. Akira Sakai and H.F. Chin to the development of cryopreservation techniques and their application to the long-term conservation of tropical crop germplasm, and to the planning and implementation of this Workshop. They also wish to thank JIRCAS, SGRP and IPGRI for financial contributions for the publication of these proceedings.

Keynote presentations 1

Keynote presentations

Development of cryopreservation techniques Akira Sakai Sapporo, 001-0045 Japan

Recent advances in cryogenic procedures For successful cryopreservation, it is essential to avoid lethal intracellular freezing which occurs during rapid cooling in liquid nitrogen (Sakai and Yoshida 1967; Sakai 1985, 1995). Thus, specimens to be preserved have to be sufficiently dehydrated to avoid intracellular freezing and thus vitrify upon rapid cooling in liquid nitrogen. Vitrification may be the only freeze-avoidance mechanism that enables hydrated cells, tissues and organs to survive at the temperature of liquid nitrogen (Sakai 1960, 1965, 1995), as suggested by Luyet as early as 1937. Vitrification, a physical process, can be defined as the phase transition of an aqueous solution from a liquid into an amorphous glassy solid, or glass, at the glass transition temperature (Tg), while avoiding ice crystallization. A glass fills spaces in a tissue, and during dehydration may contribute to preventing additional tissue collapse, solute concentration and pH alterations. Operationally, a glass is expected to exhibit a lower water vapour pressure than the corresponding crystalline solid and to thereby prevent further dehydration. As glass is exceedingly viscous and stops all chemical reactions that require molecular diffusion, its formation may lead to dormancy and stability over time (Burke 1986). Fahy et al. (1984) presented a highly concentrated vitrification solution for successful cryopreservation of animal embryos. This solution easily supercools down to a temperature below –100°C and finally solidifies into a metastable glass at the glass transition temperature (Tg: about –110°C) at a practical cooling rate. For plant cryopreservation, some simplified and valuable cryogenic procedures such as vitrification (Langis et al. 1990; Sakai et al. 1990, 1991; Towill 1990), air-drying (Uragami et al. 1990) and the encapsulation-dehydration technique (Fabre and Dereuddre 1990) have been presented, and the number of species to be cryopreserved has increased dramatically over the last 10 years or so (Engelmann 1997; Sakai 1997). At present, potentially valuable cryogenic procedures for cryopreserving apical meristems and somatic embryos include vitrification with or without encapsulation, and the encapsulation-dehydration technique. These alternative dehydration procedures, referred to as “vitrification- based techniques” (Engelmann 1997), offer practical advantages compared with classical prefreezing protocols. They are more appropriate for freezing complex organs (shoot-tips and somatic embryos) that contain a variety of cell types, each with unique requirements under conditions of freeze-induced dehydration (Withers 1979). When the vitrification technique is employed under well- optimized conditions, the whole or most of the apical meristems remain alive 2 Cryopreservation of Tropical Plant Germplasm

(Yamada et al. 1991; Matsumoto et al. 1994), thus allowing direct, organized regrowth. By contrast, classical prefreezing procedures can lead to the destruction of large zones of apical domes, and callusing only or transitory callusing is often observed before organized regrowth starts (Haskins and Kartha 1980; Touchell and Dixon 1996). By precluding ice formation in the system, vitrification-based procedures simplify the cryogenic procedure and eliminate concerns for the potentially damaging effects of intra- and extracellular crystallization, producing high levels of post-cryopreservation regrowth, and have greater potential for broad applicability, requiring only minor modification for different cell types (Engelmann 1997; Sakai 1997).

Vitrification protocol for apical meristems Vitrification can be achieved by direct immersion of samples in liquid nitrogen without a freeze-induced dehydration step, by osmotically dehydrating the cells and meristems in a highly concentrated vitrification solution. Such a technique is referred to as vitrification (complete vitrification: both cytosol and suspending solution are vitrified), distinct from conventional prefreezing methods (partial vitrification, only the cytosol is vitrified). The vitrification procedure requires a highly concentrated vitrification solution, which sufficiently dehydrates cytosols without causing injury so that they turn into a stable glass when plunged in liquid nitrogen. We have developed a glycerol-based, low-toxicity vitrification solution designated PVS2 (Sakai et al. 1990, 1991), which does not permeate into the cytosol during the dehydration process. This solution easily supercools below –100°C at practical cooling rate and finally solidifies at –115°C. More recently, we have found that a new vitrification solution (PVS4) which contains 35% glycerol (w/v) and 20% EG (w/v), in the basal medium containing 0.6M sucrose (pH 5.8) produced nearly the same recovery growth as PVS2. The acquisition of tolerance to dehydration achieved by exposure to PVS2 and the mitigation of injurious effects during the dehydration process are essential for the successful cryopreservation of excised shoot-tips by vitrification. To induce dehydration tolerance, excised shoot-tips from in vitro grown plantlets are precultured on medium with high sucrose concentrations (0.3–0.6M) for 16 h and then treated with a mixture of 2M glycerol plus 0.4M sucrose (LD solution) for 20 min before dehydration with PVS2. In our vitrification method, the injurious effects caused by the dehydration process with PVS2 are reduced or eliminated by optimizing the duration of exposure to PVS2, and by dehydrating shoot-tips gradually in two steps: LD solution at 25°C, followed by PVS2 solution at 0°C. Table 1 lists the successful reports of cryopreservation of shoot-tips from in vitro grown plants using vitrification with PVS2. These results clearly demonstrate that the vitrification protocol has a wide applicability, both in terms of species coverage, since the protocol has been successfully established for hairy roots, tubers, fruits, ornamentals and plantation crops of both temperate and tropical origins, and in terms of number of genotypes/varieties within a given species. Keynote presentations 3

Table 1. Successful cryopreservation of meristems cooled to –196°C by vitrification using PVS2 vitrification solution Plant Pretreatment† References Woody plants Apple (5 spp., cvs.) CH, PC Niino et al. 1992a Cherry (8 cvs.) CH, PC Tashiro et al. 1995 Grape (4 cvs.) PC, LD Matsumoto et al. 1998b Grevillea scapigera ‡ PC Touchell and Dixon 1996 Grevillea cirsiifolia ‡ PC Touchell and Dixon 1996 Mulberry (13 spp., cvs.) CH, PC Niino et al. 1992b Pear (5 cvs.) CH, PC Niino et al. 1992a Tea plant CH, PC Kuranuki and Sakai 1995 Herbaceous plants Garlic (12 cvs.) § None Niwata 1995 Lily (4 cvs.), Lilium japonicum CH, PC, LD Matsumoto et al. 1995b Mint (3 cvs.) CH, LD Hirai et al., unpubl. Strawberry (4 cvs.) CH, LD Hirai et al. 1998 Wasabi (4 cvs.) PC, LD Matsumoto et al. 1994 White clover (3 spp.) PC Yamada et al. 1991 Hairy roots Armoracia rusticana PC Phunchindawan et al. 1997 Panax ginseng PC, LD Yoshimatsu et al. 1996 Tropical plants Banana (6 cvs.) PG, PC, LD Thinh 1997 Cassava (2 cvs.) PC, LD Charoensub et al., unpubl. Orchid Cymbidium (2 cvs.) PC, LD Dendrodium (protocorm) ABA Thinh 1997 Pineapple PC, LD Wang et al. 1998 Tannia (2 spp.) PC, LD Thinh 1997 Taro Thinh 1997 Colocasia var. antiquorum (2 cvs.) PG, PC, LD Takagi et al. 1997; Thinh 1997 Colocasia var. esculenta (4 cvs.) PG, PC, LD Thinh 1997 Yam Dioscorea rotundata (3 cvs.) PC Kyesmu et al. 1997 † ABA: abscisic acid; CH: cold-hardening; LD: loading treatment with 2M glycerol plus 0.4M sucrose; PC: preculture; PG: pregrowth of meristem-donor plants on sucrose- enriched medium for 1 month; cv: cultivar, sp: species. ‡ Australian endangered woody plants (6 other species). § Post-dormant bulbs.

Comparative study of different cryogenic procedures Shoot formation from apical meristems of various plants or cultivars cooled to -196°C was compared for different cryogenic protocols. With most of the plants tested, the vitrification method with or without encapsulation produced much higher levels of shoot formation than the encapsulation-dehydration technique under optimal conditions (Matsumoto and Sakai 1995; Hirai et al. 1998, unpubl.). The same results were observed with endangered Australian plants (Touchell 4 Cryopreservation of Tropical Plant Germplasm

1995; Touchell and Dixon 1996). In addition, growth recovery was much quicker with vitrified meristems than with encapsulated meristems (Matsumoto and Sakai 1995; Hirai et al. 1998). Thus, the vitrification method certainly offers considerable advantages over the encapsulation-dehydration technique for the cryopreservation of apical meristems. These results suggest that the induction of dehydration tolerance by sucrose alone (0.8M sucrose for 16 h) may be insufficient for many meristems (Mandal et al. 1996). Thus, we presented a revised technique using a mixture of sucrose and glycerol (Matsumoto and Sakai 1995; Phunchindawan et al. 1997). More recently, we have found that treating apices with a mixture of 2M glycerol plus 0.6M sucrose for 90 min, followed by air-drying for 3–4 h, produced higher levels of growth recovery (94% with wasabi) than the original encapsulation-dehydration procedure (about 60%) (Sakai et al., unpubl.).

Key problems for successful cryopreservation by vitrification The key for successful cryopreservation by vitrification is to induce tolerance of specimens to dehydration with a highly concentrated vitrification solution. In the vitrification method, cells and excised meristems are usually precultured on sucrose- or sorbitol-enriched medium for 1 or 2 days to induce dehydration tolerance (Yamada et al. 1991; Matsumoto et al. 1994; Touchell 1995; Reinhoud 1996; Thinh 1997). A high level of sugar or sorbitol accumulated during preculture has been reported to be very important in improving the survival of cryopreserved cells and meristems (Uragami et al. 1990; Dereuddre et al. 1991; Reinhoud 1996; Matsumoto et al. 1998a). The accumulation of sugars increases the stability of membranes under conditions of severe dehydration (Crowe et al. 1989). Reinhoud (1996) clearly demonstrated that the development of tolerance of cultured tobacco cells to PVS2 during preculture with 0.3M mannitol solution for 1 day appeared to be a combined result of mannitol uptake and the cellular response to mild osmotic stress caused by the preculture: production of ABA, proline and certain proteins including late embryogenesis abundant ones (LEAs). With apical meristems of numerous species, preculture with sugar or sorbitol did not lead to substantial increases in growth recovery after vitrification. However, treatment with LD solution for 20 min following preculture with 0.3M sucrose for 16 h was very effective in increasing the growth recovery of vitrified wasabi shoot-tips (Matsumoto et al. 1994). The same results were observed in about 20 tropical monocotyledons (Thinh 1997). Thus, the treatment with LD solution seems to be an important step in the vitrification procedure for some plants. The LD solution was reported to be very effective in inducing dehydration tolerance to freeze-dehydration (Nishizawa et al. 1992) or to PVS2 (Nishizawa et al. 1993; Matsumoto et al. 1995a, 1995b). During treatment with the LD solution, the cells are considerably dehydrated and plasmolyzed. However, little or no permeation of glycerol into the cytosol was observed after a 20-min incubation. Thus, the protective effect of a brief incubation with LD solution might be a result of the concentration of cytosolic cryoprotectants accumulated during the preculture with sucrose, and to the protective effect of plasmolysis. The presence of LD solution in the periprotoplasmic space of plasmolyzed cells may mitigate Keynote presentations 5 mechanical stress caused by severe dehydration (Tao et al. 1983; Jitsuyama et al. 1997) and give some protective action to minimize the injurious membrane changes during severe dehydration (Steponkus et al. 1992), though the mechanism of action is not well understood. Successful cryopreservation is not only determined by the cryopreservation procedure itself, but also by the condition of plant cells (exponential phase) or apical meristems (age of growth). Thus, for successful cryopreservation, excised apices must be in a physiological state suitable for the acquisition of osmotolerance and the production of vigorous growth recovery. This should be decided empirically and species- or culture-specifically.

Prospects for cryopreservation of tropical plants Thinh (1997) succeeded in cryopreserving tropical monocotyledons such as taro, banana, pineapple and orchids, totaling about 20 species or cultivars, using vitrification with only slight modifications of the technique. More recently, yam (Kyesmu et al. 1997), cassava (Charoensub, unpublished) and protocorms of Dendrobium (Wang et al. 1998) have been successfully cryopreserved by vitrification. In view of the wide range of efficient and simple vitrification-based techniques available, many tropical plant species or cultivars could be amenable to cryopreservation, provided that the tissue culture protocols, such as apical meristem and somatic embryo culture, are sufficiently operational for the species. For further development of cryopreservation of tropical plants, studies on preconditioning for the induction of dehydration tolerance appear to be most important.

References Burke, M.J. 1986. The glassy state and survival of anhydrous biological systems. Pp. 358- 363 in Membranes, Metabolism, and Dry Organisms. A.C. Leopold, ed. Comstock, Cornell Univ. Press, Ithaca and London. Crowe, J.H., L.M. Crowe, J.F. Carpenter, A.S. Rudolph, C.A. Wistrom, B.J. Spargo and T.J. Anchordoguy. 1989. Interactions of sugars with membranes. Biochimica et Biophysica Acta 947: 367–384. Dereuddre, J., S. Blandin and N. Hassen. 1991. Resistance of alginate-coated somatic embryos of carrot (Daucus carota L.) to desiccation and freezing in liquid nitrogen. 1: Effect of preculture. Cryo–Letters 12: 125–134. Engelmann, F. 1997. Importance of desiccation for cryopreservation of recalcitrant seed and vegetatively propagated apices. Plant Genetic Resources Newsletter 112: 9–18. Fabre, J. and J. Dereuddre. 1990. Encapsulation-dehydration: A new approach to cryopreservation of Solanum shoot tips. Cryo–Letters 11:413–426. Fahy, G.M., D.R. MacFarlane, C.A. Angell and H.T. Meryman. 1984. Vitrification as an approach to cryopreservation. Cryobiology 21: 407–426. Haskins, R.H. and K.K. Kartha. 1980. Freeze-preservation of pea meristems: cell survival. Canadian Journal of Botany 58: 833–840. Hirai, D., K. Shirai, S. Shirai and A. Sakai. 1998. Cryopreservation of in vitro-grown meristems of strawberry (Fragaria x ananassa Duch.) by encapsulation vitrification. Euphytica 101:109–115. 6 Cryopreservation of Tropical Plant Germplasm

Jitsuyama, Y., T. Suzuki, T. Harada and S. Fujikawa. 1997. Ultrastructural study of mechanism of increased freezing tolerance to extracellular glucose in cabbage leaf cells. Cryo–Letters 18: 33–44. Kuranuki, Y. and A. Sakai. 1995. Cryopreservation of in vitro-grown shoot tips of tea (Camellia sinensis) by vitrification. Cryo–Letters 16: 345–352. Kyesmu, P.M., H. Takagi and S. Yashima. 1997. Cryopreservation of white yam (Dioscorea rotundata ) shoot apices by vitrification. P. 162 in Proceedings of Annual Meeting of Japan Molecular Biology, 20–12 July 1997, Kumamoto University, Kumamoto, Japan. Langis, R., B. Schnabel–Preikstas, B.J. Earle and P.L. Steponkus. 1990. Cryopreservation of carnation shoot tips by vitrification. Cryobiology 276: 658–659. Luyet, B.J. 1937. The vitrification of organic colloids and protoplasm. Biodynamica 1: 1–14. Mandal, B.B., K.P.S. Chandel and S. Dwivedi. 1997. Cryopreservation of yam (Dioscorea spp.) shoot apices by encapsulation-dehydration. Cryo–Letters 17: 165–174. Matsumoto, T. and A. Sakai. 1995. An approach to enhance dehydration tolerance of alginate-coated dried meristems cooled to –196°C. Cryo–Letters 16: 299–306. Matsumoto, T., A. Sakai and K. Yamada. 1994. Cryopreservation of in vitro-grown apical meristems of wasabi (Wasabia japonica ) by vitrification and subsequent high plant regeneration. Plant Cell Reports 13:442–446. Matsumoto, T., A. Sakai, C. Takahashi and K. Yamada. 1995a. Cryopreservation in vitro- grown apical meristems of wasabi (Wasabi japonica) by encapsulation-vitrification method. Cryo–Letters 16: 189–206. Matsumoto, T., A. Sakai and K. Yamada. 1995b. Cryopreservation in vitro-grown apical meristems of lily by vitrification. Plant Cell, Tissue and Organ Culture 41: 237–241. Matsumoto, T., A. Sakai and Y. Nako. 1998a. A novel preculturing for enhancing the survival of in vitro-grown meristems of wasabi (Wasabia japonica) cooled to –196°C by vitrification. Cryo–Letters 19: 27–36. Matsumoto, T., A. Sakai and Y. Nako. 1998b. Cryopreservation of in vitro cultured axillary shoot tips of grape (Vitis vinifera) by vitrification. Supplement, Journal of the Japanese Society of Horticultural Science 67: 78. Niino, T., A. Sakai and K. Nojiri. 1992a. Cryopreservation of in vitro-grown shoot tips of apple and pear by vitrification. Plant Cell, Tissue and Organ Culture 28: 261–266. Niino, T., A. Sakai, S. Enomoto and S. Kato. 1992b. Cryopreservation in vitro-grown shoot tips of mulberry by vitrification. Cryo–Letters 13: 303–312. Nishizawa, S., A. Sakai, Y. Amano and T. Matsuzawa. 1992. Cryopreservation of asparagus (Asparagus officinalis L.) embryogenic suspension cells and subsequent plant regeneration by a simple freezing method. Cryo–Letters 13: 379–388. Nishizawa, S., A. Sakai, Y. Amano and T. Matsuzawa. 1993. Cryopreservation of asparagus (Asparagus officinalis L.) embryogenic suspension cells and subsequent plant regeneration by vitrification. Plant Science 91: 67-73. Niwata, E. 1995. Cryopreservation of apical meristems of garlic (Allium sativum L.) and high subsequent plant regeneration. Cryo–Letters 16: 102–107. Phunchindawan, M., K. Hirata, A. Sakai and K. Miyamoto. 1997. Cryopreservation of encapsulated shoot primordia induced in horse radish (Armoracia rusticana) hairy root cultures. Plant Cell Reports 16: 469–473. Reinhoud, P.J. 1996. Cryopreservation of tobacco suspension cells by vitrification. Doctoral Thesis, Leiden University, Institute of Molecular Plant Sciences, Leiden, the Netherlands.. Sakai, A. 1960. Survival of the twig of woody plants at –196°C. Nature 185: 393–394. Sakai, A. 1965. Determining the degree of frost-hardiness in highly hardy plants. Nature 206: 1064–1065. Keynote presentations 7

Sakai, A. 1985. Cryopreservation of shoot tips of fruit trees and herbaceous plants. Pp. 135–158 in Cryopreservation of Plant Cells and Organs (K.K. Kartha, ed.). CRC Press, Boca Raton, Florida. Sakai, A. 1995. Cryopreservation of germplasm of woody plants. Pp. 53–69 in Biotechnology in Agriculture and Forestry Vol. 32: Cryopreservation of Plant Germplasm. 1 (Y.P.S. Bajaj, ed.). Springer–Verlag, Heidelberg. Sakai, A. 1997. Potentially valuable cryogenic procedures for cryopreservation of cultured plant meristems. Pp. 53–66 in Conservation of Plant Genetic Resources In Vitro. Volume 1: General Aspects. M.K. Razdan and E.C. Cocking (eds.). Science Publishers Inc., Enfield, USA. Sakai, A. and S. Yoshida. 1967. Survival of plant tissue at super-low temperatures. VI. Effects of cooling and rewarming rates on survival. Plant Physiology 42: 1695–1701. Sakai, A., S. Kobayashi and I. Oiyama. 1990. Cryopreservation of nucellar cells of navel orange (Citrus sinensis Osb. var. brasiliensis Tanaka) by vitrification. Plant Cell Reports 9: 30–33. Sakai, A., S. Kobayashi and I. Oiyama. 1991. Survival by vitrification of nucellar cells of navel orange (Citrus sinensis Osb. var. brasiliensis Tanaka) cooled to –196°C. Journal of Plant Physiology 137: 465–470. Steponkus, P.L., R. Langis and S. Fujikawa. 1992. Cryopreservation of plant tissues by vitrification. Pp. 1–61 in Advances in Low-Temperature Biology, Vol. 1. P.L. Steponkus (ed.). JAI Press Ltd., Hampton Mill, UK. Takagi, H., N.T. Thinh, O.M. Islam, T. Sendai and A. Sakai. 1997. Cryopreservation of in vitro-grown shoot tips of taro (Colocasia esculenta (L.) Schott) by vitrification. 1. Investigation of basic conditions of the vitrification procedures. Plant Cell Reports 16: 594–599. Tao, D., P.H. Li and J.V. Carter. 1983. Role of cell wall in freezing tolerance of cultured potato cells and their protoplasts. Physiologia Plantarum 58: 527–532. Tashiro, H., T. Niino, J. Magoshi and T. Akihama. 1995. Cryopreservation of in vitro– grown meristems of cherry by vitrification. In Japanese Society of Plant Tissue Culture 14th Annual Meeting Report, p.120. Thinh, N.T. 1997. Cryopreservation of germplasm of vegetatively propagated tropical monocots by vitrification. Doctoral Papers of Kobe University, Department of Agronomy, Japan. Touchell, D.H. 1995. Principles of cryobiology for conservation of threatened Australian plants. Doctoral Thesis of the University of Western Australia, Botany Division, Australia. Touchell, D.H. and K.W. Dixon. 1996. Cryopreservation for conservation of Australian endangered plants. Pp. 169–180 in In Vitro Conservation of Plant Genetic Resources (M.N. Normah, M.K. Narimah and M.M. Clyde, eds.). Plant Biotechnology Laboratory, Faculty of Life Sciences, University Kebangsaan, Malaysia. Towill, L.E. 1990. Cryopreservation of isolated mint shoot tips by vitrification. Plant Cell Reports 9: 178–180. Uragami, A., A. Sakai and M. Nagai. 1990. Cryopreservation of dried axially buds from plantlets of Asparagus officinalis L. in vitro. Plant Cell Reports 9: 328–331. Wang, J.H., J.G. Ge, F. Liu, H.W. Bian and C.N. Huang. 1998. Cryopreservation of seeds and protocorms of Dendrobium candidum. Cryo–Letters 19: 123–128. Withers, L.A. 1979. Freeze preservation of somatic embryos and clonal plantlets of carrot (Daucus carota ). Plant Physiology 63: 460–467. Yamada, T., A. Sakai, T. Matsumura and S. Higuchi. 1991. Cryopreservation of apical meristems of white clover (Trifolium repens L.) by vitrification. Plant Science 78: 81–87. Yoshimatsu, K., H. Yamaguchi and K. Shimomura. 1996. Traits of Panax ginseng hairy roots after cold storage and cryopreservation. Plant Cell Reports 15: 555–560. 8 Cryopreservation of Tropical Plant Germplasm

Importance of cryopreservation for the conservation of plant genetic resources Florent Engelmann International Plant Genetic Resources Institute, 00145 Rome, Italy

Introduction The two basic approaches to conservation of plant genetic resources are ex situ and in situ conservation. Article 2 of the Convention on Biological Diversity (UNCED 1992) provides the following definitions for these categories: ex situ conservation means the conservation of components of biological diversity outside their natural habitats. In situ conservation means the conservation of ecosystems and natural habitats and the maintenance and recovery of viable populations of species in their natural surroundings and, in the case of domesticates or cultivated species, in the surroundings where they have developed their distinctive properties. The two basic conservation strategies can be further subdivided into several specific techniques. Ex situ conservation includes seed storage, in vitro storage, DNA storage, pollen storage, field genebanks and botanical gardens, while the in situ approach encompasses genetic reserves, on-farm and home garden conservation (Maxted et al. 1997). It is now well recognized that for any given genepool, a number of different complementary approaches and methods are necessary for a safe, efficient and cost-effective conservation. The appropriate strategy and the balance depend on factors such as the biological characteristics of the plants, their present management and use by humans, available infrastructure for conservation, number of accessions in a given collection and geographic sites, the purpose of conservation, the availability of germplasm, and political and administrative policies. The extent of the utilization of a particular method may differ from one genepool to another (Withers 1993). For many plant species which produce orthodox seeds, i.e. which can be dehydrated extensively and stored dry at low temperature (Roberts 1973), the emphasis for genetic resource conservation will be on seed storage. However, three categories of crop present problems with regard to seed storage. First, there are those that do not produce seeds at all, and are propagated vegetatively, for example, banana and plantain (Musa spp.). Second, there are crops including potato (Solanum tuberosum), other root and tuber crops such as yams (Dioscorea spp.), cassava (Manihot esculenta) and sweet potato (Ipomoea batatas), and sugarcane (Saccharum spp.) that have some sterile genotypes and some that produce orthodox seed. However, like many temperate fruits, these seeds are highly heterozygous and therefore of limited utility for the conservation of gene combinations. These crops are usually propagated vegetatively to maintain clonal genotypes. Then, third, there are those crops that produce recalcitrant seeds (Roberts 1973) which include a large number of tropical fruits and timber species. Recalcitrant seeds cannot tolerate desiccation to moisture contents that Keynote presentations 9 would permit exposure to low temperatures. They are often large with considerable quantities of fleshy endosperm. Finally, recent investigations have identified species displaying an intermediate form of seed storage behaviour (Ellis et al. 1990, 1991). These seeds can tolerate desiccation down to relatively low moisture content but the dry seeds are often injured by low temperature. Compared with truly recalcitrant seeds, the storage life of these seeds can be prolonged by some drying, but it is impossible to obtain the long-term conservation of orthodox seeds (Engelmann 1997a). As regards the balance of techniques employed within complementary strategies developed for conserving the genetic resources of these problem species, the emphasis in the case of non-orthodox forest tree species will be on in situ conservation in genetic reserves, while for species which are propagated vegetatively the emphasis will be on ex situ conservation techniques, including field genebank and in vitro storage. However, it is essential to recognize that, owing to the various problems and limitations encountered with both genetic reserves and field genebanks (Withers and Engels 1990; Maxted et al. 1997), cryopreservation currently offers the only safe and cost-effective option for the long-term conservation of genetic resources of these problem species. This paper presents the various cryopreservation techniques available, their current development and problems with their application to recalcitrant seed and vegetatively propagated species.

Cryopreservation techniques available Most of the experimental systems employed in cryopreservation (cell suspensions, calluses, shoot-tips, embryos) contain high amounts of cellular water and are thus extremely sensitive to freezing injury since most of them are not inherently freezing-tolerant. Cells have thus to be dehydrated artificially to protect them from the damages caused by the crystallization of intracellular water into ice (Meryman and Williams 1985; Mazur 1984). The techniques employed and the physical mechanisms upon which they are based are different in classical and new cryopreservation techniques (Withers and Engelmann 1997). Classical techniques involve freeze-induced dehydration, whereas new techniques are based on vitrification. Vitrification can be defined as the transition of water directly from the liquid phase into an amorphous phase or glass, whilst avoiding the formation of crystalline ice (Fahy et al. 1984).

Classical cryopreservation techniques Classical cryopreservation techniques involve slow cooling down to a defined prefreezing temperature, followed by rapid immersion in liquid nitrogen. With temperature reduction during slow cooling, the cells and the external medium initially supercool, followed by ice formation in the medium (Mazur 1984). The cell membrane acts as a physical barrier and prevents the ice from seeding the cell interior and the cells remain unfrozen but supercooled. As the temperature is further decreased, an increasing amount of the extracellular solution is converted into ice, thus resulting in the concentration of intracellular solutes. Since cells 10 Cryopreservation of Tropical Plant Germplasm remain supercooled and their aqueous vapour pressure exceeds that of the frozen external compartment, cells equilibrate by loss of water to external ice. Depending upon the rate of cooling and the prefreezing temperature, different amounts of water will leave the cell before the intracellular contents solidify. In optimal conditions, most or all intracellular freezable water is removed, thus reducing or avoiding detrimental intracellular ice formation upon subsequent immersion of the specimen in liquid nitrogen. However, too-intense freeze- induced dehydration can incur different damaging events owing to concentration of intracellular salts and changes in the cell membrane (Meryman et al. 1977). Rewarming should be as rapid as possible to avoid the phenomenon of recrystallization in which ice melts and reforms at a thermodynamically favourable, larger and more damaging crystal size (Mazur 1984). Classical techniques are generally operationally complex since they require the use of sophisticated and expensive programmable freezers. In some cases, their use can be avoided by performing the freezing step with a domestic or laboratory freezer (Kartha and Engelmann 1994). Classical cryopreservation techniques have been successfully applied to undifferentiated culture systems such as cell suspensions and calluses (Kartha and Engelmann 1994; Withers and Engelmann 1997). In the case of differentiated structures, these techniques can be employed for freezing apices of cold-tolerant species (Reed and Chang 1997). However, their successful utilization with tropical species is exceptional (Escobar et al. 1997).

New cryopreservation techniques In vitrification-based procedures, cell dehydration is performed prior to freezing by exposure of samples to concentrated cryoprotective media and/or air desiccation. This is followed by rapid cooling. As a result, all factors which affect intracellular ice formation are avoided. Glass transitions (changes in the structural conformation of the glass) during cooling and rewarming have been recorded with various materials using thermal analysis (Sakai et al. 1990; Dereuddre et al. 1991; Tannoury et al. 1991; Niino et al. 1992). Dumet et al. (1993b) showed that increased survival rates for cryopreserved oil-palm somatic embryos were correlated with the progressive disappearance of ice crystallization peaks and their replacement by glass transitions. Vitrification-based procedures offer practical advantages in comparison with classical freezing techniques (Steponkus et al. 1992). Like ultra-rapid freezing (above), they are more appropriate for complex organs (shoot-tips, embryos) which contain a variety of cell types, each with unique requirements under conditions of freeze-induced dehydration. By precluding ice formation in the system, vitrification-based procedures are operationally less complex than classical ones (e.g. they do not require the use of controlled freezers) and have greater potential for broad applicability, requiring only minor modifications for different cell types (Engelmann 1997b). A common feature of all these new protocols is that the critical step to achieve survival is the dehydration step, and not the freezing step, as in classical protocols. Therefore, if samples to be frozen are amenable to desiccation down to sufficiently Keynote presentations 11 low water contents (which vary depending on the procedure employed and the type and characteristics of the propagule to be frozen) with no or little decrease in survival in comparison with non-dehydrated controls, no or limited further drop in survival is generally observed after cryopreservation (Engelmann 1997b). Seven different vitrification-based procedures can be identified: (i) encapsulation-dehydration; (ii) a procedure actually termed vitrification; (iii) encapsulation-vitrification; (iv) desiccation; (v) pregrowth; (vi) pregrowth- desiccation, and (vii) droplet freezing. The encapsulation-dehydration procedure is based on the technology developed for the production of artificial seeds. Explants are encapsulated in alginate beads, pregrown in liquid medium enriched with sucrose for 1 to 7 days, partially desiccated in the air current of a laminar airflow cabinet or with silica gel down to a water content around 20% (fresh weight basis), then frozen rapidly. Survival rates are high and growth recovery of cryopreserved samples is generally rapid and direct, without callus formation. This technique has been applied to apices of numerous species of both temperate and tropical origins (Engelmann 1997b). Its applicability to cell suspensions and somatic embryos has also been demonstrated (Tessereau et al. 1994; Bachiri et al. 1995). Vitrification involves treatment of samples with cryoprotective substances, dehydration with highly concentrated vitrification solutions, rapid freezing and thawing, removal of cryoprotectants and recovery. This procedure has been developed for apices, cell suspensions and somatic embryos of numerous different species (Sakai 1995, 1997). Encapsulation-vitrification is a combination of encapsulation-dehydration and vitrification procedures, where samples are encapsulated in alginate beads, then subjected to freezing by vitrification. It has been applied to apices of carnation (Tannoury et al. 1991) and of lily, wasabi and Armoracia (Sakai 1997). Desiccation is the simplest procedure since it consists of dehydrating explants, then freezing them rapidly by direct immersion in liquid nitrogen. This technique is mainly used with zygotic embryos or embryonic axes extracted from seeds. It has been applied to embryos of a large number of recalcitrant and intermediate seeds (Engelmann 1997a). Desiccation is usually performed in the air current of a laminar airflow cabinet, but more precise and reproducible dehydration conditions are achieved by using a flow of sterile compressed air or silica gel. Ultra-rapid drying in a stream of compressed dry air (a process called “flash drying” developed by Berjak's group in South Africa) allows freezing of samples with a relatively high water content, thus reducing the desiccation injury (Berjak et al. 1989a; Wesley–Smith et al. 1992). Optimal survival rates are generally obtained when samples are frozen with a water content of between 10 and 20% (fresh weight basis). The pregrowth technique consists of cultivating samples in the presence of cryoprotectants, then freezing them rapidly by direct immersion in liquid nitrogen. The pregrowth technique has been developed for Musa apices (Panis 1995). In a pregrowth-desiccation procedure, explants are pregrown in the presence of cryoprotectants, dehydrated under the laminar airflow cabinet or with silica gel, then frozen rapidly. This method has currently been applied only to asparagus 12 Cryopreservation of Tropical Plant Germplasm stem segments, oil-palm somatic embryos and coconut zygotic embryos (Uragami et al. 1990; Assy–Bah and Engelmann 1992; Dumet et al. 1993a). The droplet freezing technique has presently been applied to potato apices only (Schäfer–Menuhr 1996). Apices are pretreated with liquid cryoprotective medium, then placed on aluminium foil in minute droplets of cryoprotectant and frozen directly by rapid immersion in liquid nitrogen.

Cryopreservation of vegetatively propagated and recalcitrant seed species

Vegetatively propagated species Several review papers have been published recently, which provide lists of species which have been successfully cryopreserved (Bajaj 1995; Engelmann 1997a, 1997b). For vegetatively propagated species, cryopreservation has a wide applicability both in terms of species coverage – since protocols have been successfully established for roots and tubers, fruit trees, ornamentals and plantation crops of both temperate and tropical origin – and in terms of number of genotypes/varieties within a given species. The best example is potato, for which cryopreservation has been applied to 219 different accessions, with an average recovery rate of 40% (Schäfer–Menuhr 1996). With a few exceptions (e.g. potato, pear, mulberry), vitrification-based protocols have been employed. It is also interesting to note that in many cases, different protocols can be employed for a given species and produce comparable results. The only exception could be Musa, for which encapsulation-dehydration led to very low survival, owing to the extreme sensitivity of this material to high sucrose concentrations and air desiccation (Panis 1995). The survival rates obtained are generally high to very high and up to 100% survival could be achieved in some cases, e.g. Allium, yam and potato. Regeneration is rapid and direct, and callusing is observed only in cases where the technique is not optimized, such as with yam and cassava (Chabrillange et al. 1996; Mandal et al. 1996). Different reasons can be mentioned to explain these positive results. The meristematic zone of apices, from which organized growth originates, is composed of a relatively homogeneous population of small, actively dividing cells, with few vacuoles and a high nucleo-cytoplasmic ratio. These characteristics make them more tolerant of desiccation than highly vacuolated and differentiated cells. As mentioned earlier, no ice formation takes place in vitrification-based procedures, thus avoiding the extensive damages caused by ice crystals which are formed during classical procedures. Histological examination revealed that the whole or most of the apex structure is generally preserved when vitrification-based techniques are employed, thus allowing direct, organized regrowth (Gonzalez Arnao et al. 1993). By contrast, classical procedures can lead to the destruction of large zones of the apices and callusing only or transitory callusing is often observed before organized regrowth starts (Haskins and Kartha 1980; Bagniol and Engelmann 1991). Keynote presentations 13

Other reasons for the good results obtained are linked with tissue culture protocols. Many vegetatively propagated species successfully cryopreserved until now are cultivated crops, often of great commercial importance, for which cultural practices, including in vitro micropropagation, are well established. In addition, in vitro material is “synchronized” by the tissue culture and pregrowth procedures, and relatively homogeneous samples in terms of size, cellular composition, physiological state and growth response are employed for freezing, thus increasing the chances of positive and uniform response to treatments. Finally, vitrification- based procedures allow the use of samples of relatively large size (shoot-tips of 0.5 to 2–3 mm) which can regrow directly without any difficulty. In many cases, whole plants have been regenerated from cryopreserved apices, transferred in vivo and observed for genetic stability. No modification which could be attributed to cryopreservation has yet been noted, which indicates that cryopreservation is safe as regards preservation of trueness to type (Engelmann 1997b). Even though potato is still the only example of large-scale, routine utilization of cryopreservation for long-term storage of a vegetatively propagated (and of any other) crop, freezing techniques are now operational for large-scale experimentation in an increasing number of cases. In view of the wide range of efficient and operationally simple techniques available, any vegetatively propagated species should be amenable to cryopreservation, provided that the tissue culture protocol is sufficiently operational for this species.

Recalcitrant seed species Several review papers have been published in recent years which present extensive lists of plant species whose embryos and/or embryonic axes have been successfully cryopreserved (e.g. Kartha and Engelmann 1994; Pence 1995; Engelmann et al. 1995; Engelmann 1997a, 1997b). This might lead to the conclusion that freezing of embryos is a routine procedure applicable to numerous species, whatever their storage characteristics. However, careful examination of the species mentioned in these papers reveals that only a limited number of truly recalcitrant seed species are in fact included. This is partly because research in this area is recent and addressed by very few teams worldwide. Another reason is that recalcitrance is a dynamic concept (Berjak and Pammenter 1994), which evolves with research on the biology of species and improvement in classical storage procedures. For example, seeds of coffee and oil-palm were previously classified as recalcitrant (Chin and Roberts 1980) but recent research by Ellis et al. (1990, 1991) indicated that they display an intermediate storage behaviour. In comparison with results obtained with vegetatively propagated species, it is obvious that research is still at a very preliminary stage for recalcitrant seeds. The desiccation technique is mainly employed for freezing embryos and embryonic axes. The survival rates achieved are extremely variable and generally low. Most importantly, survival is often limited and regeneration frequently restricted to cal- lusing or incomplete development of plantlets. In only a limited number of cases have whole plants have been regenerated from cryopreserved material, e.g. Howea, Veitchia and coconut (Chin and Pritchard 1988; Assy Bah and Engelmann 1992). 14 Cryopreservation of Tropical Plant Germplasm

There are a number of reasons to explain the current limited development of cryopreservation for recalcitrant seed species. First of all, there is a huge number of species with recalcitrant or suspected recalcitrant seeds and the majority are wild species. As a consequence, nothing or little is known on their biology, and even less on the seed storage behaviour of many of these species. In cases where some information on seed storage behaviour is available, tissue culture protocols, including inoculation in vitro, germination and growth of plantlets, propagation and acclimatization which are needed for regrowth of embryos and embryonic axes after freezing, are often non-existent or not fully operational (Zakri et al. 1991; Dumet et al. 1996). This is particularly critical for tree species, which constitute a large part of recalcitrant seed species, especially in the tropics, and often pose great difficulties in tissue culture (Gupta 1988; Rao 1988; Harry and Thorpe 1994). Modifications – sometimes minor – in the in vitro culture conditions might lead to improvement in the recovery rates for some species. For example, modification in the hormonal balance of the culture medium could significantly improve the survival and recovery rates of cryopreserved coffee embryos (Abdelnour et al. 1992; Normah and Vengadasalam 1992). Seeds and embryos of recalcitrant species also display various characteristics which make their cryopreservation difficult. One of the characteristics of recalcitrant seeds is that there is no arrest in their development, as with orthodox seeds (Berjak et al. 1989b). It is thus very difficult to select seeds at a precise developmental stage, even though this parameter is often of critical importance to achieve successful cryopreservation (Chandel et al. 1995; Engelmann et al. 1995). Very important variations in seed moisture content and maturity stage can be observed between provenances, between and among seed lots, as well as between successive harvests (Berjak et al. 1996). This might well explain contrasting results obtained by different laboratories working on the same species, and non-reproducible results with different seed lots. Seeds of many species are too large to be frozen directly and embryos or embryonic axes have to be employed. However, embryos are often of very complex tissue composition which display differential sensitivity to desiccation and freezing, the root pole seeming more resistant than the shoot pole (Pritchard and Prendergast 1986; Pence 1992, 1995; Dussert, pers. comm.). In some species, embryos are extremely sensitive to desiccation and even minor reduction in their moisture content – down to levels much too high to obtain survival after freezing – leads to irreparable structural damage, as observed notably with cacao (Chandel et al. 1995). Finally, embryos of some species are too large to envisage using them for cryopreservation, and seeds of some species (e.g. Symphonia globulifera, Barringtonia racemosa) do not contain well-defined embryos (Berjak et al. 1996). There are various options to consider for improving storage of non-orthodox seeds. With some species such as tea, mahogany and neem, seeds are relatively small and tolerant to desiccation, and can thus be cryopreserved directly after partial desiccation under the laminar flow (Hu et al. 1994; Marzalina 1995; Berjak and Dumet 1996). With other species which are more desiccation-sensitive, very precisely controlled desiccation (e.g. using saturated salt solutions) and cooling Keynote presentations 15 conditions may allow freezing of whole seeds, as demonstrated recently with various coffee species (Dussert et al. 1997, 1998). As mentioned earlier, desiccation has been employed preferentially for freezing embryos and embryonic axes of recalcitrant seed species and there is scope for various technical improvements in the current cryopreservation protocols. Pregrowth of embryos on media containing cryoprotective substances may give the tissues increased tolerance to further desiccation and reduce the heterogeneity of the material. Berjak and co-workers have demonstrated that flash drying, followed by ultra-rapid freezing, has been very effective for cryopreservation of several species (Berjak et al. 1989a; Wesley–Smith et al. 1992). Their hypothesis is that very rapid dehydration imposes a stasis on metabolism and precludes the deleterious reactions that would take place under lower desiccation rates and that ultra-rapid freezing induces vitrification of internal solutes or the formation of ice crystals too small to disrupt cellular integrity. Even though some species have proven far too desiccation-sensitive to be cryopreserved this way (Pammenter et al. 1993), this is a potentially interesting approach which deserves further research and experimentation with additional species. Other cryopreservation techniques including pregrowth-desiccation, encapsulation-dehydration and vitrification, which have been seldom employed so far should be experimented with (Engelmann 1992; Pence 1995). Finally, it should be emphasized that selecting embryos at the right developmental stage is of critical importance for the success of any cryopreservation experiment (Chandel et al. 1995; Engelmann et al. 1995). However, in these cases, basic protocols for disinfection, inoculation in vitro, germination of embryos or embryonic axes, plantlet development, and possibly limited propagation will have to be established prior to any cryopreservation experiment. In vitro culture of mature embryos is usually achieved with simple culture media (including mineral salts and carbon source) but more complex media have to be formulated to obtain growth of immature material (Raghavan 1994). With species for which attempts to freeze whole embryos or embryonic axes have proven unsuccessful, various authors have suggested using shoot apices sampled on the embryos, adventitious buds or somatic embryos induced from the embryonic tissues (Pence 1995; Berjak et al. 1996). This might be the only solution for species which do not have well-defined embryos but this will require that more sophisticated tissue culture procedures be developed and mastered. In addition to these technical difficulties, this would reduce the range of genetic variability captured (Pence 1995; Berjak et al. 1996), especially when using somatic embryogenesis, since response to inducing treatments is generally highly genotype- specific and somatic embryo cultures might be obtained from a limited number of genotypes only (e.g. Bozkov 1995; Gana et al. 1995). It might be advisable to use this approach with species for which relevant tissue culture procedures are already available, such as rubber tree or cacao. In case apices are to be employed, it might be more practical and efficient to sample them on in vitro plantlets rather than on embryos to reduce the risks of contamination, and to use more homogeneous material. 16 Cryopreservation of Tropical Plant Germplasm

Conclusion Significant progress has been made during the past 10 years in the area of plant cryopreservation, with the development of various efficient vitrification-based freezing protocols. An important advantage of these new techniques is their operational simplicity, since they will be applied mainly in developing tropical countries where the largest part of genetic resources of problem species is located. For many vegetatively propagated species, cryopreservation techniques are sufficiently advanced to envisage their immediate utilization for large-scale experimentation in genebanks. Research is much less advanced for recalcitrant seed species. This is because of the large number of mainly wild species (with very different characteristics) which fall within this category, and the comparatively limited level of research activities aiming at improving the conservation of these species. However, various technical approaches can be explored to improve the efficiency and increase the applicability of cryopreservation techniques to recalcitrant species. In addition, research is actively performed by various groups worldwide to improve knowledge of biological mechanisms underlying seed recalcitrance. It is hoped that new findings on critical issues such as understanding and control of desiccation sensitivity will contribute significantly to the development of improved cryopreservation techniques for recalcitrant seed species.

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Freezing behaviours in plant tissues as visualized by NMR microscopy and their regulatory mechanisms Masaya Ishikawa¹, Hiroyuki Ide², William S. Price², Yoji Arata² and Tomomi Kitashima¹ ¹ Department of Genetic Resources II, National Institute of Agrobiological Resources, Kan'nondai 2-1-2, Tsukuba, Ibaraki 305-8602, Japan ² Water Research Institute, Sengen, Tsukuba, Ibaraki 305-0047, Japan

Introduction Since plants are immobile, they have to survive the various stresses that they encounter. Freezing is one of the severest stresses as it causes ice formation, dehydration and cell deformation. As plant tissues contain a large amount of water, the behaviour of cell water at subfreezing temperatures is a key to tissue survival. To withstand freezing, cold-hardy plants have developed various mechanisms to regulate the formation of ice in their tissues, which result in diverse but tissue- and species-specific freezing behaviours. For instance, wintering temperate woody plant species display diverse freezing behaviours: bark tissues undergo extracellular freezing and xylem ray parenchyma cells of many woody species deep supercool while flower and leaf buds of several genera undergo extra-organ freezing (Fig. 1) (Sakai and Larcher 1987). Whilst most of the tissues of cold-hardy herbaceous plants undergo extracellular freezing, these freezing behaviours are species- and tissue-specific. The elucidation of freezing behaviours in cold-hardy tissues aids the development of cryopreservation techniques. Traditionally, studies on freezing behaviours in plant tissues have been done using differential thermal analysis (DTA), nuclear magnetic resonance (NMR) spectrometry, scanning electron microscopy (SEM) and visual observation. However, these methods can only provide averaged information from a bulk sample or are destructive and liable to cause artifacts (Table 1). Recently, we have developed a novel non-invasive method to visualize the localization of unfrozen water in plant tissues at subfreezing temperatures using NMR microscopy (see Price 1998). We also considered the theoretical background for interpreting the images acquired at freezing temperatures (Ishikawa et al. 1997; Price et al. 1997b). Using this method, we could successfully analyze in detail the various freezing behaviours in cold-hardy woody plant tissues (Ishikawa et al. 1997; Price et al. 1997a; Ide et al. 1998). Here, we review the use of NMR microscopy for the study of freezing behaviours in plants by introducing typical examples. We also briefly review possible regulatory mechanisms involved in the freezing behaviours. Many of the plant tissues and cultures that are used for cryopreservation are non-cold-hardy or freezing-sensitive and have not evolved innate mechanisms to regulate freezing events. The behaviour of water in these tissues is also a key to the survival at liquid nitrogen temperature. Therefore, we have to mimic innate mechanisms either by using cryoprotectants to artificially provide freezing Fundamental aspects of cryopreservation 23

Ice (A) Extracellular freezing

Ice scale Ice Supercool

(B) fl fl Extra-organ PL Dehydration freezing scale 1 2 Ice

(C) R Deep 3 supercooling

Fig. 1. Diversity in the freezing behaviour of cold-hardy plant tissues. A, extracellular freezing in the cortical tissues of Sambucus racemosa subsp. sieboldiana. B, typical extra-organ freezing in flower buds of Cornus officinalis (B-1), Rhododendron dauricum (B-2) and a larch leaf bud (B-3); PL=primordial leaves, fl= floret(s). C, deep supercooling in the xylem ray parenchyma (R) of an apple twig.

A B scale extracellular freezing floret bark

bark, scales E

florets, pith pith extra-organ freezing axis +1°C –7°C axis C D bud scales

Signal intensity F

florets

intracellular freezing –14°C 3 mm –21°C G Temperature low

Fig. 2. 1H-NMR images of a Rhododendron japonicum flower bud cooled at 5°C/h and acquired at various subfreezing temperatures (A-D). Changes in the signal intensity of each tissue or each type of freezing behaviour are shown in E-G (Price et al. 1997a). 24 Cryopreservation of Tropical Plant Germplasm

Table 1. Comparison of methods (conventional and new) used for the study of freezing behaviours in plants Methods Problems and advantages DTA non-spatially specific/ averaged information/ continuous observation Visual observation invasive, destructive/ direct observation of ice, etc. SEM invasive, destructive/ observation of fine ice, etc. NMR spectroscopy non-spatially specific/ averaged information NMR microscopy spatially specific/ non-invasive, non-destructive/various

contrast mechanisms (proton density, T1, T2, diffusion, flow, chemical shift, etc.)/ expensive tolerance or by reducing water content by drying or osmotic treatment following induction of desiccation and/or osmotic tolerance. As NMR microscopy can visualize the distribution of unfrozen water, it is a powerful tool for the study of cryopreservation mechanisms as will be illustrated below.

NMR microscopy: experimental details NMR microscopy (also known as high resolution NMR imaging or NMR micro- imaging) is essentially the same as MRI (magnetic resonance imaging) used in the medical sciences, except that much higher resolutions are used (typically three or more orders of magnitude higher). This allows localized information at the tissue/cellular level to be obtained. As detailed elsewhere (Ishikawa et al. 1997; Price et al. 1997a), 1H-NMR microscopy was conducted on a Bruker DRX 300 NMR spectrometer operating at 300 MHz. Briefly, a 10-mm 1H-imaging insert was mainly used in the imaging probe for imaging the buds. Cold nitrogen gas was used for cooling. Images were acquired using specimens wrapped with Parafilm to reduce evaporation during the imaging process. The images were acquired using a multi-slice multi-echo pulse sequence to ensure that all relevant parts of the specimen were imaged in reasonable time. Normally, a field of view (FOV) of 10 mm ´ 10 mm digitized into 128 pixels in each direction (i.e. the in-plane resolution was typically 78 µm) and a slice thickness of 500 µm was used. This degree of resolution was deemed to be a suitable compromise between resolution and acquisition time (N.B. the signal-to- noise ratio is proportional to the volume element). Typical image acquisition parameters were a recycle delay (TR) of 1.2 s and an echo time (TE) of about 7 ms (the minimum possible echo time given the other acquisition parameters). Using these experimental parameters, each set of four multi-slice images required about 20 min to acquire. The contrast in the images mainly reflects the spin-spin relaxation (T2) and spin-lattice relaxation (T1) of the studied nuclei (protons in the present case). We used a long recycle delay and a short echo time and thus, the intensity in the images should predominantly reflect the density of the mobile protons (mainly from liquid water). In the winter buds studied, the (liquid) water has a sufficiently long T2 such that its signal was still measurable at the end of the echo period in the imaging pulse sequence. However, water in the ice state has a Fundamental aspects of cryopreservation 25

submillisecond T2 and consequently is no longer detected at the end of the echo period. Thus, the light areas contain liquid water whilst the areas that have become dark during cooling represent frozen areas (or very low proton density).

Typical NMR images of freezing behaviours in wintering woody plant tissues Here we present only a few but typical examples from diverse freezing behaviours that are specific to plant tissues and species.

Extracellular freezing Typical images of extracellular freezing are seen in the scales and bark tissues of Rhododendron japonicum (Fig. 2A-D) and maple flower buds (Fig. 3A-G). The intensity of the NMR signals from these tissues greatly decreased upon cooling to -7°C (Fig. 2B, Fig. 3B and F). This indicates the spontaneous freezing of these tissues, most probably extracellular freezing, which corresponds to the HTE in the DTA profiles (Fig. 4). When the buds were further cooled to -14°C, the signal intensity of these tissues further decreased, implying the tissues had little unfrozen water (Figs. 2C, 3C and G). These decreases in the signal intensity are schematically presented in Fig. 2E. This is consistent with the report by Gusta et al. (1975) which showed that 70-80% of the total tissue water freezes during cooling to –14°C during extracellular freezing.

Extra-organ freezing in shoot and flower primordia In R. japonicum flower buds, all the florets remained unfrozen at –14°C and even at –21°C for some florets (Fig. 2C and D). This was consistent with the LTEs recorded between –17 and –27°C in the DTA profile (Fig. 4A). During cooling to lower temperatures, a gradual reduction in floret size was also noted in the NMR images of this species (Fig. 2A-D). There was about a 20% decrease in the area of the image of florets during cooling from +1 to –21°C (details not shown). The slow dehydration of florets was consistent with the previous observation of flower buds dissected at subfreezing temperatures (Ishikawa and Sakai 1981, 1982). This slow dehydration of the supercooled florets results in an enhancement of supercooling and complementary ice accumulation in the bud scales (i.e. formation of an ice sink: see Fig. 1B) at naturally occurring cooling rates (Ishikawa and Sakai 1982). This complex freezing event (i.e. extra-organ freezing) could be easily seen in the NMR images of flower buds of R. japonicum (Fig. 2) and maple (Fig. 3). Typical changes in the signal intensity of the tissues during extra-organ freezing are summarized in Fig. 2F. Freezing of supercooled florets is lethal (data not shown) probably due to intracellular freezing. This event was seen in the NMR images as a dramatic decrease in the signal intensity (Fig. 2C–D, G). In maple (Acer japonicum) flower buds, the entire flower primordia (inflorescence with two surrounding leaves) remained supercooled and the size was gradually reduced during cooling to –14°C (Fig. 3). The freezing of the primordia that occurred at –20ºC (Fig. 4C) was lethal (data not shown). As shown 26 Cryopreservation of Tropical Plant Germplasm in the NMR images (Figs. 2 and 3), the frozen tissues (scales, bark, etc.) and the supercooled tissues (florets, inflorescence) are in close vicinity. However, freezing in one tissue does not necessarily propagate into other tissues. The mechanisms that prevent propagation from the frozen to the unfrozen tissues (e.g. bud axis in azalea, immature stem in maple) will be described later. NMR microscopy clearly allows the visualization of such boundaries, for example, between the frozen immature stem and supercooled inflorescence (Fig. 3F, arrow) and the gradual dehydration (arrowhead in Fig. 3G) from the basal part of the inflorescence during cooling to the adjacent already frozen tissues, which otherwise are not detectable with traditional methods.

Deep supercooling in the xylem In many temperate deciduous trees, the xylem ray parenchyma cells are generally believed to deeply supercool and thereby remain viable, whereas freezing of these cells is lethal. NMR images revealed that the mature xylem tissues of A. japonicum (Fig. 3) remained unfrozen (showing high signal intensity) even at –21°C, which is consistent with the DTA profile of the twig piece, which has a broad LTE starting around –30°C (Fig. 4B). However, with the resolution employed, it is not possible to confirm whether only the xylem ray parenchyma supercool as the images looked as if the entire xylem remained unfrozen (Fig. 3). In contrast, in azalea flower buds, deep supercooling was detected in the NMR images of the mature pith and not in the xylem (Fig. 2).

Relationship to survival at super-low temperatures In the lateral leaf buds at the base of terminal flower buds of maple, the shoot primordia remained unfrozen even at –21ºC as shown in the NMR images (Fig. 3A–D). The size of the supercooled lateral shoot primordia in the images was reduced by 58% during cooling from +1 to –21ºC and the rate was much greater than in terminal flower or shoot primordia (Fig. 3H). If the buds were cooled further with this rate of size reduction, the shoot primordia would lose most of the tissue water. In fact, DTA analysis of terminal flower buds with lateral buds detected no LTE between –21 and –40ºC (cooling rate: 5ºC/h) (data not shown). Visual determination of the viability of the same buds (cooled to –40ºC) revealed that the lateral shoot primordia were all viable (the flower primordia were killed). The lateral buds of A. japonicum have been reported to consistently tolerate –70ºC after full cold-hardening (Ishikawa et al. 1997). These facts imply that lateral shoot primordia of A. japonicum undergo Type-I extra-organ freezing (Ishikawa and Sakai 1982), in which the organ is dehydrated at a considerable rate during slow cooling and can tolerate the resultant fully dehydrated state. Since all the freezable water in the organ is removed during slow cooling, the organ can tolerate temperatures lower than the homogeneous ice-nucleation temperature (around –40 ºC) (George and Burke 1977) and sometimes even immersion in liquid nitrogen. Type-I extra-organ freezing has been observed in leaf buds of very cold-hardy spruce and larch (Fig. 1, B-3) and hydrated seeds of small size (Ishikawa and Sakai 1982; Sakai and Larcher 1987). Fundamental aspects of cryopreservation 27

scale +1 infl -7 -14 -21°C

xylem

LB

bark A B C D

+1°C -7°C -14°C 1 mm 100 H scale infl 80

60

40 Flower primordia 20 IS Terminal shoot primodia xylem % area of primordia images Lateral shoot primodia bark 0 1 -7 -14 -21 E pith F G Temperature, °C

Fig. 3. 1H–NMR images of a flower bud of Acer japonicum cooled at 5°C/h. A–D and E–G (slightly larger magnification) are images taken at different temperatures of different slices of the same bud; LB=lateral leaf bud. There was a clear boundary (indicated by the arrow in Fig. 3F) between the frozen immature stem (IS) and supercooled inflorescence (infl). H, Changes in the size of supercooled tissues measured gravimetrically from the NMR images (Ishikawa et al. 1997).

HTE Temperature,LTE ÞC

A

HTE LTE B Exothermic response HTE C LTE

0 -5 -10 -15 -20 -25 -30 -35 -40

Temperature (°C)

Fig. 4. Typical DTA profiles of a flower bud (A) of Rhododendron japonicum and of a twig piece (B) and a flower bud (C) of Acer japonicum. HTE and LTE represent high and low temperature exotherms respectively. 28 Cryopreservation of Tropical Plant Germplasm

Technical and interpretation problems In comparing the images taken at different temperatures, it is important to consider the factors that influence the signal intensity apart from that resulting directly from the water to ice transition. There are a number of factors that affect the image intensity as the temperature decreases and we have considered these factors in detail (Ishikawa et al. 1997; Price et al. 1997b). As the temperature is decreased, the T1 of the tissue water decreases, which results in an increase in image intensity as the water is able to more fully reach thermal equilibrium before the start of the next scan in the imaging sequence (N.B. the repetition delay of the imaging sequence is normally too short to allow the water spins to fully relax so that the water magnetization is always partly saturated). However, concomitantly the T2 of the water resonance decreases which leads to a loss of signal intensity. These two competing effects largely compensate for each other except at the freezing point when all signal intensity is effectively lost owing to the drastic shortening of T2. Nevertheless in some tissues such as supercooled florets (Fig. 2) the increase in intensity due to the decrease in the T1-saturation effect with decreasing temperature is observed (Ishikawa et al. 1997; Price et al. 1997b). Other factors that need to be accounted for when interpreting NMR images include signals from non-water protons, local distortions caused by magnetic susceptibility differences due to air bubbles, ghosting of images, etc. These have been detailed elsewhere (Ishikawa et al. 1997; Price et al. 1997a, 1997b).

Advantages of NMR microscopy compared with the conventional method of freezing behaviour studies NMR imaging provides spatial information that can be correlated with non- spatial information obtained from traditional techniques such as DTA. In particular it allows the identification of the tissues that produce the exotherms observed using DTA. Importantly, the tissues that are frozen can be immediately recognized by eye from the image of the whole plant specimen, which allows unambiguous determination of the organized freezing behaviour of the plant tissues. The high resolution and non-invasive nature of NMR imaging allows the freezing behaviours to be observed in small or difficult (or impossible) to excise tissue pieces (e.g. bud axis). Apart from the non-invasive nature of NMR imaging which allows visualization of ongoing physical processes in live samples, the other advantage of NMR imaging is its sensitivity to a wide range of chemical (i.e. chemical shift) (Pope et al. 1993; Ishida et al. 1996) and physical contrast mechanisms such as diffusion, flow and relaxation (Köckenberger et al. 1997; Price 1998) (Table 1). These contrast mechanisms cannot be observed by other methods. In the examples shown, the contrast in the images was provided by the decrease in relaxation times of the water molecules resulting from the reduction in (reorientational) motion as they enter the ice state. As noted above, water in different parts of the buds has different characteristics (e.g. different T1 and T2 relaxation times and activation energies, etc.). By using different imaging Fundamental aspects of cryopreservation 29 sequences and appropriate parameters, these differences can be used to provide additional sources of contrast (data not shown). Thus, the imaging technique and parameters can be 'tuned' to be sensitive to the different states of water in buds and the resulting information is ultimately useful in helping to understand the mechanisms involved in freezing behaviours and cold acclimation (Millard et al. 1995).

Regulatory mechanisms of freezing behaviours As seen in the NMR microscopy images, various fractions of water in cold-hardy plant tissues remain stably supercooled to temperatures as low as –20ºC or lower in spite of adjacent tissues freezing spontaneously at temperatures higher than -7ºC (Figs. 2 and 3). It is of interest to know how the plants regulate the freezing of their tissues. There are at least structural and biochemical factors involved.

Structural factors The deep supercooling ability of xylem ray parenchyma cells persists even in cells killed by heating or freezing; thus the cell wall structure (i.e. non-living component of cells) has been considered important (George and Burke 1977; Ashworth and Abeles 1984). Pure water in a finely dispersed state supercools to as low as –40ºC, which is the homogeneous nucleation temperature of water (George and Burke 1977). The parenchyma cells can be considered as fine droplets of water surrounded by cell walls. The cell walls should work as a barrier that excludes the growth of ice crystals into a cell and concomitantly as a barrier against water movement from the cell to extracellular ice. The cell walls should also have sufficient tensile strength to withstand the negative hydrostatic pressures resulting from the vapour pressure difference between water and ice. To meet these requirements, porosity and permeability of the cell walls have been considered important (George and Burke 1977; Wisniewski and Ashworth 1987). Wisniewski and co-workers (1991) showed a positive involvement of the pit membrane structure, especially pectic components in the deep supercooling of xylem ray parenchyma. There have been some studies show ing the involvement of vascular tissues in ice propagation and the maintenance of supercooling. In the supercooling of peach flower buds, the viability of the bud axis is critical for the supercooling flower primordia (Ashworth et al. 1989) and the prevention of ice propagation into flower primordia from the frozen tissues may be related to the non- differentiation of xylem vessel elements in the vascular tissues. This was subsequently confirmed in some species but not necessarily in some other species in the genus Prunus (Kader and Proebsting 1992). This is not the case in overwintering plants but the propagation of freezing is known to sometimes be delayed at the nodes in wheat stems when frosted during the growing season (Single 1964). The structure of vascular tissues in the stem is considered to be responsible for this delay. Physiological or biochemical factors involved in the freezing behaviour of cold-hardy plant tissues include several possible factors such as ice-nucleating 30 Cryopreservation of Tropical Plant Germplasm activities, antifreeze (ice-binding) activities, anti-nucleating activities, etc. Here the first two cases are briefly described.

Ice-nucleating activities Cold-hardy plants seem to positively control the initiation of freezing by producing ice-nucleating substances in the tissues that are designated to freeze early. To determine why the scale tissues of R. japonicum freeze spontaneously during cooling to –7ºC, the ice-nucleating activity of detached scales and other tissues was determined using an improved test tube method (Ishikawa, unpubl.) based on the method developed by Hirano et al. (1985). The results are summarized in Fig. 5. The outer and inner bud scales which freeze first and work as ice sinks had the highest ice-nucleating activity (INA) (–5.9 and –6.8ºC, respectively), while the florets which remain supercooled lacked effective INA (-13 to –16ºC). In the twigs, the bark tissues which undergo extracellular freezing had a high INA of –6.3ºC whilst the xylem and pith tissues which undergo deep supercooling had low INA (–12.8 and –12.4ºC, respectively). The INA of each tissue closely corresponded with its freezing behaviour. These INA values were affected only slightly by a 4–fold increase in the tissue amount and also by an increase in the incubation time at subfreezing temperatures (data not shown). This means that these INA are specific to the tissues. The high INA in the outer scales was unaffected by homogenization of the tissues into fine powders and subsequent extensive washings. This implies that this INA does not arise from macrostructures such as the presence of trichomes or the outer scales situated in the outermost part of the buds, but from substances tightly bound to the cell walls. The high INA in the bud scale tissues was resistant to autoclaving (121ºC for 15 min) whilst the high INA in the bark tissues was completely lost following autoclaving (data not shown). This implies that the substances responsible for the INA in each tissue have different sensitivities to heat treatment. Characterization and isolation of these ice-nucleating activities have been attempted in our laboratory, which will be detailed elsewhere (Ishikawa, unpubl.).

Antifreeze activities There has been no study on the antifreeze or ice-binding activities in relation to the freezing behaviour of bud tissues of woody plants, to our knowledge. However in some cold-hardy herbaceous plant tissues, antifreeze proteins have been detected in the apoplast. Hon et al. (1995) reported that the antifreeze activities in the apoplast of cold-hardened winter rye were confined to three pathogenesis-related (PR) proteins (Fig. 6). When isolated, these proteins were able to retard ice growth. More recently, a polygalacturonase inhibitor (another PR protein) homologue has been found to be responsible for the ice-binding activity in the apoplast of cold-hardened carrot roots (Worrall et al. 1998). The function of these antifreeze proteins in the apoplast of herbaceous plants, which undergo extracellular freezing, is not very clear since the degree of thermal hysteresis (i.e. actual prevention of initiation of freezing) generated by these proteins is small. Fundamental aspects of cryopreservation 31

Fig. 5. Ice-nucleating activities (INA) of various tissues of a Rhododendron japonicum flower bud determined using a test tube method. The values are the 50% ice-nucleating temperature of 40 replicates.

NA Cold acclimated

3 wk 1d 1wk 3wk 5wk 7wk

Fig. 6. Increases in the ability to inhibit ice crystal growth on the a axis of apoplast fluid isolated from winter rye during cold acclimation, which resulted in the bipyramidal ice crystal growth along the c axis (modified from Hon et al. 1995).

It is generally thought that they inhibit the recrystallization of ice and thus prevent the growth of ice crystals in the extracellular space during prolonged exposure to subfreezing temperatures. One problem in the study of antifreeze activities is the assay method which is rather qualitative and poorly reproducible. 32 Cryopreservation of Tropical Plant Germplasm

Worrall et al. (1998) have developed an assay method; however, since the assay mixture contains a large amount of sucrose, it has been questioned if the results represent the real ice-binding properties. Hence we are attempting to develop a new antifreeze activity assay and are also trying to isolate new classes of antifreeze activity fractions from the various tissues of wintering buds of woody plants.

Possible application of NMR microscopy to cryopreservation studies Since proton NMR microscopy non-invasively visualizes the concentration and distribution of water, the method can also be applied to cryopreservation studies. Some possible applications are summarized in Table 2. One application is to clarify the distribution and amount of water within the sample to be cryopreserved either at the preculture, desiccation, imbibition in the cryoprotectants (or vitrification solutions) or the slow prefreezing step (Fig. 7). The materials can range from the embryo axis in recalcitrant seeds, embryos, apical meristems to axillary buds which show differential survival responses to the cryogenic temperatures depending on the water content and tissue parts of the samples. The other type of applications take advantage of the different contrast mechanisms available such as the relaxation times and diffusion of water (Price 1998) and chemical shift imaging of various compounds (Pope et al. 1993; Ishida et al. 1996). Diffusion-weighted imaging and relaxation time imaging of water can differentiate the change and/or the distribution of the state of water within a sample during preculture, cold acclimation, desiccation or imbibition in the cryoprotectants. Chemical shift imaging can, for example, visualize the distribution of compounds such as sucrose, and thus the method can follow the changes in the content and distribution of sucrose during preculture with increasing concentration of sucrose. Another important study may be to follow the penetration of cryoprotectant or vitrification solution during imbibition since the location of the components of a vitrification solution in the sample to be cryopreserved is an important issue (Hagedorn 1996) and has never been studied in plant systems to our knowledge. An important application of NMR spectroscopy (not imaging) in the last decade has been the extensive studies on the behaviour of molecules in the vitrified state in dry materials and lyophilized biopolymers (Angell 1995; Stillinger 1995). NMR studies show that the transition from vitrified state to crystals is more a continuous phenomenon than an abrupt change and that this transition can occur at much lower temperatures than the devitrification (glass transition) temperatures determined by differential scanning calorimetry (DSC). Any of the methods used for cryopreservation depend upon the vitrification of water molecules except for completely desiccated samples. Since cryopreservation is normally done for long-term storage, samples with devitrification temperatures around –110 to –120ºC should preferably be stored in liquid nitrogen in consideration of the results of the recent NMR studies on vitrification. Such samples, if stored at temperatures only slightly lower than the glass transition temperature, could possibly deteriorate in the long term. Fundamental aspects of cryopreservation 33

Table 2. Possible applications of NMR microscopy (non-invasive, spatially specific tool) for the study of cryopreservation of plant genetic resources (recalcitrant seeds, meristems, buds, etc.) and in vitro cultured materials

A. Preculture step 1. Visualization of changes in the state and amount of water within minute samples. 2. Imaging changes in osmolytes (sucrose, etc.).

B. Desiccation step 1. Visualization of changes in the state and amount of water in the sample.

C. Mechanism of cryoprotectants 1. Visualization of changes in the state and amount of water in the sample during imbibition in the cryoprotectants. 2. Imaging the penetration and distribution of cryoprotectants (sucrose, DMSO).

D. Winter buds 1. Visualization of changes in the state and amount of water in the sample during cold acclimation. 2. Imaging the freezing process of the tissues during slow cooling prior to liquid nitrogen submersion.

+1°C -7°C -14°C -21°C

scale PL axis

bark

xylem

Fig. 7. 1H–NMR images of a terminal mulberry bud cooled at 5°C/h. The bud scales and bark readily froze by –7°C as indicated by the decrease in signal intensity. The scales worked as an ice sink. PL: primordial leaves. The amount of unfrozen water remaining in the tissues is probably related to the differential viability response at cryogenic temperatures depending on the tissues and pre-freezing temperatures (Yakuwa and Oka 1988). 34 Cryopreservation of Tropical Plant Germplasm

In conclusion, NMR microscopy is a powerful tool for studying the freezing behaviours in cold-hardy plant tissues and also cryopreservation mechanisms since it can non-invasively provide spatially-specific information. Cold-hardy plants seem to have developed various mechanisms to control freezing events and the deep understanding of these may lead to important advances in cryopreservation techniques.

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Millard, M.M., O.B. Veisz, D.T. Krizek and M. Line. 1995. Magnetic resonance imaging (MRI) of water during cold acclimation and freezing in winter wheat. Plant, Cell and Environment 18:535–544. Pope, J.M., D. Jonas and R.R. Walker. 1993. Applications of NMR micro-imaging to the study of water, lipid, and carbohydrate distribution in grape berries. Protoplasma 173:177–186. Price, W.S. 1998. NMR imaging. Pp. 139–216. in Annual Reports on NMR Spectroscopy. G.A. Webb (ed.). Academic Press, New York. Price, W.S., H. Ide, Y. Arata and M. Ishikawa. 1997a. Visualization of freezing behaviours in flower bud tissues of cold hardy Rhododendron japonicum by nuclear magnetic resonance micro-imaging. Australian Journal of Plant Physiology 24:599–605. Price, W.S., H. Ide, M. Ishikawa and Y. Arata. 1997b. Intensity changes in 1H–NMR micro- images of plant materials exposed to subfreezing temperatures. Bioimages 5:91–99. Sakai, A. and W. Larcher. 1987. Frost Survival of Plants. Responses and Adaptation to Freezing Stress. Ecological Studies No 62. Springer Verlag, Berlin. Single, W.V. 1964. Studies on frost injury to wheat. II. Ice formation within the plant. Australian Journal of Agricultural Research 15:869–875. Stillinger, F.H. 1995. A topographic view of supercooled liquids and glass formation. Science 267:1935–1939. Wisniewski, M. and E.N. Ashworth. 1987. The use of lanthanum to characterize cell wall permeability in relation to deep supercooling and extracellular freezing in woody plants. II. Intrageneric comparisons between Betula lenta and Betula papyrifera. Protoplasma 141:168 Wisniewski, M., G. Davis and R. Arora. 1991. Effect of macerase, oxalic acid and EGTA on deep supercooling and pit membrane structure of xylem parenchyma of peach. Plant Physiology 96:1354–1359. Worrall, D., L. Elias, D. Ashford, M. Smallwood, C. Sidebottom, P. Lillford, J. Telford, C. Holt and D. Bowles. 1998. A carrot leucine-rich-repeat protein that inhibits ice recrystallization. Science 282:115–117. Yakuwa, H. and S. Oka. 1988. Plant regeneration through meristem culture from vegetative buds of mulberry Morus bombycis Koidz. stored in liquid nitrogen. Annals of Botany 62:79–82. 36 Cryopreservation of Tropical Plant Germplasm

Ultrastructural aspects of freezing adaptation of cells by vitrification Seizo Fujikawa¹ and Yutaka Jitsuyama² ¹ Institute of Low Temperature Science, Hokkaido University, Sapporo 060-0819, Japan ² Faculty of Agriculture, Hokkaido University, Sapporo 060-0819, Japan

Introduction Vitrification procedures for long-term biological cryopreservation provide a vitrified state through entire samples, that is, including all solutions both inside and outside of cells, by pretreatment of samples with a vitrification solution (VS) followed by cooling with liquid nitrogen (Steponkus et al. 1992). Conversion of water to a vitrified state, at least inside the cells, is also a prerequisite for adaptation of biological materials to freezing conditions, especially at extremely low temperatures, in both natural freezing and artificial cryopreservation. Vitrification of water in the cells prevents lethal effects of intracellular freezing as well as lethal effects of excess dehydration. In this presentation, based mainly upon studies with cryo-scanning electron microscopy (Cryo-SEM) and freeze- fracture replica electron microscopy (FFR), we first present ultrastructural evidence concerning the causes of freezing injury, and then suggest a wide variety of possible vitrification states capable of preventing freezing injury.

Method for observing frozen biological samples by combination of Cryo-SEM and FFR Samples of biological materials for observation were frozen in conditions controlled with respect to cooling rates and freezing temperatures in the presence or absence of pretreatments, and then cryofixed from the freezing conditions to -150 oC at a cooling rate exceeding 10 000 oC/min and stored in liquid nitrogen (LN). We routinely use a cryo-unit for electron microscopy that allows observation of the freezing pattern of samples over the whole area within 3 mm2 at magnifications from ×20 to more than ×10 000 by means of a secondary emission Cryo-SEM image, and then observation of part of the sample with higher magnification by transmission electron microscopy, using FFR obtained from Cryo-SEM samples by dissolution to leave a replica (Fujikawa et al. 1988; Fujikawa 1991). The final images of the frozen fixed samples were obtained using this cryo- unit at temperatures between –100 and –160 oC. These processes allow for observation of the intrinsic structural changes due to the controlled freezing conditions, without effects of thawing or warming, at levels from whole tissue to the molecular assembly. Fundamental aspects of cryopreservation 37

Ultrastructural evidence of occurrence of freezing injury

Freezing injury by intracellular freezing Intracellular freezing is fatal for cells in all biological tissues. In plant cells, FFR showed apparent plasma membrane rupture by the formation of a large amount of intracellular ice in the frozen state. The intracellularly frozen cells are subjected to further ultrastructural changes during a thawing process, resulting in serious damage of cells after thawing (Fujikawa 1994a).

Freezing injury by extracellular freezing In many plant tissues subjected to equilibrium slow freezing, FFR revealed ultrastructural changes in the plasma membranes of cells that produced freezing injury. These reflect the formation of aparticulate (intramembrane particle-free) domains with accompanying lamellar-to-hexagonal II (HII) phase transitions or accompanying fracture-jump lesions (Gordon-Kamm and Steponkus 1984; Fujikawa and Miura 1986; Steponkus and Lynch 1989; Webb and Steponkus 1993; Steponkus et al. 1993; Fujikawa 1994b). Fracture-jump lesions have been shown to be sites of membrane fusion (Fujikawa 1995). These plasma membrane ultrastructural changes were produced only at sites where at least two membranes were in close proximity to each other (Fujikawa and Miura 1986; Steponkus and Lynch 1989). The cells producing these ultrastructural changes under freezing were seriously disrupted on thawing. There was a strong association between occurrence of freezing injury and formation of these ultrastructural changes in the plasma membranes as a result of the freezing-induced close approach of membranes (Fujikawa 1987). It is important to note that the close approach of membranes, leading to injury, develops not only by osmotic shrinkage of cells due to freezing-induced dehydration, but also by mechanical deformation of cells due to growth of extracellular ice. Many plant cells are more seriously damaged by actual freezing than by osmotic treatment with the corresponding osmolarity (Tao et al. 1983; Fujikawa and Miura 1986; Murai and Yoshida 1998). Cryo-SEM and FFR observation showed that freezing resulted in a high frequency of close membrane approach due to both cellular shrinkage and cellular deformation, while osmotic treatment resulted in a lower frequency of the close membrane approach, due to cellular shrinkage alone. It is also noted that deformation of cell walls due to growth of extracellular ice is strongly related to the occurrence of close membrane approach by facilitating cellular deformation.

Various factors for avoiding freezing injury When we consider that the close approach of membranes is one of the main factors in occurrence of freezing injury, it is reasonable to suggest that the survival of cells may be obtained by avoiding conditions that produce close approach of membranes during freezing. It is suggested that many cellular changes that occur during cold acclimation in nature are related to prevention of the close membrane approach. 38 Cryopreservation of Tropical Plant Germplasm

These protection mechanisms include an increased number of hydrated phospholipids in the polar head groups on the surface of plasma membranes (Steponkus and Webb 1992), accumulation of compatible osmolytes, such as soluble carbohydrates, in cytoplasm (Sakai and Yoshida 1968), stiffening of the cell walls (Rajashekar and Lafta 1996), accumulation of anti-freeze proteins (AFP) in the sap of apoplastic spaces (Griffith and Antikainen 1996), and secondary changes in endoplasmic reticulum (ER) (Fujikawa and Takabe 1996). Among these, there is a strong possibility that accumulation of soluble carbohydrates can prevent injurious close membrane approach during freezing at lower temperatures, such as LN temperatures, probably by forming a vitrification state between membranes.

Evidence of vitrification survival in embryogenic cells incorporating sugar Suspension cultures of embryogenic cells of asparagus have a constitutive freezing tolerance (tolerance to equilibrium slow freezing) of –8oC (LT50). Pre- incubation of these culture cells in the presence of 0.8M sucrose for 2 d resulted in increased freezing tolerance of –25 oC (LT50). Confocal laser scanning microscopy (CLSM), using Lucifer Yellow–CH as a tracer of incorporated sucrose (Oparka et al. 1990), verified the incorporation of sugars inside the cells during incubation. When these sugar-loaded cells were slowly frozen to –20oC and then immersed directly in LN, the survival at –20 oC (60%) remained unchanged after rapid thawing. However, when samples were slowly frozen to –20oC and immersed in LN, then kept at –20oC for sufficient time during thawing from LN, the ability to survive after rapid thawing was almost lost. The samples frozen slowly to –20 oC and immersed in LN did not show evidence of intracellular ice. On the other hand, the samples frozen in the same way but kept at –20 oC during thawing exhibited formation of large intracellular ice. It is suggested that large intracellular ice is produced as a result of devitrification of intracellular solutions that remained in the liquid state during slow freezing to –20oC, and then converted to a vitrification state upon freezing in LN. The survival of slowly frozen cells (not being immersed in LN) gradually reduced in parallel with reduction of final freezing temperature, decreasing, for example, from 60% at –20 oC to 40% at –40 oC. From these results, it is suggested that at least a part of survival after immersion in LN of cells frozen slowly to -20oC is obtained by the conversion of residual water to a vitrification state during freezing in LN. The frequency of injurious ultrastructural changes of plasma membranes by close approach of membranes is significantly lower in cells that were slowly frozen to –20oC and immersed in LN, than in cells that were slowly frozen to -40oC. It is suggested that the vitrification state, which may be produced by concentrated sucrose as a result of dehydration during slow freezing to –20 oC, prevents close apposition of membranes and consequently reduces occurrence of freezing injury. Fundamental aspects of cryopreservation 39

Various forms of vitrification survival Various forms of survival, probably by vitrification, are seen in a wide variety of natural and artificial freezing. In nature, cortical parenchyma cells in cold-hardy woody plants have a high freezing tolerance, even below –40 oC in winter. In these woody cells, abundant soluble carbohydrates are accumulated as a result of seasonal cold acclimation. With slow temperature reduction during winter, extracellular freezing takes place in these tissues. The resultant dehydration by extracellular freezing concentrates cellular solutes, and a vitrified state is produced even at comparatively high freezing temperatures or slow cooling rates. This phenomenon is referred to as high-temperature vitrification and has been verified by differential scanning calorimetry (Hirsh et al. 1985). FFR showed that in these woody plant cells with extremely high freezing tolerance in winter, the close approach of membranes is completely inhibited. We suppose that the formation of a vitrified state is also possible in artificial freezing of biological materials to LN temperature for the purpose of cryopreservation. During freezing with controlled cooling rates and without addition of cryoprotectants, yeast cells (Bank and Mazer 1973) and human red blood cells (Fujikawa 1981) can survive at an optimum cooling rate, which corresponds to the fastest rate permitting extracellular freezing. It is shown that at optimum cooling rates, these cells are larger than cells subjected to slower cooling, which show higher injury rates (Bank and Mazer 1973). The greater size of surviving cells at the optimum cooling rate is apparently due to decreased dehydration, suggesting the presence of vitrified water in these cells. In a routine method of conventional cryopreservation, samples are loaded first with dilute cryoprotectants, frozen at optimum cooling rates to below (at least) -30 oC and then immersed in LN. Concentration of cryoprotectants as a result of dehydration during slow freezing may produce a vitrified state during slow freezing or during immersion in LN. The presence of cryoprotectants in the extracellular spaces may also reduce formation and growth of a large extracellular ice in size, which may reduce the degree of cellular deformation due to growth of extracellular ice. We showed above that reduction of cellular deformation may reduce freezing injury by reducing the close approach of membranes. Some dried seeds and pollens can survive direct immersion from room temperature into LN (Sakai and Larcher 1987). Such organs with constitutively high dehydration tolerance may convert all water to a vitrification state on freezing in LN, without dehydration occurring during freezing. In these cells, FFR showed no evidence of ice crystals.

Diverse contributions allowing survival in cryopreservation procedures by promoting vitrification In vitrification procedures for cryopreservation, all solutions, both inside and outside of the cells, are converted to a vitrified state by immersion in LN. For this purpose, samples were first loaded with dilute cryoprotectants and then subjected to immersion into vitrification solution (VS) before immersion in LN. Because VS itself converts to a vitrified state upon immersion in LN, we may 40 Cryopreservation of Tropical Plant Germplasm easily fall into the misunderstanding that, in every case, almost all solutions inside of the cells may be substituted with VS. However, the situation inside the cells that allows for survival by vitrification is produced in various ways depending upon the combination of the kind of sample and the kind of VS used. In protoplasts isolated from rye leaves (Fujikawa and Steponkus 1991; Steponkus et al. 1992), treatment with VS (7M ethylene glycol, 0.88M sorbitol, 6% bovine serum albumin) produces mainly only cellular dehydration without penetration of VS inside the cells. The vitrification state inside the cells will be produced by concentration of pre-loaded dilute cryoprotectants (in non- acclimated samples) or by concentration of endogenously accumulated cryoprotectants (in cold-acclimated samples), as a result of dehydration by immersion in VS. In these cases, dehydration with non-penetrating sugars, instead of VS, resulted in similar shrinkage of cells as well as survival rates similar to those with VS treatment. Thus, the situation is similar to the case of conventional cryopreservation, only differing in the manner of dehydration, by slow freezing and by VS, respectively. In bovine blastocysts (Kuwayama et al. 1994), a vitrification state allowing for higher survival may be produced by partial penetration of VS (20% calf serum, 22.5% glycerol, 22.5% 1,2–propanediol) inside the cells. In these samples, step- wise application of VS in 16 steps resulted in reduction of cellular shrinkage as well as increased survival, as compared with treatments of less than 8 steps. It is suggested that higher survival may be produced by more penetration of VS inside the cells as a result of step-wise treatment with VS. In mouse blastocysts (Fujikawa et al. 1992; Valdez et al. 1992), higher survival was obtained with VS (20% ethylene glycol, 20% dimethylsulfoxide, 10% 1,3– butanediol), which can penetrate very quickly inside the cells without evidence of conspicuous cellular shrinkage. In a sample of these cells, pre-loaded with dilute cryoprotectants and treated with non-penetrating sugars (with osmolality corresponding to that of VS), severe damage and distinct cellular shrinkage were observed. Thus it is suggested that in these samples, higher survival may result from vitrification, mostly of VS that penetrated the cells. As a result of the penetration of a large amount of VS inside the cells, serious cellular damage was produced by chemical toxicity when samples remained in VS for too long.

Conclusions The present study shows that conversion of cellular solutions to a vitrification state during freezing to lower temperatures, such as LN temperature, is an important factor for freezing survival of biological materials. This is so not only for the purpose of cryopreservation but also for all categories of freezing. The vitrified state inside cells may prevent freezing injury produced by close apposition of membranes. In this regard, presence of an extracellular vitrified state during cryopreservation may also have a role in preventing or reducing freezing injury, by inhibiting the cellular deformation that is produced by growth of extracellular ice. Fundamental aspects of cryopreservation 41

Acknowledgements Part of this presentation was based on collaborative work with Dr C.A. Valdez, Dr M. Kuwayama and Dr P.L. Steponkus. This study was supported by a grant from the Ministry of Education, Culture and Science of Japan, and from the Cooperative Research Funds of the Institute of Low Temperature Science.

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The use of physical and biochemical studies to elucidate and reduce cryopreservation-induced damage in hydrated/desiccated plant germplasm Dominique Dumet and Erica E. Benson Plant Conservation Biotechnology Group, Division of Molecular and Life Sciences, School of Science and Engineering, University of Abertay-Dundee, Dundee, DD1 1HG, Scotland

Introduction Successful cryopreservation is dependent on a number of practical and applied factors. However, the development of improved cryopreservation strategies, (e.g. for recalcitrant tissues) can be greatly assisted through increasing our fundamental understanding of cryoprotective mechanisms and by elucidating the basis of cryopreservation-associated injuries. This paper will first introduce the established theories of cryo-injury and cryoprotection and present them in terms of the injurious physical and biochemical events associated with freezing phenomena. This will be followed by an overview of two analytical themes, which can be used to profile freezing and cryo-injury. The first concerns the use of thermal analysis [specifically Differential Scanning Calorimetry (DSC)] and cryomicroscopy to study cryopreservation. The second explores the potential of using fundamental biochemical studies of free radical mediated oxidative stress to develop improved cryopreservation strategies. Physical and biochemical techniques can be used to profile the different components of cryopreservation protocols and discern the magnitude of injurious events. Thermal analyses can profile changes in moisture content status, ice-nucleation and vitrification and similarly, biochemical markers of oxidative damage and antioxidants can be used to indicate cellular responses to cryoprotection and freezing injury. Finally, this paper discusses the application of fundamental approaches to develop improved cryopreservation protocols, with emphasis on their use in the study of desiccation stress, and in the application of novel strategies to ameliorate injury in cryopreserved plant germplasm. The paper has been largely structured for readers who are considering, for the first time, the use of fundamental and/or analytical approaches for the study of cryopreservation injury.

The “two-factor” hypothesis of cryo-injury: the role of water Water is one of the most important components of living systems, conferring structural order and regulating every life process. The control of water status during cryopreservation is the key factor in developing successful cryoprotective strategies and limiting cryo-injury. According to Crowe et al. (1988), 95% of the water present in biological tissues is “free” and will convert to ice during freezing, causing irreversible damage. Mazur (1970, 1977), proposed a “two- factor” hypothesis to account for cryo-injury based on the physical effects of ice crystal formation and the dynamic effects of freezing rate. Thus, at fast cooling 44 Cryopreservation of Tropical Plant Germplasm rates, large intracellular ice crystals cause mechanical damages; ice formation is, therefore, the first factor of the hypothesis. The second factor is attributed to dehydration damage arising from ice crystal formation. Thus, at a slow cooling rate and in the event where crystallization is first induced in the extracellular compartment, damage is attributed to the extreme osmotic dehydration which occurs when intracellular, unfrozen water moves from inside to outside the cell to compensate for the water vapour deficit as water freezes in the extracellular component (Steponkus et al. 1992). Different researchers have attributed the damaging effect of osmotic stress (also termed “solution effects”) to: a volumetric/area contraction, a concentration of intra- and extra-cellular compounds, eutectic crystallization, or possible changes in pH and enzyme activities (Steponkus 1984). Figure 1 summarizes the physical and biochemical events that can occur when plant cells are exposed to freezing injury.

Fig. 1. The physical and biochemical factors involved in cryopreservation damage and the analytical approaches that can be used to study them. Fundamental aspects of cryopreservation 45

The biophysical basis of cryoprotection To date, two basic cryoprotective strategies have been developed for plant tissues. These are characterized by the physical properties of water, tissue moisture content status, and the application, or not, of chemical additives, termed cryoprotectants. The first strategy involves the addition of cryoprotective compounds and the second, the removal of most (if not all) of the freezable water through evaporation prior to freezing. Both approaches have a common goal, which is to reduce and/or completely inhibit ice formation in the tissues. The ability to preserve tissues in liquid nitrogen in the absence of ice is now considered one of the most important approaches to plant cryoprotection and it is termed “vitrification”. In this situation the solidification of the intracellular solution is due to an extreme elevation of cell viscosity during cooling (Fahy et al. 1984). Under these conditions water cannot crystallize as the translational molecular motion of the water molecules is arrested and an amorphous metastable glass is formed. As a consequence, cryo-injury imposed by intra- and extracellular freezing and the associated colligative or “solution” effect is circumvented. There are many theories which describe the cryoprotectant mode of action. In the case of traditional approaches to cryopreservation, which involve controlled rate cooling and the application of chemical additives, cryoprotection is attributed to colligative effects (Withers 1980). Other hypotheses include: osmotic desiccation, reduction in ice crystal growth, improvement of water efflux from the cytosol, as well as the stabilization of macromolecules. In the case of desiccation-based methods, success depends on the natural or induced desiccation tolerance of the system to be cryopreserved. Indeed, it is well known that some plant tissues are adapted to extreme desiccation, such as pollen and orthodox seeds (Leopold 1990). However, others are particularly sensitive to a water loss which can result in a series of damaging events involving the “solution effects” described above (Sun and Leopold 1997). Consequently, it might be necessary to improve the tolerance of the tissue prior to desiccation and high sucrose treatments have been reported beneficial, in this regard, for various tissues grown in vitro (Dereuddre et al. 1991; Dumet et al. 1993a; Paulet et al. 1993). Two major hypotheses exist concerning the mode of action of sucrose in desiccation tolerance. First, it could replace the water molecules involved in the maintenance of macromolecular structure (Crowe et al. 1988); secondly, it could induce vitrification of the intracellular medium at biological temperatures (Williams and Leopold 1989; Koster 1991). It is important to note that whilst cryoprotective additives or desiccation treatment have a central role in cryopreservation, they can also damage the tissue and their application must be optimized.

Biochemical basis of cryo-injury and desiccation stress: the significance of free radicals Physical damage is not the only cause of lethal injury and damage can also be expressed at the biochemical level. Freezing injury promotes many sublethal changes such as metabolic uncoupling, which can then lead to the production of toxic free radicals (see Fig. 1). These molecules have unpaired electrons and they 46 Cryopreservation of Tropical Plant Germplasm can readily cause damage by extracting electrons from essential macromolecules such as lipids, proteins and DNA. Under standard conditions, tight metabolic coupling and efficient antioxidant protection ensure that free radical damage does not occur and that essential free radical reactions such as those that take place in electron transport systems proceed efficiently. Stresses imposed on tissues during cryopreservation can promote the production of free radicals (Benson 1990), the most important being the hydroxyl radical (.OH), and the - superoxide radical (O2 ). In addition other toxic, activated oxygen species can be 1 produced such as hydrogen peroxide (H2O2) and singlet oxygen (O 2 ). Free radicals attack the lipid fraction of membranes and this results in the formation of lipid peroxides (Fig. 1) which, being unstable, break down to form toxic secondary oxidation products (Esterbauer et al. 1988). The aldehydic products of lipid peroxidation are especially cytotoxic and some of the most damaging are aldehydes (Fig. 1) which include malondialdehdye (MDA) and the hydroxyalkenals (e.g. hydroxy–2–nonenal, HNE). These compounds are known to impair cell function (Esterbauer et al. 1988) as they cross-link to macromolecules such as DNA and proteins and their production can be mutagenic in animal cells (Esterbauer et al. 1988; Yang and Scaich 1996). There is now considerable evidence to suggest that free radical mediated oxidative stress occurs during cryopreservation and in seed germplasm exposed to low-temperature storage (Benson 1990; Hendry 1993). It can also occur in the other steps of cryopreservation protocols that are associated with desiccation and tissue culture manipulations (Benson 1990; Bailey et al. 1994; Robertson et al. 1995). This is exemplified by the direct (Magill et al. 1994) and indirect (Benson and Withers 1987; Benson and Norhona-Dutra 1988) detection of free radical species in plant cells which have undergone freezing treatments, cryopreservation and/or desiccation. Secondary lipid peroxidation products have also been detected in cryopreserved plant cells (Benson et al. 1992, 1995) and in stressed plant tissue cultures (Robertson et al. 1995; Benson et al. 1997b).

Analytical approaches for elucidating the physical and biochemical basis of cryopreservation-induced damage There have been considerable advances in the use of analytic approaches to enhance our current knowledge of the damage induced in biological tissues by cryopreservation (for a summary of methods see Benson 1997). Techniques which offer considerable potential are those which allow direct, “real-time” visualization of freezing events, as is the case of cryomicrosopy, and those which use thermal analysis (DSC) to detect the presence of freezable water and to determine tissue water phase status. Biochemical studies of freezing injury can be more complex as investigative techniques frequently require tissue extraction in order to analyze for cellular markers of stress. However, wherever possible non- invasive methods for detecting biochemical “markers” of cryo-injury should be used. This section explores some of the techniques available, which the authors (together with collaborating colleagues) have applied to their own systems. Fundamental aspects of cryopreservation 47

Cryomicroscopy Cryopreservation is a dynamic process comprising a complex sequence of events. If this can be visualized in “real time” at the cellular and structural level, using cryomicroscopy, there exists considerable potential for accumulating information regarding injury and survival mechanisms. This is a technique which utilizes a specially adapted light microscope to which a cooling stage has been attached. The temperature of the cryostage can be regulated using liquid nitrogen to effect temperature gradients; modern systems are programmed by electronic instrumentation. By using cryomicroscopy it is possible to expose samples to specifically defined chilling, freezing and warming rates and at the same time visualize changes in water phase status and assess the consequences of these physical changes on cell structure (Fleck 1998). This approach has been used to determine the basis of freezing damage in the cryopreservation-recalcitrant alga Vaucheria sessilis. This organism is coenocytic and its cellular structure makes it particularly vulnerable to cryo-injury (Fleck et al. 1997a, 1997b). Thus, cryomicroscopy was used to pinpoint those components of the cryopreservation protocol which were associated with intra- and extracellular ice formation.

Thermal analysis by differential scanning calorimetry Dependent upon the presence of freezable water in biological tissues, it is possible to detect thermal transitions in samples of tissues using the technique of differential scanning calorimetry (DSC). This is based on the fact that whenever a material undergoes a physical change of state, heat is either liberated or absorbed. Differential scanning calorimeters are instruments which can precisely detect the heat flow in a sample which is exposed to thermal gradients. DSC can also be used to measure the thermal properties of samples, sample purity and water content status (McNaughton and Mortimer 1975). This is achieved by measuring the enthalpy change which occurs in a test sample as compared with a reference material. In DSC the sample (for example a piece of plant tissue) and the reference are placed in small chambers which are each provided with individual heaters/coolers. The temperature of the sample holder and the reference holder are kept the same by the continuous adjustment of the instrument's thermal regulator. Concomitantly, a signal proportional to the difference between the heat input to the sample and that of the reference holder is fed to the recorder which produces a thermogram (McNaughton and Mortimer 1975). It is therefore possible to measure heat changes associated with freezing and melting of the water of biological tissues and to use this information to develop cryopreservation strategies which are based, for example, on vitrification protocols (see below). Moreover, DSC can be used to detect the presence or absence of freezable water in plant tissues and this approach is an invaluable aid in the development of cryoprotective strategies based on desiccation. 48 Cryopreservation of Tropical Plant Germplasm

Free radical markers of cryopreservation stress Two main approaches may be used to determine free radical stress in cryopreserved systems (Benson 1990). The first is via direct free radical detection using Electron Paramagnetic Resonance Spectroscopy (EPR). Thus, the production of free radicals has been observed in plant germplasm exposed to desiccation and or freezing injury (Hendry 1993; Magill et al. 1994). This method can be very useful for fundamental studies; however, the interpretation of EPR spectra is problematic (Goodman et al. 1995) as EPR spectra are influenced by tissue moisture content and physiological status. It can, therefore, be difficult to interpret EPR spectra derived from biological samples and the identification and correlation of specific free radical processes with injurious events is not easily achieved. In the applied context, EPR spectroscopy is expensive and requires specialist input for instrumentation and data interpretation; it is not likely therefore to be a technique which is amenable for routine genebank use. Free radicals can also be monitored using indirect methods and a simple gas chromatography technique based on the sampling of head space marker gases evolved by tissues contained in culture vessels has been applied to monitor cryopreservation damage in plants (Benson and Withers 1987; Harding and Benson 1995; Benson et al. 1997b). This method is non-invasive and non- destructive, and involves the addition of low concentrations of dimethyl sulphoxide (DMSO) to viable samples (contained in airtight culture vessels) just before they are exposed to stress treatments. DMSO penetrates the cells where it “traps” hydroxyl radicals and breaks down to form a methyl radical. This abstracts hydrogen evolving the highly volatile “marker gas” methane. Head space samples are withdrawn from the culture vessels and injected into a Gas Chromatograph; the levels of methane production can be correlated with stress and it is possible to monitor the indirect production of free radicals during the different steps of a cryopreservation protocol. It is also feasible, using the same approach, to monitor the production of other markers gases of cryo-injury such as the plant stress hormone ethylene and the volatile lipid peroxidation products ethane and pentane. Aldehydic lipid peroxidation products can also be used as markers of cryopreservation damage. However, in this case the tissues must be extracted and thus destroyed. Lipid peroxidation profiles have been constructed for cryoperserved plant cells using the thiobarbituric acid (TBA) assay which reacts with aldehydes forming thiobarbituric acid reactive substances (TBARS). These are chromophores and can be detected using simple colorimetric or fluorimetric techniques (Fig. 1). The coloured TBARS are attributed to secondary oxidation products formed when lipid peroxides break down to form aldehydes in the cell; these then react with TBA. Whilst this approach has been successfully used to monitor freezing damage in cryopreserved plant systems (Benson et al. 1992, 1995; Fleck 1998) it is important to caution that the assay can be subjected to interference from cryoprotective additives and sugars. However, more stringent methods of measuring oxidative stress in plants are becoming available (Deighton et al. 1997). Finally, it is possible to monitor changes in the antioxidant Fundamental aspects of cryopreservation 49 status of cryopreserved plant tissues (Benson 1990) and this approach could be particularly useful when applied in combination with assays which monitor free radical and TBAR production (Benson et al. 1997b; Fleck 1997b).

The development of improved cryopreservation methods using physical and biochemical studies One of the most important applications of fundamental cryopreservation research is in the development of improved cryopreservation protocols; this can be achieved in two ways. First, analytical tools can be used to profile the injurious physical and biochemical events which occur when tissues are cryopreserved. In this respect, all the different components of a cryopreservation protocol can be assessed, including pregrowth, cryoprotection and the associated tissue culture manipulations. Such profiles allow the detection of those components of a cryopreservation method which cause the most damage. Usually these studies are correlated with survival responses and viability testing (Harding and Benson 1995). Once damaging events have been elucidated for each step of a protocol, it is possible to target specific measures which will reduce injury and enhance survival. The following section highlights examples of how physical and biochemical studies have been used to develop improved cryopreservation methods for specific plant systems.

Using thermal analysis to develop cryoprotective desiccation strategies for oil- palm somatic embryos As previously mentioned the success of a dessication-based cryopreservation protocol depends on the desiccation tolerance of the tissue. Indeed, if a tissue withstands total removal of its freezable water, cryopreservation in the desiccated state will not result in ice-induced freezing injury, and consequently high survival rates after cryopreservation are expected. The removal of water from plant tissues using a range of different desiccation treatments is now considered to be one of the most simple and effective cryoprotective strategies. Depending on the presence of freezable water in the biological tissue, ice- nucleation or glass transitions will occur upon cooling, resulting in either ice crystal or amorphous glass formation. While ice crystals will invariably melt upon cooling, the behaviour of the glass depends on its stability. The less stable the glass, the greater the probability of devitrification occurring during thawing with the possible result of lethal ice formation. Conversely, very stable glasses will undergo glass transitions upon thawing, thus avoiding ice crystal formation. DSC analysis has been performed with oil-palm somatic embryos at different stages of a desiccation-based cryopreservation protocol. For each stage, the corresponding water content and survival rate of the polyembryonic cultures were determined. In this experiment, embryo clumps were, or were not, pretreated for 7 days on a high-sucrose medium (0.75M) before being desiccated over silica gel from 0 to 16 h. Freezing and thawing were performed quickly (see Dumet et al. 1993b for details). Table 1 shows the different parameters recorded for the two extreme desiccation durations: 0 and 16 h. The high sucrose 50 Cryopreservation of Tropical Plant Germplasm pretreatment was shown to be responsible for an osmotic desiccation of the polyembryonic cultures as their water content, initially of 14 g/g DW, dropped to 4 g/g after such a treatment. A direct consequence is the decrease in quantity of ice formed within the embryo clumps during cooling, and the ability of 40% of them to withstand cryopreservation. When embryo clumps were desiccated for 16 h in the presence of silica gel, 100% of the sucrose-treated ones survived in comparison with 20% of the non-pretreated embryos. This was despite the fact that they both had very similar water contents: 0.5 and 0.7 g/g DW. At such water contents, crystallizations no longer occurred in the former category and their tolerance to cryopreservation was optimal (80%). By comparison, ice- nucleations were still recorded and were lethal to the non-sucrose-pretreated embryos. Despite the detection of a large ice-nucleation (166 J/g FW) in the non- desiccated sucrose-pretreated embryo clumps, 40% of them withstood the freeze/thaw cycle. However, decreased ice formation (66 J/g FW in average) was lethal for the 16-h desiccated non-sucrose-pretreated embryo clumps. Our hypothesis is that the presence of sucrose limits the ice crystal growth, resulting in multiple microcrystallization events being less damaging than the formation of a few larger crystals, which is the case in absence of the sucrose. Only glass transitions were recorded in the sucrose-pretreated embryo clumps with water contents of 0.8g/g DW; in contrast, ice crystallizations still occurred in the non-sucrose-pretreated embryos at 0.7 g/g of DW water content. Therefore, it would appear that the high sucrose pretreatment increases the fraction of unfreezable water in the embryo clump. The DSC analysis indicates the efficiency of the sucrose pretreatment in terms of ice desiccation tolerance improvement and ice-nucleation reduction.

Table 1. Water content, enthalpy variation (DE)† upon cooling and survival rate after 0 or 16 hours desiccation and/or cryopreservation of polyembryonic culture pretreated or not on 0.75M sucrose medium Not sucrose treated Sucrose treated Water content (G/g DW ‡) 0 h DH § 14.6±0.7 4.0±1 16 h DH 0.7±0.4 0.5±0.4 DE (J/g FW ‡) upon cooling 0 h DH§ 252±6 156±14 16 h DH 66±54 0 Survival rate (%) after 0 h DH 100 100 16 h DH 20 100 0 h DH +LN ¶ 0 40 16 hDH +LN 0 80 † D E: Enthalpy variation (variation greater than 0 indicates ice formation). ‡ DW = dry weight; FW = fresh weight. § DH = Desiccation. ¶ LN = Cryopreservation. Fundamental aspects of cryopreservation 51

The application of thermal analysis to validate a vitrification protocol used for the cryopreservation of Ribes nigrum shoot-tips A DSC analysis was performed on black currant apices before and after vitrification in a 0.4M sucrose PVS2 cryoprotectant solution which comprised ethylene glycol/dimethylsulfoxide/glycerol (see Benson et al. 1996 for details). In this protocol, shoot-tips were frozen in the presence of the PVS2 solution, and consequently thermal analysis was performed on four different samples: the apices in the presence of the vitrification solution, the meristems before and after PVS2 treatment, and the PVS2 solution alone. The cryoprotectant treatment was found to be slightly toxic as 80% of the meristems survived after exposure. Crystallization/melting events were always recorded upon cooling in the non- PVS2-treated meristems as well as in the meristems frozen in the presence of the PVS2 solution. However, while cryopreservation was lethal for the former category, 20% of the latter withstood the freeze/thaw cycle. When the DSC analysis was performed on isolated apices after equilibration in PVS2, glass transitions only were recorded upon cooling and thawing, reflecting glass stability. The PVS2 solution frozen on its own invariably underwent a glass transition upon cooling, whilst devitrification was occasionally recorded upon thawing, although optimal vitrification conditions were not reached (as ice crystallization still occurred) when apices were frozen in the presence of PVS2 and were lethal for 80% of them. As no ice-nucleation event was recorded in the apices frozen on their own after PVS2 treatment, it would appear that the vitrification solution was less stable upon thawing than the isolated apices (suggesting that the ice crystallization/melting recorded when PVS2 is combined with the meristem is likely to happen in the vitrification solution only). It would be interesting to determine the survival rate of the apices frozen in such conditions. However, in the experimental conditions, freezing and thawing are performed quickly by plunging the cryotube into either liquid nitrogen or a hot water bath. The resulting freezing/thawing rates are generally estimated to be many hundred degrees per minute. The limit of resolution of the DSC does not allow the use of such freezing/thawing rates and most often the rates are between 10 and 20 degrees per minute. Consequently when freezable water is still present in the samples the ice formation and localization may be different from what can be suggested by the DSC analysis. Using another approach of cryopreservation (encapsulation/dehydration), higher survival rates for black currant meristems were obtained after cryopreservation (up to 80%) (Benson et al. 1996).

Storage survival enhancement using free radical studies A number of studies have demonstrated that activated oxygen species (Benson and Norhona–Dutra 1988), free radicals (Magill et al. 1994) and lipid peroxidation products (Benson et al. 1992) accumulate in plant cells and tissues exposed to cryopreservation treatments. One of the most useful outputs of monitoring the progress of oxidative stress in these systems has been the identification of those components of a cryopreservation strategy which predispose the cells to the most damage. Thus, certain pretreatment regimes were found to be more stressful to 52 Cryopreservation of Tropical Plant Germplasm rice cells being prepared for cryopreservation than for others (Benson et al. 1992). Stresses incurred by pre-cryopreservation treatments can, in some cases, cause incipient damage and by using biochemical “markers” to monitor sublethal injury it is possible to improve overall post-storage survival. Studies of singlet oxygen production by plant tissues recovering from cryopreservation have demonstrated that the formation of this toxic, activated species is greater in pigmented cells where it is produced via photoxidation (Benson and Norhona– Dutra 1988). For certain systems it may, therefore, be useful to perform the initial stages of post-storage recovery under subdued lighting. Mammalian cryobiologists have developed a wide range of strategies to improve the functional metabolism of transplant organs and tissues held at low temperatures. These systems are particularly prone to ischemic damage and it is essential that on reperfusion they be able to overcome free radical mediated oxidative stress (McAnulty and Huang 1997). One common approach of medical cryobiologists is the application of antioxidant drugs to transplant organs prior to and during low-temperature storage (Benson et al. 1995). These treatments have been found to enhance organ recovery. A similar approach has been used for cryopreserved rice cells in which the chelating drug desferrioxamine has been applied just prior to and during cryopreservation. Modifications of the pregrowth and recovery medium by adding the iron-chelating agent desferrioxamine enhances the recovery of cryopreserved rice cell cultures. This is further enhanced if cations are removed from the culture medium during the period of application. Desferrioxamine has been marketed as the drug, Desferral, by Ciba Geigy and can be obtained from the Ciba Laboratories, Horsham, UK. Its mode of action involves the reduction in oxidative stress via the removal of cations which enhance the Fenton reaction and hydroxyl radical production. Similar positive effects of desferrioxamine have been found when the drug is applied to unicellular algal cells which are highly sensitive to cryopreservation damage (Benson et al. 1997a; Fleck 1998).

Solving recalcitrance problems using fundamental research: potential possibilities Our current understanding of low-temperature stress in plants is increasing rapidly, not only in applied cryopreservation research, but also within the wider field of whole plant stress physiology. Outputs of fundamental research may offer valuable and novel approaches for the development of cryopreservation protocols for extremely storage-recalcitrant species. Thus, it may be prudent for applied cryopreservation researchers to maintain a proactive interest in whole plant, low-temperature physiology. Thomashow (1998) has recently reviewed freezing tolerance mechanisms and suggests several possibilities for conferring natural freeze-resistance in plants: · Prevention of freeze-induced protein denaturation · Prevention of molecules precipitating · Prevention of intracellular ice formation · Stabilization of membranes Fundamental aspects of cryopreservation 53

· Fatty acid desaturation · Stabilization of membranes by hydrophillic polypeptides · Expression of specific cold-tolerance genes · Accumulation of sucrose and other simple sugars.

Specific genes expressed during the acquisition of freezing tolerance include those that produce the newly discovered COR (cold-regulated proteins) and the Late-Embryogenesis Abundant (LEA) proteins. The latter are of interest to cryobiologists as they confer resistance to both dehydration and cold stress (Thomashow 1998). Desiccation sensitivity is a limiting factor for the cryopreservation of recalcitrant seed germplasm and the study of developmental seed physiology in these systems may be of considerable value. Thomashow (1998) also reports that a number of cold-responsive genes encode for proteins which have enzymatic activities known to confer cold tolerance, and this has been attributed to the enzymatic modification of membrane lipid composition. It has recently been reported that plants have anti-freeze proteins (AFPs) (Griffith et al. 1997) and that some AFPs affect the shape of ice crystals and are potent inhibitors of ice-nucleation, thus preventing the accumulation of large crystals in the cell (Thomashow 1998). Plant cryobiologists utilize vitrification as a cryoprotective strategy, which in reality constitutes cryogenic storage without ice. As glasses are meta-stable it may be interesting to explore the relationship between AFP production and glass stabilization in cryopreserved systems. Thermal analysis of these systems may be especially interesting. Many of the natural cold-tolerance mechanisms described above have already inspired plant cryoconservationists to adapt and apply them as protective strategies. In doing so there exists considerable potential to enhance the survival of cryogenically stored plant germplasm. To date, recent specific examples include the application of proline and abscisic acid responsive proteins as a pre-growth treatment in cryopreserved Ribes shoot-tips (Luo and Reed 1997), the use of free radical inhibitors to stabilize membranes (Benson et al. 1995; Fleck 1998) and the application of sucrose pregrowth treatments (see Dumet et al., this vol., p. 172).

Conclusions and future prospects Advances in fundamental cryobiological research involving the application of physical and biochemical studies to elucidate cryopreservation-induced damage have resulted in a number of findings which have practical applications in plant cryopreservation. Importantly, these studies have been underpinned by basic investigations of cold tolerance performed on whole plant systems. However, the future challenge will be to broaden these fundamental studies and explore the physical and biochemical basis of recalcitrance in tropical plant germplasm. Undoubtedly this will require concerted action regarding collaborative exchanges between fundamental researchers and germplasm curators. It will also be important to bear in mind that any fundamental research performed is well targeted and that potential outputs have direct, cost-effective and practical benefits. 54 Cryopreservation of Tropical Plant Germplasm

Acknowledgements Dr Dominique Dumet gratefully acknowledges the support of the European Commission for a Marie Curie Research Fellowship. The authors gratefully acknowledge the contributions of all collaborative colleagues who have been involved in the research programmes mentioned in this review. The authors thank the International Plant Genetic Resources Institute (IPGRI), Rome and the Japan International Research Center for Agricultural Sciences (JIRCAS), Japan, for their invitation and support in presenting this paper at the International Workshop on “Cryopreservation of tropical germplasm: Current research progress and applications”, Tsukuba, Japan, 20–23 October 1998.

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Physiological and molecular changes in tobacco suspension cells during development of tolerance to cryopreservation by vitrification Poula J. Reinhoud, Isabella Versteege, Ilona Kars, Frank van Iren and Jan W. Kijne Institute of Molecular Plant Sciences, Leiden University, 2300 RA Leiden, The Netherlands

Introduction Plant cell suspensions are widely used in cellular and molecular research and for commercial purposes. However, suspension-cultured plant cells are genetically unstable and the costs for maintaining cell lines are high. Therefore, a safe method for long-term storage of plant cell suspensions is highly desirable. Storage of biological material at ultra-low temperature (liquid nitrogen, –196°C) called cryopreservation, presents an attractive tool because it enables storage for infinite time periods with retention of viability. Plant cell suspensions have been successfully cryopreserved by the vitrification procedure (Langis et al. 1989; Uragami et al. 1989; Reinhoud et al. 1995a). During vitrification, cells are dehydrated at non-freezing temperatures by exposure to a highly concentrated mixture (5–8M) of so-called cryoprotectants (for example glycerol, DMSO, ethylene glycol), followed by ultra-rapid freezing. The dehydration step is necessary to reduce the chance of intracellular ice crystal formation during freezing and thawing. Rapid cooling rates are required to facilitate vitrification (formation of a glass-like structure) of the cells and the surrounding medium. After storage in liquid nitrogen, cells are rapidly thawed to avoid ice crystal formation and/or growth during warming. Previously, we and others have shown that plant cells or protoplasts, which can tolerate the dehydration step of the vitrification procedure, can also survive after rapid freezing and thawing (Langis and Steponkus 1991; Steponkus et al. 1992; Reinhoud et al. 1995b). Thus, it can be hypothesized that tolerance to dehydration rather than freezing tolerance is sufficient for cultured plant cells to survive vitrification. Tolerance to vitrification in tobacco (line LT) cells can be induced by a preculture period of 24 h in medium supplemented with mannitol (Reinhoud et al. 1995b). Most protocols for cryopreservation of cultured plant cells include such a preculture period (Withers 1985). However, up to now insight on the physiological and molecular mechanisms of induction of tolerance to cryopreservation by a mannitol preculture period was virtually lacking (Pritchard et al. 1986a, 1986b, 1986c; Göldner et al. 1991). Knowledge of these mechanisms is important for the development of improved cryopreservation procedures. The mannitol preculture provides mild osmotic stress to the tobacco suspension cells (Reinhoud et al. 1995b). Such a treatment may activate an adaptive mechanism that can render the cells tolerant to more severe treatments. 58 Cryopreservation of Tropical Plant Germplasm

We studied physiological and molecular changes in the tobacco cells during the mannitol preculture and their role in the development of tolerance to vitrification.

Exposure to osmotic stress is sufficient to render tobacco cells tolerant to vitrification A preculture period of 24 h in culture medium supplemented with mannitol causes mild osmotic stress and renders 55% of the tobacco suspension cells (line LT) tolerant to severe dehydration and subsequently to rapid freezing and thawing in the vitrification procedure (Reinhoud et al. 1995b). Without preculture, tobacco LT cells are not able to deplasmolyze after vitrification, indicating that cell membranes are damaged in such a way that they have irreversibly lost their semipermeable properties (Reinhoud 1996). Induction of tolerance to vitrification in the tobacco cells is not specific for mannitol. Other compounds (PEG6000 and NaCl), causing similar osmotic stress, can also induce tolerance, but to lower levels than when using a preculture with mannitol (Reinhoud 1996). PEG6000 is not supposed to enter plant cells because it can not penetrate cell walls (Carpita et al. 1979). Na + and Cl- ions can enter the cells, but are not expected to provide much protection because high concentrations of ions in the cell are toxic, especially upon dehydration (McCue and Hanson 1990). During the mannitol preculture, the tobacco cells also take up mannitol (Reinhoud 1996). Mannitol can act as an osmoprotectant. For example, transgenic plants that produce mannitol can tolerate high salinity better than do control plants (Tarczynski et al. 1993; Thomas et al. 1995). Thus, exposure of tobacco LT cells to mild osmotic stress appears to be sufficient to induce tolerance to vitrification, but it is conceivable that uptake of mannitol by the tobacco cells improves survival rates.

Mannitol preculture induces increased production of ABA and proline In response to the mannitol preculture, the tobacco cells appeared to produce and secrete increased amounts of ABA. Furthermore, after 8 h of mannitol preculture the tobacco cells started to accumulate proline, resulting in a 10–fold increase after 24 h (Reinhoud 1996). These compounds are typically involved in dehydration tolerance. A preculture with ABA also induced proline accumulation and could induce some level of tolerance to vitrification, which suggests that increased production of ABA in the tobacco LT cells is sufficient for the development of tolerance to vitrification. Werner et al. (1991) and Tetteroo et al. (1995) were able to induce desiccation tolerance in Funaria and carrot somatic embryos, respectively by a treatment with ABA, and Lee et al. (1992) could increase freezing tolerance of potato cells by ABA treatment. These results demonstrate that uptake of mannitol during preculture with mannitol is not essential for induction of tolerance to vitrification, but may improve survival rates. Fundamental aspects of cryopreservation 59

To investigate whether production of ABA during mannitol preculture is necessary for induction of tolerance to vitrification, we tested the effect of fluridone, an inhibitor of the carotenoid biosynthesis pathway. However, treatment with fluridone had no effect on mannitol-induced production of ABA (Reinhoud 1996). Apparently, the tobacco cells contain a sufficient amount of ABA precursors, so that inhibition of carotenoid synthesis does not immediately result in inhibition of ABA production (Li and Walton 1987). Both a mannitol and an ABA preculture induced accumulation of proline. To investigate whether proline is sufficient for induction of tolerance, we tested preculturing with proline. Proline preculture appears to be a potent alternative for the mannitol preculture, because 4 h of preculture with proline yielded only 10% lower survival rates (Reinhoud 1996). Compounds such as proline may function in protecting cellular components during stress (Le Rudulier et al. 1984). Transgenic plants that overproduce proline show increased tolerance to osmotic stress (Kishor et al. 1995). The mechanism by which proline provides protection is not fully understood (Naidu et al. 1992). Proline is a compatible (or non- perturbing) osmolyte that can balance osmotic pressures (Morgan 1984) and counteract the concentration of electrolytes within the cell during exposure to water stress (Kwon and Handler 1995). Proline may also function as an osmo- or cryoprotectant by protecting membranes and proteins during freezing, as shown in in vitro studies (Rudolph and Crowe 1985; Carpenter and Crowe 1988) by preferential exclusion (Carpenter et al. 1990) or by hydrophobic interaction (Schobert and Tschesche 1978; Anchordoguy et al. 1987). In contrast, Hare and co- workers (1998) suggest that the flux through the proline biosynthesis pathway (resulting in replenishment of NADP+ and redox cycling) is more important than the free proline concentration. By testing precultures with other amino acids, we showed that induction of tolerance to vitrification is not specific for the molecular structure of proline. Each amino acid tested, whether amphipathic or non- amphipathic, was able to induce tolerance to vitrification (Reinhoud 1996). In contrast, Anchordoguy et al. (1987) showed that liposome fusion during freezing and thawing could be inhibited by adding proline but not by hydroxyproline, showing a correlation between the amphipathic structure and the ability to protect the vesicles during freezing. The difference in results may be related to the method of freezing or to the fact that proline interacts differently with artificial small unilamellar vesicles than with more complex natural membranes. Since uptake of proline appears to suffice for survival to vitrification and induction of tolerance is not specific for the molecular structure of proline, non- structure-specific mechanisms, such as balancing osmotic pressures and/or preferential exclusion, are more likely to play a role in protecting cellular components during vitrification. For example, protection by preferential exclusion can be attained by solutes from different chemical classes, such as sugars, polyols, amino acids, methylamines and lyotropic salts (Carpenter and Crowe 1988). We also investigated whether the mannitol preculture induced changes in membrane composition typically related to cold-hardening and increased 60 Cryopreservation of Tropical Plant Germplasm chilling/freezing tolerance. These changes include an increase in the ratio of unsaturated to saturated fatty acid of membrane phospholipids (Lynch and Steponkus 1987; Palta et al. 1993). Murata et al. (1992), Wolter et al. (1992) and Kodama et al. (1994) showed that, by increasing the degree of unsaturation of fatty acids by genetic engineering, the chilling sensitivity of the material could be decreased. Membranes containing more unsaturated fatty acids have a lower liquid-crystalline to gel phase transition temperature (Hoekstra et al. 1992). During cold-hardening the amount of phospholipids increases (Lynch and Steponkus 1987; Palta et al. 1993). In rye leaves, there is a 30% increase in phosphatidylcholine (PC) upon cold-hardening (Steponkus et al. 1988). PC is the most hydrated lipid species in plant membranes (Uemura and Steponkus 1994). Steponkus and co-workers (1988) showed that the cryobehaviour of protoplasts is influenced by the amount of PC in the membrane. Non-acclimated protoplasts, artificially enriched with PC, showed exocytotic vesiculation during osmotic contraction instead of endocytotic vesiculation, resulting in increased freezing tolerance. Vitrification also involves major osmotic contraction and expansion; thus increased amounts of PC may be beneficial. However, in the cultured tobacco LT cells, we could not detect an increase in the level of unsaturation of membrane lipids or in the amount of PC after 24 h of mannitol preculture (Reinhoud 1996). Apparently, these changes in membrane are not required in the tobacco cells to survive cryopreservation by vitrification, confirming the hypothesis that vitrification is primarily a matter of dehydration tolerance.

Mannitol preculture induces expression of a gene encoding a zinc-finger protein and of a lea5 gene Comparison of proline content in mannitol-, ABA- and proline-precultured cells showed that the intracellular proline concentrations could not easily be correlated with the degree of tolerance to vitrification. The amount of proline in mannitol- or ABA-precultured cells is not sufficient to render cells precultured in proline tolerant to vitrification (Reinhoud et al., unpubl.). Duncan and Widholm (1991) came to the same conclusion with respect to the induction of chilling tolerance in maize callus cultures by mannitol, ABA or proline. In addition, we noticed that accumulation of proline in mannitol-precultured cells does not perfectly correlate with the development of tolerance because cells precultured with mannitol for 8 h already show increased tolerance, whereas production of proline has not started yet (Reinhoud 1996). These results suggest that other factor(s) must play a role in the development of tolerance under these conditions. Other factors that may contribute to the development of tolerance in mannitol- and ABA-precultured cells include the induction of certain genes, which may function in protecting plants from environmental stresses (Skriver and Mundy 1990; Chandler and Robertson 1994; Zhu et al. 1997). Preliminary results of experiments using the protein synthesis inhibitor cycloheximide indicated that for induction of tolerance in tobacco suspension cells by mannitol, protein synthesis is necessary. Interestingly, when the same concentration of cycloheximide was used in combination with a proline preculture, survival rates Fundamental aspects of cryopreservation 61 were hardly affected. This indicates that protein synthesis is not required with use of a proline preculture. However, one has to take into account that the mannitol-precultured cells were unable to synthesize proteins for 24 h and the proline-precultured cells for only 4 h. Therefore, the general condition of the mannitol-precultured cells compared with that of proline-precultured cells may be worse, which may contribute to lower survival rates. Xin and Li (1993) also found that induction of chilling tolerance by ABA in maize required protein synthesis, whereas proline-induced tolerance did not require protein synthesis. Using differential display (DD) (Liang and Pardee 1992), we compared transcripts of non-precultured and mannitol-precultured cells, in order to identify cDNA clones which represent genes that are differentially expressed during mannitol preculture. This resulted in the isolation of two cDNA clones (clone c20 and c24) which are differentially expressed during the mannitol preculture. The gene represented by c20 was rapidly induced after addition of mannitol to the tobacco cells. Even in cells precultured with mannitol for 30 min the transcript appeared to be present. After 8 h of mannitol preculture, the gene was down-regulated. Clone c20 was also rapidly induced during proline preculture. However, after 2 h the amount of transcript had decreased below detection levels. These results are consistent with the observation that addition of proline causes less osmotic stress to the tobacco cells than does addition of mannitol (Reinhoud 1996). The predicted amino acid sequence of cDNA clone c20 showed the presence of two zinc-finger domains of the Cys2/His2 -type and a domain of basic amino acids which can act as a nuclear localization signal. Comparison of the predicted amino acid sequence of c20 with known sequences revealed the highest homology (78% identity) with a flower-specific zinc-finger DNA-binding protein (PETZFBP2) of Petunia (Takatsuji et al. 1994). This gene is also induced in osmostressed leaves of Petunia (Van der Krol, pers. comm.). Furthermore, c20 shows homology with an STZ gene of Arabidopsis (55% identity on amino acid level). This gene can complement calcineurin mutants of yeast (mutants lacking this phosphatase have increased sensitivity to Na + ions) and can increase salt tolerance of wild-type yeast (Lippuner et al. 1996). These authors hypothesize that STZ regulates steps in control of Na + ion balance in plant cells. c20 also shares 55% identity with a cold-inducible zinc-finger protein (scof-1) of Glycine max (Kim et al., unpubl.). Thus, c20 most probably encodes a zinc-finger DNA- binding protein, is rapidly induced during mannitol preculture and may be involved as a transcription factor in ion-mediated adaptation to osmostress. The gene represented by c24 also showed a transient expression pattern during the 24-h mannitol preculture period. Expression of c24 in tobacco cells during mannitol preculture positively correlated with the development of tolerance to vitrification, except that the amount of lea5 transcripts decreased after 8 h of mannitol preculture, whereas survival rates were still increasing (Reinhoud et al. 1995b). Also during proline preculture, c24 was up-regulated, albeit to a lesser extent than during the mannitol preculture, probably because 62 Cryopreservation of Tropical Plant Germplasm addition of proline to the tobacco cells causes less osmotic stress. Since uptake of proline appears to suffice for rendering tobacco LT cell tolerant to vitrification (Reinhoud 1996), high expression of lea5 may not be necessary in these cells. Comparison of the predicted amino acid sequence of c24 with known sequences revealed that c24 belongs to a family of genes which include lea5 (late embryogenesis abundant) genes from cotton and citrus (49% identity with c24) (Galau et al. 1993; Naot et al. 1995). These genes are expressed during the post- abscission stage of embryo development, during water-related stress in an embryo culture (cotton), in a salt-stressed cell suspension and in plants exposed to drought and heat (citrus). The family also includes AtDi21, an ABA- and drought-inducible gene from Arabidopsis (43% identity with c24) (Gosti et al. 1995). The increase in expression of c24 in tobacco LT cells during mannitol preculture positively correlated with the increased production of ABA (Reinhoud 1996). When ABA is applied exogenously to the tobacco cells, lea5 is transiently induced. Members of the LEA protein family have been identified in several plant species including cotton, carrot, rice and barley (Dure et al. 1989), and accumulation correlated with the development of desiccation tolerance (Chandler et al. 1988; Close et al. 1989; Dure et al. 1989; Lane 1991). In general, LEA proteins are very hydrophilic and are suggested to bind or replace water during osmotic stress (Baker et al. 1988; Hughes and Galau 1989). Xu et al. (1996) demonstrated that transgenic rice plants, in which a barley hva1 gene (group 3 LEA protein) is constitutively and highly expressed, show increased tolerance to water deficit and salt stress. In contrast, Rab17 (group 2 LEA protein) is suggested to be involved in nuclear protein transport. Lea5 has been described as an atypical lea gene, because the N-terminal half of the encoded protein is rather hydrophobic (Galau et al. 1993). So far, the function of LEA5 proteins is unknown. Based on a homology with a small, acid-soluble spore protein of Bacillus megaterium which binds to double-stranded DNA and protects it from chemical and enzymatic cleavage in the dormant spore, Burns et al. (1996) suggested that LEA5 may play a similar role in protecting DNA in desiccated tissues. However, further evidence is required. To investigate whether expression of lea5 is sufficient for tobacco LT cells to survive vitrification, we produced transgenic tobacco cells in which lea5 is expressed under control of the CaMV 35S promoter. Cryopreservation experiments using these transgenic cell lines indicated the absence of a correlation between expression of lea5 and survival after vitrification without preculture. Apparently, higher expression levels of lea5 are necessary and/or other factors determine tolerance in mannitol-precultured cells, such as the presence and/or location of compatible solutes. Without excluding other factors we summarized the changes in the tobacco suspension cells as a result of the mannitol preculture in Figure 1. In conclusion, the development or tolerance to vitrification during the mannitol preculture in tobacco LT cells correlates with the uptake of mannitol, an increase in production of ABA and proline and an increase in expression of a zinc finger transcription factor and a lea5 gene. Changes in membrane composition typical for the develop- Fundamental aspects of cryopreservation 63

? ?

zinc-finger LEA5 protein

mannitol osmostress ABA proline tolerance

0 0.5 2 8 24

Duration of mannitol preculture (h)

Fig. 1. Schematic representation of changes detected in tobacco suspension cells during the mannitol preculture which results in tolerance to cryopreservation by vitrification. ment of freezing tolerance are not required. Proline accumulation may be important but cannot be the only factor explaining the development of tolerance to vitrification. However, when proline is applied exogenously, uptake of proline appears to be sufficient to render the tobacco cells tolerant to cryopreservation by vitrification. The role of increase expression of the gene encoding the zinc-finger protein and lea5 gene in the development of tolerance to vitrification remains to be studied. Our results can contribute to the design of improved protocols for cryopreservation of plant cells.

Acknowledgements This work was supported by the Innovation-Oriented Research Program Biotechnology (IOB-p), Department of Economic Affairs, The Netherlands.

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Molecular mechanisms of freezing and drought tolerance in plants Kazuko Yamaguchi–Shinozaki¹, Mie Kasuga¹, Qiang Liu¹, Yoh Sakuma¹, Hiroshi Abe¹, Setsuko Miura¹ and Kazuo Shinozaki² ¹ Biological Resources Division, JIRCAS, Tsukuba, Ibaraki 305-8686, Japan ² Laboratory of Plant Molecular Biology, Tsukuba Life Science Center, The Institute of Physical and Chemical Research, 3-1-1 Koyadai, Tsukuba, Ibaraki 305-0074, Japan

Introduction Drought and freezing are environmental conditions that cause adverse effects on the growth of plants and the productivity of crops. Plants respond to these stresses at the molecular and cellular levels as well as at the physiological level. Expression of a variety of genes has been demonstrated to be induced by these stresses (reviewed in Thomashow 1994; Shinozaki and Yamaguchi–Shinozaki 1996). The products of these genes are thought to function not only in stress tolerance but also in the regulation of gene expression and signal transduction in stress response (Shinozaki and Yamaguchi–Shinozaki 1997). Thus, these gene products can be classified into two groups. The first group includes proteins that probably function in protecting cells from dehydration. The second group of gene products contains protein factors that are involved in further regulation of gene expression and signal transduction and that function in stress response (Shinozaki and Yamaguchi–Shinozaki 1997). Genetic engineering is thought to be useful for improving the stress tolerance of plants. Recently, several different gene transfer approaches have been employed to improve the stress tolerance of plants (Holmberg and Bulow 1998). The transferred genes included those encoding enzymes required for the biosynthesis of various osmoprotectants (Tarczynski and Bohnert 1993; Kavi Kishor et al. 1995; Hayashi et al. 1997) or those encoding enzymes for modifying membrane lipids, LEA proteins and detoxification enzymes (Kodama et al. 1994; Ishizaki–Nishizawa et al. 1996; McKersie et al. 1996; Xu et al. 1996). In all these experiments, a single gene for a protective protein or an enzyme was overexpressed under the control of the 35S cauliflower mosaic virus (CaMV) constitutive promoter in transgenic plants, although a number of genes have been shown to function in environmental stress tolerance and response. The genes encoding protein factors that are involved in the regulation of gene expression and signal transduction and function in stress response seem to be useful to improve the tolerance of a plant to stresses by gene transfer as they can regulate many stress-inducible genes involved in stress tolerance. The plant hormone abscisic acid (ABA) is produced under environmental stress and plays important roles in tolerance against drought, high salinity and cold. Many genes that respond to drought and/or cold stress are also induced by exogenous application of ABA. It appears that dehydration triggers the production of ABA, which, in turn, induces various genes. Several reports have described genes that are induced by dehydration and cold stress, but are not 68 Cryopreservation of Tropical Plant Germplasm responsive to exogenous ABA treatments. These findings suggest the existence of ABA-independent as well as ABA-dependent signal-transduction cascades between the initial signal of drought or cold stress and the expression of specific genes (Shinozaki and Yamaguchi–Shinozaki 1996). To understand the molecular mechanisms of gene expression in response to drought and cold stress, cis- and trans-acting elements that function in ABA-independent and ABA-responsive gene expression by drought and cold stress have been precisely analyzed (Shinozaki and Yamaguchi-Shinozaki 1996). In this article, we summarize recent progress of our research on cis- and trans-acting factors involved in ABA- independent gene expression in drought and cold stress response. We also report stress tolerance of transgenic plants that overexpress a single gene for a stress- inducible transcription factor using Arabidopsis as a model.

Function of drought / cold stress inducible agents A variety of genes are induced by drought and/or cold stress in various plant species, and functions of their gene products have been predicted from sequence homology with known proteins. Genes induced under stress conditions are thought to function not only in protecting cells from water deficit or temperature change by the production of important metabolic proteins but also in the regulation of genes for signal transduction in the stress responses (Shinozaki and Yamaguchi–Shinozaki 1996, 1997). Thus, these gene products are classified into two groups (Fig. 1). The first group includes proteins that probably function in stress tolerance (Shinozaki and Yamaguchi–Shinozaki 1996): water channel proteins involved in the movement of water through membranes, the enzymes required for the biosynthesis of various osmoprotectants (sugars, proline and betaine), proteins that may protect macromolecules and membranes (LEA proteins, osmotin, antifreeze protein, chaperon and mRNA-binding proteins), proteases for protein turnover (thiol proteases, Clp protease and ubiquitin) and the detoxification enzymes (glutathione S-transferase, soluble epoxide hydrolase, catalase, superoxide dismutase and ascorbate peroxidase). Some of the stress-inducible genes that encode proteins, such as a key enzyme for proline biosynthesis, were overexpressed in transgenic plants to produce a stress-tolerant phenotype of the plants (Kavi Kishor et al. 1995). The second group contains protein factors involved in further regulation of signal transduction and gene expression that probably function in stress response: protein kinases, transcription factors and enzymes in phospholipid metabolism (Shinozaki and Yamaguchi–Shinozaki 1996). Now it becomes more important to elucidate the role of these regulatory proteins for further understanding of plant responses to the stress and for improving the tolerance of plants by gene transfer. The existence of a variety of stress-inducible genes suggests complex responses of plants to environmental stress. Fundamental aspects of cryopreservation 69

Functional Proteins Regulatory Proteins

Membrane proteins (Water channel protein, Transporters)

Transcription factors Detoxification enzymes (MYC, MYB, bZIP, DREB) (GST, ascorbate peroxidase superoxide dismutase, sEH)

drought Protection factors Protein kinases of macromolecules and/or (MAPK,MAPKKK, (LEA protein, chaperon, CDPK, S6K) antifreeze protein) cold stress

Key enzymes for osmolyte biosynthesis PI turnover (proline, betaine, sugars) (Phospholipase C PIP5K, DGK, PAP) Proteinases (thiol proteases, CLP protease, and ubiquitin)

Fig. 1. Drought- and cold-stress inducible genes and their possible functions in stress tolerance and response. Gene products are classified into two groups. The first group includes proteins that probably function in stress tolerance (Functional proteins), and the second group contains protein factors involved in further regulation of signal transduction and gene expression that probably function in stress response (Regulatory proteins).

Regulation of gene expression by drought and cold stress The expression patterns of genes induced by drought were analyzed by RNA gel- blot analysis. Results indicated broad variations in the timing of induction of these genes under drought conditions. Most of the drought-inducible genes respond to treatment with exogenous ABA, whereas some others do not (Shinozaki and Yamaguchi–Shinozaki 1996, 1997). Therefore, there are not only ABA-dependent but also ABA-independent regulatory systems of gene expression under drought stress. Analysis of the expression of ABA-inducible genes revealed that several genes require protein biosynthesis for their induction by ABA, suggesting that at least two independent pathways exist between the production of endogenous ABA and gene expression during stress. As shown in Figure 2, we identified at least three independent signal pathways which function under drought conditions: two are ABA-dependent (pathways II and III) and one is ABA-independent (pathways IV). One of the ABA-dependent pathway requires protein biosynthesis (pathway II: Shinozaki and Yamaguchi–Shinozaki 1996, 1997). There are also at least two independent cold stress signal transduction pathways: one is ABA-dependent (pathway III) 70 Cryopreservation of Tropical Plant Germplasm and the other is ABA-independent (pathway IV). These pathways overlap with those of the drought response. In addition, two other signal-transduction pathways may function only in drought response (pathway I) or in cold response (pathway V: Shinozaki and Yamaguchi–Shinozaki 1996). Therefore, at least five independent signal transduction pathways mediate drought or cold responses (Fig. 2). The existence of complex signal transduction pathways in the responses of plants to drought and cold stress gives a molecular basis for these complex physiological responses.

DROUGHTSTRESS COLDSTRESS

SIGNALPERCEPTION SIGNALPERCEPTION

ABA independent ABA ABA independent I II III IV V

? PROTEIN SYNTHESIS bZIP DREBs (AP2/ERBP) ? (MYC, MYB)

? MYCRS,MYBRS ABRE DRE ? ( erd1 ) ( rd22 ) ( rd29B ) (rd29A ) ( DREB1A) GENE EXPRESSION GENE EXPRESSION GENE EXPRESSION

WATER STRESS RESPONSE AND TOLERANCE

Fig. 2. Signal-transduction pathways between initial drought and cold stress signals and gene expression. At least five signal transduction pathways exist (I–V): two are ABA- dependent (I and II), and three are ABA-independent (I, IV and V). Protein synthesis is required in one of the ABA-dependent pathways (II). At least four signaling pathways (I, II, II and IV) function under drought conditions (indicated by solid arrows), and three pathways (III, IV and V) function under cold stress (indicated by dashed arrows).

Identification of a cis-acting element, DRE, involved in drought- and cold-responsive expression A number of gene are induced by drought, salt and cold in aba (ABA-deficient) or abi (ABA-insensitive) Arabidopsis mutants. This suggests that these genes do not require ABA for their expression under cold or drought conditions. Among these genes, the expression of a drought-inducible gene for rd29A/lti78/cor78 was extensively analyzed (Yamaguchi–Shinozaki and Shinozaki 1994). At least two separate regulatory systems function in gene expression during drought and cold stress; one is ABA-independent (Fig. 2, pathway IV) and the other is ABA- dependent (pathway III). Fundamental aspects of cryopreservation 71

To analyze the cis-acting elements involved in the ABA-independent gene expression of rd29A, we constructed chimeric genes with the rd29A promoter fused to the ß-glucuronidase (GUS) reporter gene and transformed Arabidopsis and tobacco plants with these constructs. The GUS reporter gene driven by the rd29A promoter was induced at significant levels in transgenic plants by conditions of dehydration, low temperature or high salt, or by treatment with ABA (Yamaguchi–Shinozaki and Shinozaki 1993). The deletion, the gain-of- function and the base substitution analysis of the promoter region of rd29A gene revealed that a 9-bp conserved sequence, TACCGACAT (DRE, Dehydration Responsive Element), is essential for the regulation of the expression of rd29A under drought conditions. Moreover, DRE has been demonstrated to function as a cis-acting element involved in the induction of rd29A by either low-temperature or high-salt stress (Yamaguchi–Shinozaki and Shinozaki 1994). Therefore, DRE seems to be a cis-acting element involved in gene induction by dehydration, high salt or low temperature, but does not function as an ABA-responsive element in the induction of rd29A.

Important roles of the DRE-binding proteins during drought and cold stress Two cDNA clones that encode DRE-binding proteins, DREB1A and DREB2A, were isolated by using the yeast one-hybrid screening technique (Liu et al. 1998). The deduced amino acid sequences of DREB1A and DREB2A showed significant sequence similarity with the conserved DNA-binding domains found in the EREBP and APETALA2 proteins that function in ethylene-responsive expression and floral morphogenesis, respectively (Okamuro et al. 1997; Ohme–Takagi and Shinshi 1995). Each DREB protein contained a basic region in its N–terminal region that might function as a nuclear localization signal and an acidic C– terminal region that might act as an activation domain for transcription. These data suggest that each DREB cDNA encodes a DNA-binding protein that might function as a transcriptional activator in plants. The ability of the DREB1A and DREB2A proteins expressed in Escherichia coli to bind the wild-type or mutated DRE sequences was examined using the gel retardation method (Liu et al. 1998). The results indicate that the binding of these two proteins to the DRE sequence is highly specific. To determine whether the DREB1A and DREB2A proteins are capable of transactivating DRE-dependent transcription in plant cells, we performed transactivation experiments using protoplasts prepared from Arabidopsis leaves. Coexpression of the DREB1A or DREB2A proteins in protoplasts transactivated the expression of the GUS reporter gene. These results suggest that DREB1A and DREB2A proteins function as transcription activators involved in the cold- and dehydration-responsive expression, respectively, of the rd29A gene (Fig. 3; Liu et al. 1998). We isolated cDNA clones encoding two DREB1A homologs (named DREB1B and DREB1C). The DREB1B clone was identical to CBF1 (Stockinger et al. 1997). We also isolated cDNA clones encoding a DREB2A homolog and named it DREB2B. Expression of the DREB1A gene and its two homologs was induced by 72 Cryopreservation of Tropical Plant Germplasm

Cold Drought, Salt

ABA ABA Independent Independent ABA

Transcription Transcription Modification

Transcription DREB1/CBF1 DREB2 bZIP factors

rd29A promoter DRE ABRE TATA Cis-elements Cis-elements

Fig. 3. A model of the induction of the rd29A gene and cis- and trans-acting elements involved in stress-responsive gene expression. Two cis-acting elements, DRE and ABRE, are involved in the ABA-independent and ABA-responsive induction of rd29A, respectively. Two types of different DRE-binding proteins, DREB1 and DREB2, separate two different signal-transduction pathways in response to cold and drought stress, respectively. DREB1s/CBF1 are transcriptionally regulated whereas DREB2s are controlled post-translationally as well as transcriptionally. ABRE-binding proteins encode bZIP transcription factors. low-temperature stress, whereas expression of the DREB2A gene and its single homolog was induced by dehydration (Liu et al. 1998; Shinwari et al. 1998). These results indicate that two independent families of DREB proteins, DREB1 and DREB2, function as trans-acting factors in two separate signal-transduction pathways under low-temperature and dehydration conditions, respectively (Fig. 3; Liu et al. 1998).

Analysis of the in vivo roles of DREB1A and DREB2A by using transgenic plants We generated transgenic plants in which DREB1A or DREB2A cDNAs were introduced to overproduce DREB proteins to analyze the effects of overproduction of DREB1A and DREB2A proteins on the expression of the target rd29A gene. Arabidopsis plants were transformed with vectors carrying fusions of the enhanced CaMV 35S promoter and the DREB1A (35S:DREB1A) or DREB2A (35S:DREB2A) cDNAs in the sense orientation (Mituhara et al. 1996; Liu et al. 1998). All of the transgenic plants carrying the 35S:DREB1A transgene (the 35S:DREB1A plants) showed growth-retardation phenotypes under normal Fundamental aspects of cryopreservation 73 growth conditions. The 35S:DREB1A plants showed variations in phenotypic changes in growth retardation that may have been due to the different levels of expression of the DREB1A transgene for the position effect (Liu et al. 1998). To analyze whether overproduction of the DREB1A protein caused the expression of the target gene in unstressed plants, we compared the expression of the rd29A gene in control plants carrying the pBI121 vector. Transcription of the rd29A gene was low in the unstressed wild-type plants but high in the unstressed 35S:DREB1A plants, and the level of the rd29A transcripts under the unstressed control condition was found to depend on the level of the DREB1A transcripts (Liu et al. 1998). To analyze whether overproduction of the DREB1A protein caused the expression of other target genes, we evaluated the expression of its target stress- inducible genes. In the 35S:DREB1A plants the kin1, cor6.6/kin2, cor15a, cor47/rd17 and erd10 genes were expressed strongly under unstressed control conditions, as was the rd29A gene (Kiyosue et al. 1994; Ishizaki–Nishizawa et al. 1996). In contrast, the transgenic plants carrying the 35S:DREB2A transgene (the 35S:DREB2A plants) showed little phenotypic change. In 35S:DREB2A transgenic plants, the rd29A mRNA did not accumulate significantly, although the DREB2A mRNA accumulated even under unstressed conditions (Liu et al. 1998). Expression of the DREB2A protein is not sufficient for the induction of the target stress-inducible gene. Modification, such as phosphorylation of the DREB2A protein, seems to be necessary for its function in response to dehydration (Fig. 3). However, DREB1 proteins can function without modification.

Drought, salt and freezing stress tolerance in transgenic plants The tolerance to freezing and dehydration of the transgenic plant was analyzed using the 35S:DREB1A plants grown in pots at 22°C for 3 weeks. When plants were exposed to a temperature of –6°C for 2 days, returned to 22°C and grown for 5 days, all of the wild-type plants died (Table 1), whereas the 35S:DREB1A plants survived at high frequency (Liu et al. 1998; Kasuga et al. 1999). Freezing tolerance was correlated with the level of expression of the stress-inducible genes under unstressed control conditions (Liu et al. 1998; Kasuga et al. 1999). To test whether the introduction of the DREB1A gene enhances tolerance to dehydration stress, we did not water the plants for 2 weeks. Although all of the wild-type plants died within 2 weeks, between 70 and 20% of the 35S:DREB1A plants survived and continued to grow after rewatering. Drought tolerance was also dependent on the level of expression of the target genes in the 35S:DREB1A plants under unstressed conditions (Liu et al. 1998; Kasuga et al. 1999). Overexpression of the DREB1A cDNA, driven by the constitutive 35S CaMV promoter in transgenic plants, activated strong expression of the target stress- inducible genes under unstressed conditions, which, in turn, increased tolerance of freezing, salt and drought stresses (Liu et al. 1998; Kasuga et al. 1999). Jaglo– Ottosen et al. (1998) reported that CBF1 overexpression also enhances freezing tolerance. However, the overexpression of stress-inducible genes controlled by the DREB1A protein caused severe growth retardation under normal growth conditions (Liu et al. 1998; Kasuga et al. 1999). 74 Cryopreservation of Tropical Plant Germplasm

Table 1. Survival rates of the 35S:DREB1Ab, 35S:DREB1Ac and rd29A:DREB1Aa transgenic plants. After exposing to freezing (A) , drought (B) and high-salinity (C) stress treatments (see experimental protocol), the number of surviving plants that continued to grow were counted. Experiments were repeated five times; 5 to 50 plants were tested in each experiment. Survival Total %† A. Freezing tolerance rd29A:DREB1A 143 144 99.3 35S:DREB1Ab 47 56 83.9** 35S:DREB1Ac 15 42 35.7** wild type (control) 0 55 0.0** B. Drought tolerance rd29A:DREB1A 52 80 65.0 35S:DREB1Ab 15 35 42.9* 35S:DREB1Ac 6 28 21.4** wild type (control) 0 25 0.0** C. Salt stress tolerance rd29A:DREB1A 119 149 79.9 35S:DREB1Ab 4 24 16.7** wild type (control) 4 29 13.8** † Statistical significance, compared with the value of rd29A:DREB1A, was determined by C2 test (*P<0.005, **P<0.005).

To resolve the problem of growth retardation, we used the stress-inducible rd29A promoter to cause overexpression of DREB1A in transgenic plants (rd29A:DREB1A plants; Kasuga et al. 1999). Because the rd29A promoter was stress-inducible and contained binding sites for the DREB1A protein, it did not cause expression of the DREB1A transgene at high levels under unstressed conditions. Instead, it rapidly amplified expression of the DREB1A transgene only under dehydration, salt and low-temperature stress. The rd29A:DREB1A plants revealed strong stress tolerance even though their growth retardation under normal growing conditions was not significant. Moreover, the growth and the productivity of these plants were almost the same as those of the wild-type plants under normal growing conditions (Kasuga et al. 1999). On the contrary, the rd29A:DREB1A transgenic plants are more tolerant to the stresses than the 35S:DREB1A plants that exhibited growth retardation under normal growing conditions (Kasuga et al. 1999). As the rd29A gene is one of the target genes of the DREB1A protein, the rd29A promoter is more suitable for the tissue-specific expression of the DREB1A gene in plants than the 35S CaMV promoter. In the rd29A:DREB1A plants, the target gene products seem to be strongly accumulated in the same tissues that express the products under stress conditions. These results indicate that combination of the DREB1A cDNA with the rd29A promoter would be quite useful for improving drought, salt and freezing-stress tolerance in transgenic plants. In previous studies, we showed that DRE also functions in gene expression in response to stress in tobacco plants (Yamaguchi–Shinozaki and Shinozaki 1993, Fundamental aspects of cryopreservation 75

1994), which suggests the existence of similar regulatory systems in tobacco and other crop plants. DRE-related motifs have been reported in the promoter region of cold-inducible Brassica napus and wheat genes (Jiang et al. 1996; Ouellet et al. 1998). These observations suggest that both the DREB1A cDNA and the rd29A promoter can be used to improve the dehydration, salt and freezing tolerance of crops by gene transfer.

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Ohme–Takagi, M. and H. Shinshi. 1995. Ethylene-inducible DNA binding proteins that interact with an ethylene-responsive element. Plant Cell 7: 173–182. Okamuro, J.K., B. Caster, R. Villarroel, M. Van Montagu and K.D. Jofuku. 1997. The AP2 domain of APETALA2 defines a large new family of DNA binding proteins in Arabidopsis. Proceedings of the National Academy of Sciences USA 94: 7076–7081. Ouellet, F., A. Vazquez–Tello and F. Sarhan. 1998. The wheat wcs120 promoter is cold- inducible in both monocotyledonous and dicotyledonous species. FEBS Letters 423: 324–328. Shinozaki, K. and K. Yamaguchi–Shinozaki. 1996. Molecular responses to drought and cold stress. Current Opinions in Biotechnology 7: 161–167. Shinozaki, K. and K. Yamaguchi–Shinozaki. 1997. Gene expression and signal transduction in water-stress response, Plant Physiology 115: 327–334. Shinwari, Z.K., K. Nakashima, S. Miura, M. Kasuga, M. Seki, K. Yamaguchi–Shinozaki and K. Shinozaki. 1998 An Arabidopsis gene family encoding DRE/CRT binding proteins involved in low-temperature-responsive gene expression. Biochemistry and Biophysics Research Communications 250: 161–170. Stockinger, E.J., S.J. Gilmour and M.F. Thomashow. 1997. Arabidopsis thaliana CBF1 encodes an AP2 domain-containing transcriptional activator that binds to the C- repeat/DRE, a cis-acting DNA regulatory element that stimulates transcription in response to low temperature and water deficit. Proceedings of the National Academy of Sciences USA 94: 1035–1040. Tarczynski, M. and H. Bohnert. 1993. Stress protection of transgenic tobacco by production of the osmolyte mannitol. Science 259: 508–510. Thomashow, M.F. 1994. Arabidopsis thaliana as a model for studying mechanisms of plant cold tolerance. Pp. 807–834 in Arabidopsis. Meyrowitz, E. and Somerville, C. (eds.). Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, pp. 807–834. Xu, D., X. Duan, B. Wang, B. Hong, T.H.D. Ho and R. Wu. 1996. Expression of a late embryogenesis abundant protein gene, HVA1, from barley confers tolerance to water deficit and salt stress in transgenic rice. Plant Physiology 110: 249–257. Yamaguchi–Shinozaki, K. and K. Shinozaki. 1993. Characterization of the expression of a desiccation-responsive rd29 gene of Arabidopsis thaliana and analysis of its promoter in transgenic plants. Molecular Gene Genetics 236: 331–340. Yamaguchi–Shinozaki, K. and K. Shinozaki. 1994. A novel cis-acting element in an Arabidopsis gene is involved in responsiveness to drought, low-temperature, or high- salt stress. Plant Cell 6: 251–264. Fundamental aspects of cryopreservation 77

Cryopreservation of medicinal plant resources: retention of biosynthetic capabilities in transformed cultures Kayo Yoshimatsu, Kaori Touno and Koichiro Shimomura Tsukuba Medicinal Plant Research Station, National Institute of Health Sciences, Tsukuba, Ibaraki 305-0843, Japan

Introduction Plants biosynthesize structurally diverse secondary metabolites such as alkaloids, terpenes, phenolics, etc., which are commonly utilized in our daily life as pharmaceuticals, flavours, dyes and food additives. Often a certain group of secondary metabolites is restricted to a particular taxonomic group (species, genus, family or closely related group of family). Since most secondary metabolites have a rather complex structure, it is not easy to synthesize them at a low price. Therefore plants in the field or in their natural habitat remain a main source of useful secondary metabolites. However, supply of these secondary metabolites depends on the environmental (weather, pests, etc.) and/or social (economic, political, etc.) conditions of the producing country, and hence the stable supply of secondary metabolites might be threatened. Thus plant biotechnology is one of the more desirable systems to efficiently produce the useful secondary metabolites. Many researchers have investigated the production of target secondary metabolites from different tissue cultures such as calluses, cell suspensions, adventitious shoots and adventitious root cultures. In many cases, fast-growing unorganized cell suspensions failed to accumulate significant amounts of secondary metabolites. The biosynthesis of the secondary metabolites seems to be related to the differentiation of the cells or plant organs such as shoots or roots. However, organized tissues are, in most cases, either phytohormone- dependent or slow growing (Sauerwein et al. 1992). Transformed plant cells with Agrobacterium rhizogenes produce morphologically characteristic cultures such as hairy roots. In most cases, these cultures exhibit phytohormone independency, rapid and vigorous growth, and the capability of producing the same kind of secondary metabolites as intact plants. Profitability for use of transformed cultures for the study on biosynthesis of medic-inally important secondary metabolites has been reported (Shimomura et al. 1997). The transformation mechanism by Agrobacterium rhizogenes is shown in Fig. 1. Agrobacterium rhizogenes, a gram-negative soil bacterium, is a plant pathogen and the causative agent of hairy root disease at wound sites on dicotyledonous plants. Two regions, the transferred DNA, called T-DNA region and virulence region in the megaplasmid, Ri plasmid, are associated with hairy root formation (Armitage et al. 1988). Contact of the Agrobacterium with compounds released from wounded plant tissue results in the transcription of the vir genes. The vir genes' products catalyze T-DNA transfer into the plant cell and T-DNA is stably inserted into the plant nuclear DNA. In the T-DNA, there are genes for opine production and bacterial strains are characterized by their particular opines. In addition, there are genes that code for auxin synthesis. These genes derived from 78 Cryopreservation of Tropical Plant Germplasm the bacterium are successfully expressed and function, and then morphologically characteristic hairy roots form. The site of integration of T-DNA into plant nuclear DNA is apparently random. In general, a number of transformed clones are induced by infection and their biosynthetic capabilities differ. They are valuable for differential screening. Therefore stable long-term preservation of transformed cultures is advantageous for the progress of the study because maintenance of these cultures requires extensive labour, space and facilities. Benson and Hamill (1991) first reported the cryopreservation of hairy roots for Beta vulgaris and Nicotiana rustica. However, the survival (regrowth after cryopreservation) ratio was below 20%. Recently, the vitrification procedure for cryopreservation has been applied to a wide range of plant meristems (Sakai 1993). This method may be profitable for routine preservation of hairy root clones because it eliminates the need for controlled slow freezing and permits cryopreservation of cells and meristems by direct transfer into liquid nitrogen. We investigated the cryopreservation of hairy roots using the vitrification method (Fig. 2). If the cells were sufficiently dehydrated or concentrated and then supercooled to a very low temperature, they would be vitrified and could avoid lethal intracellular freezing. Therefore cells have to be sufficiently dehydrated with a vitrification solution and completely vitrified in liquid nitrogen. However, it is necessary to control the procedures to prevent injury by chemical toxicity or excessive osmotic stresses. In the method based on the two- step vitrification procedure developed by Matsumoto et al. (1994), the plant cells are first treated with a less toxic loading solution (2M glycerol and 0.4M sucrose) and subsequently dehydrated with PVS2 because direct exposure of cultured cells to a highly concentrated vitrification solution may lead to harmful effects. The cryotubes containing the plant cells are then directly immersed in liquid nitrogen and preserved. For reculture, the cryotubes are rapidly rewarmed at 40°C to thaw the solution and the plant cells are washed with 1M sucrose and then recultured. Several studies revealed that the preculture conditions, such as relatively high sucrose concentration or cold-hardening, greatly influence the freezing or dehydration tolerance.

Cryopreservation of Panax ginseng hairy roots The roots of ginseng, Panax ginseng C.A. Meyer, are used for the treatment of anemia, diabetes, gastritis and various conditions arising from aging (Evans 1989). In recent years, it has also become a popular tonic and health food in the Western countries, and therefore the demand for the plant has increased dramatically worldwide. The medicinal activity appears to reside in a number of saponins termed ginsenosides (Shibata 1977) (Fig. 3). Ginseng is very expensive because of its long-term conventional cultivation (5–7 years, Choi 1988). As an alternative, the production of ginsenosides by transformed root cultures (hairy root cultures) was attempted by Yoshikawa and Furuya (1987); however, the hairy roots required phytohormones for satisfactory growth. We also established P. ginseng hairy root culture by infecting Agrobacterium rhizogenes ATCC 15834 onto petiole segments of field-grown plants Fundamental aspects of cryopreservation 79

Fig. 1. Transformation mechanism by Agrobacterium rhizogenes.

3. Dehydration by PVS2 2. Loading · 1. Preculture 30% w/v glycerol · 2M glycerol · · 0.3M sucrose 15% w/v ethylene glycol · 0.4M sucrose · 15% w/v DMSO · 0.4M sucrose

4. Cryopreservation · in LN (-196°C)

7. Reculture 6. Washing 5. Thawing · 1M sucrose · 40°C, 1 min

Fig. 2. Procedure for cryopreservation of medicinal plant hairy roots. 80 Cryopreservation of Tropical Plant Germplasm

R1 R2 R3 ginsenoside Rb1 Glc(b162)Glc– H Glc(b166)Glc– ginsenoside Rc Glc(b162)Glc– H Ara(f)(a166)Glc– ginsenoside Rd Glc(b162)Glc– H Glc– ginsenoside Re H Rha(a162)Glc–O– Glc– ginsenoside Rg1 H Glc–O– Glc–

Fig. 3. Ginsenosides of Panax ginseng quantified by HPLC. and found that our hairy roots grew vigorously and produced much more ginsenosides than the hairy roots previously reported. Although cryopreservation of P. ginseng cell cultures has been reported (Butenko et al. 1984; Seitz and Reinhard 1987), there has been no report on cryopreservation of P. ginseng hairy roots. Therefore, we first investigated the duration effect of preculture and PVS2 treatment on the viability without immersion into liquid nitrogen using P. ginseng hairy roots cultured on a half-strength macrosalts MS (½ MS) solid medium. Hairy root tips (about 1 mm) including root meristems were excised and cultured on phytohormone-free (HF) ½ MS medium containing 0.3M sucrose at 25°C in the dark for 1, 2 or 3 days. The root tips were loaded with 2M glycerol and 0.4M sucrose at 25°C for 10 min, and then dehydrated with PVS2 at 25°C for 5, 7, 10 or 12 min. After the dehydration, the root tips were immediately washed with 1M sucrose at 25°C for 10 min and directly subjected to FDA staining for viability testing. Over 50% viability and a relatively higher percentage of stained cells and/or stronger fluorescence were observed with FDA staining in the three treatments; the combinations of preculture (days) – PVS2 treatment (min) were 1–10, 2–10 and 3–7. The segments were then treated with the same three combinations with a slightly prolonged PVS2 treatment (11 or 9 min), immersed into liquid nitrogen and preserved for 4 days. After rapid rewarming and washing, viability was immediately determined by FDA staining and recovery was estimated from root regeneration and elongation after 3 weeks of reculture. Although the data differed slightly between viability and recovery, the best Fundamental aspects of cryopreservation 81 results (33% viability and 50% recovery) were obtained with 3-d preculture and 9-min PVS2 treatment (Yoshimatsu et al. 1996). We found that it is the root meristem that was successfully cryopreserved. Therefore, we investigated the effect of auxin added to the preculture medium on the survival rate of cryopreserved hairy roots. This is because auxin generally enhances cell division. TIBA (2,3,5–tri–iodobenzoic acid, auxin polar transport inhibitor) was also used because the addition of TIBA in the culture medium increased endogenous auxin levels in cultured root segments of Cephaelis ipecacuanha (Yoshimatsu and Shimomura 1994). The hairy root tips precultured with either 5 mg/L indole acetic acid (IAA), 0.1 mg/L 2,4–dichlorophenoxyacetic acid (2,4–D) or 0.5 mg/L TIBA or without phytohormone for 3 days were dehydrated with PVS2 for 8 min and cryopreserved for 4 days or 15 weeks. Among the phytohormones tested, the best recovery (root regeneration and elongation) was observed in the segments precultured with 0.1 mg/L 2,4–D. In this condition, there were survival rates of 60% after 4 days of cryopreservation and 54% after 15 weeks of cryopreservation. This enhancement of recovery by addition of 2,4–D was significant (P<0.01, by analysis of variance) compared with HF treatment. The survival rate fluctuated when the segments were precultured without phytohormone (20% after 4 days of cryopreservation, 0% after 15 weeks of cryopreservation). It was observed that Beta vulgaris hairy root tips in the early stages of growth were more amenable to cryopreservation than those in the stationary growth stage (Benson and Hamill 1991). Therefore the fluctuation of recovery in HF treatment was thought to be due to inherent physiological differences among hairy root tips and pretreatment with auxin or TIBA might overcome this difficulty by promoting cell viability before cryopreservation (IAA, 27% after 4 days of cryopreservation and 28.6% after 15 weeks of cryopreservation; TIBA, 20% after 4 days and 24.6% after 15 weeks of cryopreservation). There are two T-DNAs (TL-DNA and TR-DNA) in the Ri plasmid of Agrobacterium rhizogenes strain ATCC 15834. The value of hairy root cryopreservation depends on the molecular stability of the T-DNA. Therefore we examined the existence of T-DNAs by PCR analysis (Aoki et al. 1997) as a preliminary investigation for genetic stability, and confirmed the existence of T- DNAs (TL- and TR-DNAs) in the hairy roots regenerated from cryopreserved root tips as well as in the untreated hairy roots. No band was detected in the non-transformed roots, as expected. It is particularly important that cryopreserved plant cells remain capable of producing cells or tissues identical with the non-treated ones. The growth of hairy roots regenerated from cryopreserved root tips was compared with that of untreated ones. Although the regenerated roots proliferated fewer new lateral roots than the control, they elongated similarly to the control. Suppression of root proliferation in hairy roots regenerated from cryopreserved root tips might be attributed to the direct effect arising from the cryogenic procedure. However, they grew well and no differences were observed compared with the control when further transferred into HF ½ MS liquid medium and cultured at 25°C in the dark. 82 Cryopreservation of Tropical Plant Germplasm

The regrown roots were transferred into ½ MS liquid medium and cultured at 25°C in the dark for 6 weeks and their ginsenosides were analyzed by HPLC. The untreated hairy root cultures produced much more ginsenoside Rb1 than field-grown ginseng, which produced ginsenoside Re as a main saponin. This might be one of the differences between field-grown roots and cultured roots. The regenerated hairy roots from cryopreserved root tips precultured without phytohormone or with IAA, TIBA or 2,4–D produced almost the same levels of ginsenosides, showing the same patterns as those in the untreated control. This result indicates that cryopreservation of root tips did not influence the biochemical capabilities of hairy roots. Initially, hairy roots grown on solid medium were used for cryopreservation because of their reduced water content, and the above results were obtained. Hairy roots grow much more rapidly and vigorously in the liquid culture condition than on the solid. Therefore cryopreservation of P. ginseng hairy roots cultured in the liquid medium was studied. The root tips (about 1 mm) of hairy roots in HF ½ MS liquid medium were excised and precultured on 0.3M sucrose medium without phytohormone or with either 2,4–D (0.1 or 0.5 mg/L) or TIBA (0.1 or 0.5 mg/L) for 1 day. After the preculture, the root tips (10 segments per tube) were transferred into a cryotube (2 ml), loaded at 25°C for 10 min, dehydrated with PVS2 at 25°C for 10 min and then cryopreserved. As a result, high recovery (regrowth) rates of over 70% were obtained with these preculture conditions (HF, 83.3%; 0.1 mg/L 2,4–D, 70.7%; 0.5 mg/L 2,4–D, 85.9%; 0.1 mg/L TIBA, 17.4%; 0.5 mg/L TIBA, 43.7%).

Cryopreservation of Angelica acutiloba hairy roots Angelica acutiloba belongs to the Umbelliferae family and its roots are commonly prescribed in Traditional Medicine for women's diseases such as menstrual disorders (The Japanese Pharmacopoeia 1996). Hairy root cultures of A. acutiloba were established by infecting Agrobacterium rhizogenes MAFF 03-01724 onto hypocotyl segments and maintaining in HF Gamborg B5 (B5) liquid medium at 20°C in the dark. For the cryopreservation of the A. acutiloba hairy roots, the root tips (about 3–5 mm) grown in HF B5 liquid medium were treated similarly to the P. ginseng hairy roots [precultured on 0.3M sucrose medium without phytohormone or with either 2,4–D (0.1 or 0.5 mg/L) or TIBA (0.1 or 0.5 mg/L) at 20°C in the dark for 1 day, loaded at 25°C for 10 min, dehydrated with PVS2 at 25°C for 10 min and then cryopreserved]. These conditions appeared to be excessive for A. acutiloba hairy roots and recovery rates were low (5.6–11.8%). Angelica acutiloba hairy roots are thinner and more fragile than P. ginseng hairy roots. Therefore, the temperature for the loading and dehydration was lowered to 0°C and the effect of preculture medium and duration of PVS2 treatment on the recovery after cryopreservation was investigated. The medium used for preculture (B5 basal, 20°C in the dark) was a combination of sucrose (3%, 0.3M or 0.5M) and phytohormone (HF or 0.1 mg/L 2,4–D). The root tips were loaded for 10 min and dehydrated with PVS2 for either 10, 15 or 20 min. The root tips Fundamental aspects of cryopreservation 83 precultured with the combination of 0.3M sucrose and 0.1 mg/L 2,4–D gave high recovery rates; the best recovery was by root tips dehydrated with PVS2 for 15 min (PVS2 for 10 min, 89.3%; PVS2 for 15 min, 96.3%; PVS2 for 20 min, 88.6%). Regrowth was not observed in the root tips precultured with 3% sucrose. The root tips precultured with the combination of 0.5M sucrose and 0.1 mg/L 2,4–D gave much lower recovery (PVS2 for 10 min, 59.6%; PVS2 for 15 min, 66.7%; PVS2 for 20 min, 50.9%). Regenerated hairy roots from cryopreserved root tips grew similarly to the untreated hairy roots.

Cryopreservation of Atropa belladonna hairy roots Atropa belladonna is one of the most famous European medicinal plants belonging to the Solanaceae family. It contains tropane alkaloids such as hyoscyamine and scopolamine, which act on the human autonomic nervous system, parasympathetic nervous system as a blocking agent (Evans 1989) (Fig. 4). There are several reports describing transformation of A. belladonna by Agrobacterium (Suzuki et al. 1993). A high alkaloid-producing clone of hairy roots was established by infection of A. rhizogenes MAFF 03-01724 and maintained in HF ½ MS liquid medium at 25°C in the dark. Cryopreservation of cell suspensions, pollen embryos and mesophyll protoplasts has so far been reported (Bajaj and Simola 1991); however, there has been no report on cryopreservation of hairy roots. For the cryopreservation, cryogenic conditions for A. acutiloba hairy roots were applied to A. belladonna hairy roots (Touno et al. 1997). The root tips (3–5 mm) were precultured on 0.3M sucrose medium with 0.1 mg/L 2,4–D or without phytohormone at 25°C in the dark for 1 day, loaded at 0°C for 10 min, dehydrated with PVS2 at 0°C for 15 min and then cryopreserved in liquid nitrogen for different durations (1 day, 1 week, 1 month or 3 months). Higher recovery rates (63.0–96.3%) were obtained by the preculture with 0.1 mg/L 2,4–D (HF, 51.9–73.5%). Regrown hairy roots subcultured into HF ½ MS liquid medium grew similarly well compared with untreated hairy roots. To confirm the conservation of T-DNA derived from A. rhizogenes strain MAFF 03-01724, existence of T-DNA in the regenerated hairy roots was examined by PCR analysis. Amplification of T-DNA bands was clearly observed in the regenerated hairy roots from cryopreserved root tips as well as in the untreated hairy roots. Genetic stability is crucially important to evaluate the success of hairy root cryopreservation. Therefore RAPD analysis of genome DNA of A. belladonna hairy roots was performed using seven kinds of primers (10 mer) for the preliminary experiment of genetic stability. Almost no difference in the RAPD pattern was observed between the untreated roots and regenerated roots after cryopreservation. Atropa belladonna hairy roots biosynthesize tropane alkaloids, hyoscyamine, scopolamine, littorine and 6ß–hydroxyhyoscyamine (Aoki et al. 1997) (Fig. 4). To confirm the biosynthetic capability, the alkaloids in the hairy roots cultured in 84 Cryopreservation of Tropical Plant Germplasm

HF ½ MS liquid medium were quantified by HPLC for several culture passages. The main tropane alkaloid in the regenerated hairy roots as well as the untreated hairy roots was hyoscyamine. However, its concentration among the roots independently regenerated after cryopreservation fluctuated (the untreated roots, 0.35% dry wt.; the regenerated hairy roots, 0.04–0.38% dry wt.) after three subcultures in liquid culture medium. Therefore, two clones, one already recovered to the levels of untreated roots and another with lower concentration, were further subcultured. After four and five subcultures, even the clone that showed low productivity in the third subculture recovered to the levels of untreated roots.

Fig. 4. Tropane alkaloids quantified by HPLC.

Cryopreservation of Papaver somniferum transformed calli Opium poppy, Papaver somniferum, has been one of the most well-known medicinal plants since ancient times. It is a plant which causes narcosis, but its alkaloids such as morphine, codeine, papaverine and noscapine are still therapeutically valuable (Evans 1989) (Fig. 5). Many studies have been conducted to produce these alkaloids in vitro (Kamo and Mahlberg 1988). Transformed cultures of P. somniferum were established by infection of A. rhizogenes MAFF 03-01724 (Yoshimatsu and Shimomura 1992, 1996). Although plant cells transformed with A. rhizogenes generally produce morphologically characteristic cultures such as hairy roots, embryogenic and non-embryogenic callus were induced by infection with A. rhizogenes instead of hairy roots. Fundamental aspects of cryopreservation 85

Fig. 5. Opium alkaloids quantified by HPLC.

Cryopreservation of P. somniferum cell suspension cultures by controlled freezing was reported by Friesen et al. (1991), but no reports on the cryopreservation of P. somniferum cells by vitrification have been found. The transformed P. somniferum cells maintained on HF MS solid medium at 22°C in the dark were precultured in 50% loading solution (1M glycerol and 0.2M sucrose) at 20°C in the dark for 1 day, dehydrated with PVS2 at 25°C for 35 min without loading, and then cryopreserved in liquid nitrogen. After rapid thawing and washing, the cells were recultured on HF MS solid medium at 22°C in the dark. Four clones – M-1 (embryogenic), M1-2 (embryogenic, subclone of M-1), M2 (non-embryogenic) and M3 (non-embryogenic) – were used for the cryopreservation. All clones successfully regenerated after cryopreservation, showing the same morphological characteristics as the untreated cultures. To confirm the conservation of T-DNA derived from A. rhizogenes strain MAFF 03-01724, the existence of T-DNA in the regenerated cells was examined by PCR analysis. Amplification of T-DNA bands was clearly observed in the regenerated cells as well as in the untreated ones and no band corresponding to T-DNA was detected in the non-transformed cells. Preliminary evaluation of genetic stability using RAPD analysis of the genome DNA of P. somniferum cells regrown after cryopreservation was performed using two kinds of primers (10 mer). Almost no difference of the RAPD pattern was observed between the untreated cells and regrown cells. 86 Cryopreservation of Tropical Plant Germplasm

Transformed embryogenic cells of P. somniferum could synthesize morphinans, especially in the light, whereas non-embryogenic cells did not (Yoshimatsu and Shimomura 1992, 1996). The highest-alkaloid producing clone M1-2, alkaloids in the somatic embryos and regenerated shoots derived from the untreated cells and the cryopreserved cells were analyzed by HPLC. Codeine and thebaine were detected both in somatic embryos and regenerated shoots of cryopreserved cells with enhanced concentration of alkaloids in the shoots (the shoots from the untreated cells, approx. 20 µg/g dry weight codeine and 60 µg/g dry weight thebaine; the shoots from cryopreserved cells, ca. 15 µg/g dry weight codeine and 30 µg/g dry weight thebaine).

Conclusion Vitrification procedure using PVS2 is apparently feasible for the long-term conservation of transgenic cultures of medicinal plants. Hairy root cultures of P. ginseng and A. acutiloba, and A. belladonna and P. somniferum transformed calli were successfully cryopreserved in liquid nitrogen (–196°C) using the vitrification method under individually optimized cryogenic protocols (Table 1). Preculture with 0.3M sucrose and 0.1 mg/L 2,4–D raised survival rates in the case of hairy roots. Transformed cells of P. somniferum precultured in 50% loading solution (1M glycerol and 0.2M sucrose) were successfully regenerated after cryopreservation. The cryopreserved cultures retained the capability for organogenesis and secondary metabolite biosynthesis. The results obtained are promising for the progress of research on transformed cultures of medicinal plants. The immediate utilization of transformed cultures will become possible without the laborious inconvenience of maintaining large numbers of cultures.

Table 1. Summary of cryogenic conditions for the successful recovery of transformed cultures after cryopreservation Plant Preculture Loading PVS2 treatment Panax ginseng 0.3M sucrose + 0.1 mg/L 2,4–D, 25°C, 10 min 25°C, 10 min 25°C, 1 day Angelica acutiloba 0.3M sucrose + 0.1 mg/L 2,4–D, 0°C, 10 min 0°C, 15 min 20°C, 1 day Atropa belladonna 0.3M sucrose + 0.1 mg/L 2,4–D, 0°C, 10 min 0°C, 15 min 25°C, 1 day Papaver somniferum 1M glycerol + 0.2 M sucrose, – 25°C, 35 min 20°C, 1 day

Acknowledgements The authors thank Ms Wendy Shu Soo Ching for her critical reading of the manuscript. This study was supported in part by Ministry of Health and Welfare, Health Sciences Research Grants, Special Research. Fundamental aspects of cryopreservation 87

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Cryopreservation of undifferentiated plant cells Poula J. Reinhoud, Frank Van Iren and Jan W. Kijne Institute of Molecular Plant Sciences, Leiden University, 2300 Leiden, The Netherlands

Introduction Plant cell suspensions and calli are cultures of undifferentiated plant cells and are widely used in cellular and molecular research and for commercial purposes. However, these types of cultures are known for their genetic instability. They are subject to (epi-) genetic changes leading to, for instance, loss of regeneration potential, loss of embryogenic nature and changes in the pattern of production of secondary metabolites (Meijer et al. 1991; Skirvin et al. 1994; Wan and Vasil 1996; Van Iren, unpubl.). In addition, costs of continued culture are high and the risk of losing material because of disease, technical or human errors is always present. Furthermore, patenting requires deposition in a publicly accessible culture collection. Therefore, deposition of these materials at cryogenic temperatures with retention of viability, called cryopreservation, is an attractive option for long-term storage. Liquid nitrogen (–196°C) is routinely used for cryogenic storage, since it is relatively cheap and safe, requires little maintenance and is widely available. Below –120°C the rate of chemical or biophysical reactions is too slow to cause biological deterioration (Kartha 1985). Only in the long term might there be a small risk of ionising radiation causing genetic changes in materials stored at cryogenic temperatures (Grout 1995).

Damage to plant cells due to freezing and thawing The water-rich cells in a suspension culture or callus, like most cells in a plant, will not survive exposure to the temperature of liquid nitrogen. During freezing and thawing, plant cells may be damaged as a result of (i) exposure to low temperatures, (ii) formation of ice crystals, (iii) severe dehydration, and/or (iv) formation of free radicals. Below follows a brief outline of types of damage associated with freezing and thawing.

Exposure to low temperatures Upon cooling, lipids in membranes will undergo phase transition from liquid- crystalline to gel-phase. Co-existence of liquid-crystalline and gel phases in a membrane will cause leakage and therefore cell damage (Hammoudah et al. 1981). Because not every lipid has the same phase transition temperature, phase separation may occur, resulting in the formation of gel-phase rich domains ('aparticulate domains'). Upon warming, these domains may give rise to the formation of non-lamellar structures, which will cause leakage and cell damage (Quinn 1985). Exposure to low temperature may also result in inactivation of proteins that are sensitive to cold (Usami et al. 1995). 92 Cryopreservation of Tropical Plant Germplasm

Formation of ice crystals Cultured plant cells consist of approximately 95% water. When the temperature is lowered below 0°C, water will usually turn into ice. Formation of intracellular ice in plant cells is mostly lethal (Burke et al. 1976; Steponkus et al. 1992) and needs to be avoided, at least in most cells, for successful cryopreservation. By dehydrating the cells, the chance of formation of ice crystals in the cells can be strongly reduced. In principle, plant cells can tolerate extracellular ice. For example, the two-step freezing procedure uses the formation of extracellular ice during a slow freezing step to osmotically dehydrate the cells (see below). But, formation of extracellular ice may cause damage as well, for instance, due to the mechanical forces put upon the cells by the growing extracellular ice crystals, adhesion of ice crystals to the membrane surface, differences in solubility of ions in ice and liquid phases, or due to the formation of intracellular gas bubbles (see Grout 1995).

Dehydration A dehydration step, necessary to avoid the formation of intracellular ice crystals, results in concentration of solutes in cells and in strong plasmolysis of cells. Removal of water can lead to 'solute effects' in the cell, such as pH changes, increased electrolyte concentrations and macromolecular interactions (Towill 1991). Problems associated with plasmolysis may especially arise when cells have to deplasmolyze after thawing. For example, osmotic contraction can result in irreversible endocytotic vesiculation, which will result in lysis during osmotic expansion, because new membrane material is not rapidly enough available to facilitate deplasmolysis (Steponkus 1984). In addition, dehydration increases the gel-to-liquid crystalline phase transition temperature (Tm) of lipids (Crowe et al. 1989). As a result, membrane lipids undergo phase transition and phase separation at higher temperatures. Gordon-Kamm and Steponkus (1984) showed that loss of osmotic responsiveness of isolated protoplasts as a result of osmotic dehydration is associated with lateral phase separations, formation of aparticulate lamellae and lamellar-to-hexagonalII phase transitions. Membranes may even undergo fusion when they are brought into close contact during plasmolysis (Steponkus et al. 1993).

Formation of free radicals Stress to the cells caused by freezing and thawing may result in the formation of free radicals. These compounds can cause further damage, for example by lipid peroxidation, denaturation of proteins and mutations in DNA (Cadenas 1989; Benson et al. 1992; Smirnoff 1995).

Cryopreservation of cultured plant cells The objective of cryopreservation procedures is to reduce freeze-thaw damage to sublethal levels. Quatrano (1968) and Nag and Street (1973) reported the first successful experiments on cryopreservation of plant cells. Since then a large number of cell suspension and calli cultures have been successfully Cryopreservation techniques 93 cryopreserved (Table 1). In general, callus cultures are more difficult to cryopreserve than cell suspensions, because of the relative volume of the callus, its slow growth rate and the cellular heterogeneity (Withers 1987). One successful cryopreservation procedure that is applicable to all different cell suspensions or calli cultures has not been developed yet. Research focuses on optimizing the factors on which successful cryopreservation of plant cells suspensions and calli depends, such as: (i) starting material, (ii) pretreatment, (iii) cryopreservation procedure, and (iv) post-thaw treatment.

Starting material An important factor for plant cell suspensions to survive cryopreservation is the growth phase in which the cells are at the start of the procedure. Exponentially growing plant cells are more tolerant to freezing than cells harvested during the lag or stationary phases (Sugawara and Sakai 1974; Withers 1985; Yoshida et al. 1993; Reinhoud et al. 1995). Such cells have the smallest volume in the culture period, have small vacuoles and contain relatively little water, which obviously is advantageous for surviving. Also the initial cell density can influence regrowth after cryopreservation. Adjustment of the sedimented cell volume to 50% (v/v) resulted in maximum regrowth of Picea abies cultures (Find et al. 1998).

Pretreatment In nature, many plants have developed mechanisms to tolerate exposure to environmental stress such as low temperature and water shortage. In addition, desiccation tolerance is a normal feature of certain plant organs (seeds and pollen). Tolerance to these conditions correlates with a number of physiological and molecular changes such as changes in membrane composition (Lynch and Steponkus 1987; Palta et al. 1993), accumulation of small organic molecules (Yancey et al. 1982; Le Rudulier et al. 1984; Koster and Leopold 1988; Popp and Smirnoff 1995) and certain proteins (Skriver and Mundy 1990; Chandler and Robertson 1994). The capacity of plant cells to adapt to environmental stress can be employed in a preculture period prior to the cryopreservation procedure. Most common pretreatments for cryopreservation of suspension cells are preculture for several days in low concentrations (0.01–0.3M) of sugars (sucrose, melibiose, trehalose), sugar alcohols (mannitol, sorbitol) or amino acids (asparagine, alanine, proline) (Butenko et al. 1984; Withers 1985; Göldner et al. 1991). For callus cultures the pretreatments are prolonged up to several weeks. As a result of these precultures, tolerance to cryopreservation increases (Reinhoud et al. 1995). Pritchard et al. (1986) and Gnanapragasam and Vasil (1992) showed that preculturing of plant cells in mannitol or sorbitol can cause a reduction in the vacuolar volume by redistribution of the large central vacuole into a number of smaller vesicles. In tobacco suspension cells, the development of tolerance to cryopreservation by vitrification during a mannitol preculture appeared to be the combined result of mannitol uptake and the response of the tobacco cell to osmotic stress cause by the addition of mannitol to the cells (Reinhoud 1996). 94 Cryopreservation of Tropical Plant Germplasm

Table 1. Examples of cell suspension and calli cultures that have been successfully cryopreserved by two-step freezing, vitrification or encapsulation-dehydration Species Tissue Procedure Cryoprotectants† Reference Monocotyledons Bromus inermis cell suspension vitrification G+EG+D+Sucr Ishikawa et al. (PVS2) 1996 Hordeum vulgare cell suspension 2-step D+G+P; Fretz and Lörz (embryogenic) D+G+Sorb 1995 Festuca cell suspension 2-step D+sorb Wang et al. (embryogenic) 1994 Lolium cell suspension 2-step D+sorb Wang et al. (embryogenic) 1994 Oryza sativa cell suspension 2-step Sucr+D+G+P Meijer et al. 1991 Triticum aestivum callus 2-step D+G+P; Fretz and Lörz D+G+Sor 1995 cell suspension 2-step D+sucr/sorb Chen et al. 1985 Dicotyledons Asparagus cell suspension vitrification G+Sucr (PVS3) Nishizawa et al. officianalis (embryogenic) 1993 cell suspension vitrification G+EG+PG+sorb Uragami et al. (PVS1) 1989 Brassica cell suspension vitrification EG+sorb+BSA Langis et al. campestris 1989 Catharanthus cell suspension 2-step S+G+PG Van Iren et al. roseus 1995 cell suspension encapsulation- S Bachiri et al. dehydration 1995 Citrus sinensis callus vitrification G+EG+D+Sucr Sakai et al. (PVS2) 1990 callus 2-step G+sucr Sakai et al. 1991 cell suspension 2-step D+sucr Engelmann et (embryogenic) al. 1994 callus 2-step D+sucr Engelmann et al. 1994 Grape cell suspension 2-step D+malt Dussert et al. (embryogenic) 1992 Musa cell suspension 2-step D; G Panis et al. 1990 Nicotiana tabacum cell suspension 2-step S+G+PG Van Iren et al. 1995 cell suspension vitrification G+EG+D+Sucr Reinhoud et al. (PVS2) 1995 Ipomoea batatas callus encapsulation Sucr Blakesley et al. (embryogenic) 2-step 1995 Strawberry cell suspension 2-step G+EG+D (PVS2); Wu et al. 1997 G+Sucr (PVS3), etc. Trifolium repens callus vitrification G+EG+D+Sucr Yamada et al. (meristematic) (PVS2) 1993 † G=glycerol, EG=ethylene glycol, PG=propylene glycol, D=DMSO, P=proline, Sorb=sorbitol, Sucr=sucrose, Malt=maltose. Cryopreservation techniques 95

Other classes of compounds used in pretreatments are plant hormones and anti-oxidants. Abscisic acid (ABA) is a plant hormone involved in the response of plants to environmental stress. It can induce tolerance to cryopreservation in suspension cells of tobacco (Reinhoud 1996), wheat (Chen et al. 1985) and bromegrass (Reaney et al. 1989). Application of vitamin C, an antioxidant, in the preculture medium prior to cryopreservation of calli of Hordeum murinum decreased lipid peroxidation and increased survival and regeneration rates (Fretz and Lörz 1995). Exposure to non-lethal temperature stress prior to the cryopreservation procedure also can enhance survival rates. Heat shock treatment induces cryotolerance in Saccharomyces cerevisiae cells (Kaul et al. 1992) and improves survival rates after cryopreservation of mannitol-precultured tobacco cells in a non-optimal growth phase (Reinhoud et al. 1995). Cold-hardening, the ‘natural way’ to induce freezing tolerance, has been successfully applied in protocols for cryopreservation of wheat (Triticum aestivum L.) (Chen et al. 1985).

Cryopreservation procedure Three different procedures have been used for cryopreservation of plant cells: two-step freezing, vitrification and encapsulation-dehydration. These procedures use a dehydration step to avoid intracellular ice-crystal formation and so-called cryoprotectants (e.g. glycerol, dimethylsulfoxide (DMSO), ethane- or propane- diol, sugars) to limit damage during freezing and thawing. Some of these cryoprotectants will enter the cells whereas others appear to act outside the cells (McGann 1978). They function by altering certain physical properties of water, such as decreasing the freezing point of water, lowering the rate of ice crystal growth and changing the shape of ice crystals. In addition, they have certain protective properties. But, the mechanisms by which they protect cellular components during freezing and thawing are far from completely understood (Fink 1986; Anchordoguy et al. 1987; Kruuv et al. 1990; MacFarlane and Forsyth 1990) (for examples of cryoprotectant mixtures see Table 1). Most of the successful mixtures of cryoprotectants have been composed empirically. Cryoprotectants can also be toxic to plant cells, especially when used at higher concentrations or at higher temperatures (Arakawa et al. 1990; Fahy et al. 1990).

Two-step freezing The traditional method for cryopreservation of cultured plant cells is the two- step or equilibrium freezing method developed in the late 1970s (Withers and King 1980; see Withers 1985). This procedure includes an incubation of cells in a mixture of cryoprotectants (total concentration of 1–2M), which causes moderate dehydration of the cells, followed by a slow freezing step (for example, 1°C/min down to approximately –35°C). During slow freezing the cells are further dehydrated due to induction of crystallization (and thus concentration) of the external medium resulting in osmotic removal of water from the cells. Upon further (quick) cooling to the temperature of liquid nitrogen, the cell contents solidify (vitrify, that is form a glass) without ice formation (Meryman and 96 Cryopreservation of Tropical Plant Germplasm

Williams 1985). After storage, rapid warming rates are used to prevent regrowth of ice crystals during thawing. Results from our laboratory and from others have shown that this procedure is successful for a large number of cell lines from widely different species (Withers 1985; Schrijnemakers and Van Iren 1995; Van Iren et al. 1995). The two-step freezing procedure requires a programmable freezer, although alternatives for such an expensive apparatus have been used (Maddox et al. 1993; Schrijnemakers and Van Iren 1995).

Vitrification In 1985, a new method for cryopreservation of animal cells was reported (Rall and Fahy 1985). This so-called vitrification procedure is based on severe dehydration at non-freezing temperatures by direct exposure to concentrated cryoprotectants (total concentration ranging from 5–8M), followed by rapid freezing. The rapid cooling rates prevent nucleation and growth of ice crystals and facilitate vitrification of the surrounding medium as well as cell contents. After storage, rapid warming rates are used to prevent devitrification during thawing. The first successful vitrification procedures for plant cell suspensions were developed in 1989 (Langis et al. 1989; Uragami et al. 1989). The main advantage of the vitrification procedure is that an expensive programmable freezer is not required. Furthermore, vitrification uses ultra-rapid cooling rates. Therefore tolerance to vitrification is primarily a matter of dehydration tolerance (Langis and Steponkus 1991; Steponkus et al. 1992; Reinhoud 1996). Thus, for cryopreservation of plant cell cultures which are sensitive to chilling the vitrification procedure is the method of preference. In general, we obtain higher survival rates using vitrification than with two-step freezing (Reinhoud et al. 1995; Van Iren et al. 1995).

Encapsulation-dehydration The encapsulation-dehydration technique also does not require the use of a programmable freezer and slow freezing. Furthermore, this technique does not use high concentrations of (putatively toxic) cryoprotectants. The encapsulation- dehydration procedure was developed for cryopreservation of shoot-tips (Dereuddre et al. 1990) but also has been successfully applied for the cryopreservation of plant suspension cells (Bachiri et al. 1995). During the encapsulation-dehydration technique, cells are encapsulated in alginate beads, cultured for several days on medium with increased sucrose concentration, air- dried using silica gel or the airflow of a flow cabinet and directly transferred to liquid nitrogen. After storage the beads are slowly warmed. The encapsulation also has been used in combination with the two-step freezing procedure (Blakesley et al. 1995).

Post-thaw treatments After thawing, suspension cultures are generally cultured on filter paper placed on (semi-) solid medium. This enables easy transfer of the cells to fresh medium and slow diffusion of the cryoprotectants, which appears to be beneficial for Cryopreservation techniques 97 recovery of the cells (Withers 1979). Once regrowth has started (usually within 2- 4 weeks) the suspension cells can be transferred to liquid medium. The composition of the recovery medium can influence the recovery after cryopreservation. Dussert et al. (1992) and Maddox et al. (1993) showed that reduction of the auxin concentration could improve regrowth. Kuriyama et al. (1996) showed that the presence of ammonium ions in the post-thaw medium is deleterious for recovery after cryopreservation. Addition of activated charcoal to post-thaw medium enhances survival rates of cryopreserved plant cells (Kuriyama et al. 1990; Schrijnemakers and Van Iren 1995). Activated charcoal may absorb substances that are deleterious to the recovering cells. However, Dussert et al. (1992) reported that the presence of activated charcoal reduced regrowth of cryopreserved grape cells. Benson et al. (1995) showed that for the post-thaw recovery of rice cells, desferrioxamine had a positive effect. Desferrioxamine is an iron-chelating drug that has been used to reduce free radicals in animal tissues exposed to low temperature. In pigmented cultures, the use of dimmed light during recovery, to avoid photo-oxidation and free radical formation, can be beneficial for regrowth (Benson and Noronha-Dutra 1988).

Conditions for successful cryopreservation The condition for successful cryopreservation is not only that the stored culture can start regrowth after thawing, but also that the culture has retained the same characteristics as it had before cryopreservation. Many publications show that characteristics such as growth rate, regeneration capability, ploidy levels and RAPD markers are retained after cryogenic storage (Kobayashi et al. 1990; Meijer et al. 1991; Ward et al. 1993; Wang et al. 1994; Ribeiro et al. 1996). In contrast, Wan and Vasil (1996) report that efficiency of regeneration of napier grass (Pennisetum purpureum Schum.) plants from cryopreserved suspension cells was reduced in comparison with the controls. The freeze-thaw cycle may also result in selection. A cell suspension or callus culture consists of a heterogeneous cell population. One type of cell, for example highly cytoplasmic cells, may be more tolerant to cryopreservation than other types of cells. By freezing and thawing a cell culture, the more tolerant cells may be selected above other cells. Aguilar and colleagues (1993) obtained a higher production of somatic embryos after cryopreservation than with unfrozen cells. Gnanapragasam and Vasil (1992) showed that protoplast yield and plating efficiencies of cryopreserved cells were higher than for unfrozen cells. Furthermore, several freeze-thaw cycles can result in the isolation of a culture with enhanced cryotolerance (Kendall et al. 1990; Watanabe et al. 1990, 1992). Thus, although the basic properties of the cell lines may not change, there is a change of selection as a result of cryopreservation. To minimize the risk of selection, the cryopreservation procedure with the highest survival rates should be chosen. 98 Cryopreservation of Tropical Plant Germplasm

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Cryopreservation of banana embryogenic cell suspensions: an aid for genetic transformation Bart Panis, Hilde Schoofs, Serge Remy, László Sági and Rony Swennen Laboratory of Tropical Crop Improvement, Catholic University of Leuven, 3001 Heverlee, Belgium

Introduction Many pests and diseases are threatening banana cultivation. These are caused by fungi (Mycosphaerella spp., Fusarium spp.), viruses (bunchy top, bract mosaic, cucumber mosaic and streak viruses), nematodes (Radophulus similis, pratylenchus spp., etc.) and bacteria (Pseudomonas solanacearum). Resistance to these pathogens can be introduced through classical breeding or biotechnology. Since most of the cultivated banana varieties are highly sterile, classical breeding programmes are very slow and labour-intensive. Furthermore, against some pathogens such as banana viruses, no source of resistance is available in the banana genepool. Genetic engineering might be a welcome alternative. In monocots, embryogenic cell suspensions are often the material of choice for transformation, particularly in sterile crops like banana where zygotic embryos are not available. Embryogenic cell suspensions are until now the only source of regenerable protoplasts in banana (Panis et al. 1993). When subjected to electroporation, they give rise to high transient expression frequencies of an introduced marker gene (Sagi et al. 1994). Walled suspension cells are successfully transformed by means of particle bombardment (Sagi et al. 1995) and Agrobacterium (Hernandez et al. 1998). In this way, genes coding for new types of antifungal proteins as well as virus resistance genes are introduced in banana. The main bottleneck for the application of transformation remains the initiation of cell suspensions of good quality, i.e. homogeneous embryogenic cell suspensions with high regeneration frequency. The initiation of these suspension cultures is difficult and time-consuming, irrespective of the starting material used (immature male flowers, immature zygotic embryos or proliferating in vitro meristems). Once established, these valuable cell suspensions are subject to somaclonal variation and microbial contamination. Moreover, a prolonged culture period may result in a decrease and finally in a total loss of morphogenic capacity. In 1990, we developed a cryopreservation technique for 'ideal' cell suspensions which involves cryoprotection with 7.5% DMSO (dimethyl sulphoxide) during 1 h at 0°C, followed by slow freezing at 1°C/min to –40°C and plunging into liquid nitrogen (Panis et al. 1990). An ‘ideal’ embryogenic cell suspension contains a high proportion of cells which are isodiametric and characterized by a relatively large nucleus, small multiple vacuoles and tiny starch and protein grains. In this report we describe a further optimization of the cryopreservation protocol in order to apply it to less 'ideal' but also highly regenerable banana cells. Less ‘ideal’ suspensions are more heterogeneous and can contain, besides embryogenic cell clumps, cells which are highly vacuolated and elongated, cells with very dense but granular cytoplasm, cells with large starch grains and organized globules. 104 Cryopreservation of Tropical Plant Germplasm

Material and methods

Plant material Embryogenic banana (Musa spp.) cell suspensions were initiated starting from proliferating meristem cultures of different banana cultivars as described by Schoofs (1997). Cauliflower-like meristem clusters are obtained on semi-solid MS medium containing 1 µM indole acetic acid (IAA) and 10 or 100 µM benzyl adenine (BA), depending on the proliferation rate of the cultivar under investigation. These meristem clusters are transferred to a semi-solid ZZ medium which contains ½ MS macroelements and iron, MS microelements, 5 µM 2,4- dichlorophenoxyacetic acid (2,4-D), 1 µM zeatine, standard MS vitamins, 10 mg/L ascorbic acid, 30 g/L sucrose and 2 g/L Gelrite. After 2 to 6 months, embryogenic calli are formed which are introduced in liquid ZZ medium. These cell cultures are kept on a rotary shaker at about 70 rpm and at 25±2°C under continuous light of 25 µE m-2 sec -1 PAR (Photosynthetic active radiation).

Preculture In those experiments where pregrowth was applied, cells were cultured for 1 day in liquid ZZ medium supplemented with 100 g/L sucrose.

Cryoprotection Cell suspensions were always cryopreserved 10 days after the last subculture when they were in their exponential growth phase. Cells were allowed to settle in a graduated centrifuge tube and the old medium was removed. New liquid ZZ medium with 30 (ZZ30) or 180 (ZZ180) g/L sucrose was added until a final settled cell volume of 30% was obtained. An equal volume of sterile ZZ30 or ZZ180 medium containing 15% dimethylsulfoxide (DMSO) was gradually transferred to the concentrated cell suspension during 1 h at room temperature.

Freezing, storage and thawing Samples of 1.5 ml of the cryoprotected suspensions were transferred to 2-ml cryotubes, sealed with teflon tape and placed in a stirred methanol bath. This methanol bath cools at a rate of 1°C/min. As soon as a temperature of –7.5°C was obtained, cryotubes were immersed for 3 s in liquid nitrogen to initiate ice crystallization. Then, they were further cooled to –40°C. After 30 min at –40°C, cryotubes were stored for at least 1 h in liquid nitrogen (–196°C). Alternatively, cryotubes containing the cryoprotected cell suspensions were placed in a NalgeneTM cryo 1°C Freezing Container (Cat. No. 5100-0001). This is a plastic container holding 250 ml of propanol. If this container is placed in a –70°C freezer, cooling rates of about 1°C/min are obtained. After overnight incubation, the cryotubes were transferred to liquid nitrogen. After storage, cryotubes were rapidly thawed in a beaker filled with sterile water of 40°C for about 1.5 min until most of the ice melted. In a second series of experiments, cells were also stored for 4 months in liquid nitrogen. Cryopreservation techniques 105

Recovery Thawed cells were plated on semi-solid medium ZZ or RD1 medium in 90-mm Petri dishes. RD1 medium contains MS macroelements and iron, MS microelements, 1 µm BA, standard MS vitamins, 100 mg/L myo-inositol, 10 mg/L ascorbic acid, 30 g/L sucrose and 2 g/L Gelrite. During the first week following cryopreservation, Petri dishes were always placed in the dark. Chemical cell viability was determined by the fluorescein diacetate (FDA) test whereby surviving cells fluoresce very brightly under ultraviolet illumination (Widholm 1972).

Results and discussion In a first set of experiments the influence of the cooling method and the addition of 180 g/L to the cryoprotective solution was investigated for different banana cell lines (Table 1).

Table 1. Viability rates, estimated according to the FDA test (%) and regrowth in Petri dishes after freezing (each observation represents two repetitions with each four frozen cryotubes) Methanol bath Methanol bath Nalgene box Nalgene box Cell line† 7.5 D‡ 7.5 D + 180 S 7.5 D 7.5 D + 180 S THP 70 (++)§ 70 (+++) 55 (0) 60 (0) Will G.001 45 (0) 55 (+) 40 (0) 40 (0) Will E4000 8b 50 (0) 55 (+) 50 (0) 55 (0) Will E4000 8 80 (++) 90 (++) 65 (0) 70 (0) † THP=Three Hand Planty; Will=Williams. ‡ 7.5 D: Cryoprotection with 7.5% DMSO; 7.5 D + 180 S: cryoprotection with 7.5% DMSO plus 180 g/L sucrose. § +++ : growth is comparable with non-frozen control, ++ : growth is less than the non- frozen control but the inoculated surface is still covered with regrowing cells, + : isolated regrowing cell masses, 0 : no growth, all cells became white.

As indicated in earlier reports (Panis et al. 1990), the results of the FDA viability test need to be interpreted with care. When the FDA viability rates were considered, only small differences existed between different treatments and accessions. However, we observed that despite comparable FDA values, regrowth was obtained only using the methanol bath and not with the Nalgene boxes. There are two possible reasons which might account for the lack of regrowth after freezing in Nalgene boxes. Cells might be too dehydrated since slow freezing proceeds until –70°C instead of –40°C before plunging the cryovial into liquid nitrogen. Or, the lack of regrowth might also be due to insufficient dehydration since in the Nalgene boxes no ice-crystallization phase could be applied. This could result in supercooling of the extracellular solutions down to -20°C. As such the positive effect of protective dehydration is almost completely lost. The addition of higher sucrose levels to the cryoprotective solution has for most suspensions a positive effect on FDA viability rates and, what is more 106 Cryopreservation of Tropical Plant Germplasm important, on post-thaw regrowth. This was also observed in sugarcane embryogenic callus (Martinez-Montero et al. 1998). Only the cell suspension which contained the highest ratio of embryogenic cells, Three Hand Planty, showed growth comparable to the non-frozen control. In a second series of experiments, an additional pregrowth phase was included for different cell lines (Table 2). Pregrowth in liquid medium supplemented with 6% mannitol during 2 or 7 days did not affect FDA viability nor regrowth of control as well as frozen suspensions (Panis 1995). Therefore, we applied a 1-day treatment with 100 mg/L sucrose since the addition of this compound can result in a cryoprotective metabolism as shown for proliferating banana meristems (Panis et al. 1996).

Table 2. Regrowth in Petri dishes after freezing (values between brackets represent recovery rates after 4 months of storage in liquid nitrogen) Cell line† 7.5 D‡ 7.5 D + 180 S P +7.5 D + 180 S THP 7+8 +++ (++)§ +++ (+++) + (+) THP 7+8 (2) ++ (++) +++ (++) + (+) THP C.1002 ++ (+++) ++ (+++) ++ (+) Will G.0001 + (+) ++ (++) + (+) Will E4000 NS + (++) ++ (+++) + (+) Will E4000 8b 0 (0) 0 (0) 0 (0) Will E4000 0 (0) 0 (0) 0 (0) † THP=Three Hand Planty; Will=Williams. ‡ 7.5 D: Cryoprotection with 7.5% DMSO; 7.5 D + 180 S: cryoprotection with 7.5% DMSO plus 180 g/L sucrose; P + 7.5 D + 180 S: preculture for 1 day with 100 g/L sucrose followed by cryoprotection with 7.5% DMSO plus 180 g/L sucrose. § +++ : growth is comparable with non-frozen control, ++ : growth is less than the non- frozen control but the inoculated surface is still covered with regrowing cells, + : isolated regrowing cell masses, 0 : no growth, all cells became white.

No consistent difference existed between growth of cells stored for 1 h and 4 months in liquid nitrogen. All the Three Hand Planty suspensions again resulted in high recovery rates since they contain a higher frequency of embryogenic cells than the Williams suspensions. The two Williams suspensions which were not able to recover after freezing were totally lacking embryogenic cells. Preculture with sucrose had a negative effect on regrowth of frozen cells. Since precultured non-frozen cells showed growth comparable to non- precultured, non-frozen cells, this low survival could be attributed to the intracellular accumulation of sucrose which is strengthened by freeze- dehydration. In that case, toxic concentration might be reached. Regrowing cells on semi-solid RD1 medium were able to regenerate through somatic embryogenesis into normal plants. If cells were transferred after cryopreservation onto semi-solid ZZ medium, the embryogenic callus thus obtained could be inoculated into liquid ZZ medium in order to again form an embryogenic cell suspension (Table 3). Cryopreservation techniques 107

Table 3. List of regrowing banana suspensions which are currently stored in liquid nitrogen for long-term storage. Data represent number of 2-ml cryotubes stored in a specific year. Cultivar Line Group 1993 1995 1996 1997 1998 Total M. balb. tani - BB 1 1 M. acc. Malaccensis Orsay AA 5 5 Bluggoe Serre ABB 8 8 Bluggoe - ABB 2 2 Bluggoe 10 ABB 1 1 Bluggoe 9 ABB 1† 6 7 Monthan - ABB 2 2 Bise Egome-1 pb 13 AABp 14† 14 Bise Egome-1 pb 16 AABp 8† 8 Dominico Harton - AABp 6 6 French Sombre CIRAD AABp 2 2 Three Hand Planty 7+8 AABp 28 11† 8 47 Three Hand Planty lijn1 AABp 5 5 Three Hand Planty lijn2 AABp 8 8 Three Hand Planty - AABp 1 8 9 Three Hand Planty C10002 AABp 1 1 Three Hand Planty MAL AABp 8 8 Grande Naine Mexico AAA 8 6 14 Grande Naine Jim Dale AAA 6 6 Williams E4000 AAA 2 22 8† 32 Williams G0001 AAA 3 5 8 Williams G0002 AAA 3 5 8 Williams BSJ AAA 12 12 Nakitengwa pb 15 AAAh 14 14 Nakitengwa pb 23-erlen 3 AAAh 14† 14 Total 242 † Cell lines screened for their capacity to give rise to an embryogenic cell suspension after freezing.

Such post-thaw re-initiated cell suspensions were the source of regenerable protoplasts in rice (Meijer et al. 1991), Festuca and Lolium species (Wang et al. 1994). Moreover, cryopreserved rice callus (Cornejo et al. 1995) and maize cell cultures (Gordon-Kamm et al. 1990) proved to be a constant source of regenerable cell cultures for the production of transgenic plants. Using the optimized cryopreservation protocol described above we are now able to store 25 cell lines belonging to 11 different banana cultivars in liquid nitrogen (Table 3). The fact that some cell suspensions are not able to withstand cryopreservation might be considered as a reason for further optimization of the cryopreservation protocol. In addition to the more conventional procedure involving slow freezing in the presence of a cryoprotective solution often containing DMSO, successful cryopreservation of cell suspensions is also reported after vitrification (Sakai et al. 1990; Nishizawa et al. 1993; Huang et al. 108 Cryopreservation of Tropical Plant Germplasm

1995; Watanabe and Steponkus 1995), encapsulation-dehydration (Bachiri et al. 1995; Swan et al. 1998), encapsulation-vitrification (Gazeau et al. 1998), encapsulation combined with slow freezing (Gazeau et al. 1998) and vitrification combined with slow freezing (Wu et al. 1997). However, since the cell suspensions which are recalcitrant to the above-described cryopreservation protocol are not regenerable, they will not be used in genetic engineering. As such their preservation might be more of scientific than of practical value.

Acknowledgements The author thanks INIBAP (International Network for the Improvement of Banana and Plantain), the Belgian Administration for Development Cooperation (BADC/ABOS) and the ‘Onderzoeksfonds’ of the Catholic University of Leuven for their financial support. Also SGRP (System-wide Genetic Resources Programme) is gratefully acknowledged for the travel funds to participate in the workshop.

References Bachiri, Y., C. Gazeau, J. Hansz, C. Morisset and J. Dereuddre. 1995. Successful cryopreservation of suspensions cell by encapsulation-dehydration. Plant Cell Tissue and Organ Culture 43: 241-248. Cornejo, M.-J., V.L. Wong and A.E. Blechl. 1995. Cryopreserved callus: a source of protoplasts for rice transformation. Plant Cell Reports 14: 210-214. Gazeau, C., H. Elleuch, A. David and C. Morisset. 1998. Cryopreservation of transformed Papaver somniferum cells. Cryo-Letters 19: 147-159. Gordon-Kamm, W., T. Spencer, M.L. Mangano, T.R. Adams, R.J. Daines, W.G. Tart, J.V. O’Brien, S.A. Chambers, W.R. Adams, N.G. Willetts, T.B. Rice, C.J. Mackey, R.W. Krueger, A.P. Kausch and P.G. Lemaux. 1990. Transformation of maize cells and regeneration of fertile transgenic plants. Plant Cell 2: 603-618 Hernandez, J.B., S. Remy, V. Galán Saúco, R. Swennen and L. Sági. 1998. Chemotactic movement and attachment of Agrobacterium tumefaciens to single cells and tissues of banana. Journal of Plant Physiology (in press). Huang, C.-N., J.-H. Wang, Q.-S. Yan, X.-Q. Zhang and Q.-F. Yan. 1995. Plant regeneration from rice (Oryza sativa L.) embryogenic cell suspension cells cryopreserved by vitrification. Plant Cell Reports 14: 730-734. Martinez-Montero, M.E., M.T. Gonzáles-Arnao, C. Borroto-Nordelo, C. Puentes-Díaz and F. Engelmann. 1998. Cryopreservation of sugarcane embryogenic callus using a simplified freezing process. Cryo-Letters 19: 171-176. Meijer, E.G.M., F. van Iren, E. Schrijnemakers, L.A.M. Hensgens, M. van Zijderveld and R.A. Schilperoort. 1991. Retention of the capacity to produce plants from protoplasts in cryopreserved cell lines of rice (Oryza sativa L.). Plant Cell Reports 10: 171-174. Nishizawa, S., A. Sakai, Y. Amano and T. Matsuzawa. 1993. Cryopreservation of asparagus (Asparagus officinalis L.) embryogenic suspension cells and subsequent plant regeneration by vitrification. Plant Science 91: 67-73. Panis, B. 1995. Cryopreservation of banana (Musa spp.) germplasm. Dissertationes de Agricultura 272, Catholic University of Leuven, Belgium. Panis, B., A. Van Wauwe and R. Swennen. 1993. Plant regeneration through direct somatic embryogenesis from protoplasts of banana (Musa spp). Plant Cell Reports 12: 403-407. Cryopreservation techniques 109

Panis, B., L.A. Withers and E. De Langhe. 1990. Cryopreservation of Musa suspension cultures and subsequent regeneration of plants. Cryo-Letters 11: 337-350. Panis, B., N. Totté, K. Van Nimmen, L.A. Withers and R. Swennen. 1996. Cryopreservation of banana (Musa spp.) meristem cultures after preculture on sucrose. Plant Science 121: 95-106. Sagi, L., B. Panis, S. Remy, H. Schoofs, K. De Smet, R. Swennen and B.P.A. Cammue. 1995. Genetic transformation of banana and plantain (Musa spp.) via particle bombardment. Bio/Technology 13: 481-485. Sagi, L., S. Remy, B. Panis, R. Swennen and G. Volckaert. 1994. Transient gene expression in electroporated banana protoplasts (Musa spp. cv. 'Bluggoe', ABB group) isolated from embryogenic cell suspensions. Plant Cell Reports 13: 262-266. Sakai, A., S. Kobayashi and I. Oiyama. 1990. Cryopreservation of nucellar cells of navel orange (Citrus sinensis Osb. var. brasiliensis Tanaka) by vitrification. Plant Cell Reports 9: 30-33. Schoofs, H. 1997. The origin of embryogenic cells in Musa. Dissertationes de Agricultura 330, Catholic University of Leuven, Belgium. Swan, T.W., E.A. Deakin, G. Hunjan, G.R. Souch, M.E. Spencer, A.M. Stafford and P.T. Lynch. 1998. Cryopreservation of cell suspensions of Polygonum aviculare using traditional controlled rate freezing and encapsulation/dehydration protocols, a comparison of post-thaw cell recovery. Cryo-Letters 19: 237-248. Wang, Z.Y., G. Legris, J. Nagel, I. Portykus and G. Spangenberg. 1994. Cryopreservation of embryogenic cell suspensions in Festuca and Lolium species. Plant Science 103: 93- 106. Watanabe, K. and P.L. Steponkus. 1995. Vitrification of Oryza sativa L. cell suspensions. Cryo-Letters 16: 255-262. Widholm, J.M. 1972. The use of fluorescein diacetate and phenosafranin for determining viability of cultured plant cells. Stain Technology 47: 189-194. Wu, Y., F. Engelmann, A. Frattarelli, C. Damiano and L.A. Withers. 1997. Cryopreservation of strawberry cell suspension cultures. Cryo-Letters 18: 317-324. 110 Cryopreservation of Tropical Plant Germplasm

Cryopreservation of sugarcane embryogenic callus Marcos E. Martínez-Montero1, María Teresa González-Arnao2, C. Borroto- Nordelo1, C. Puentes-Díaz 1 and Florent Engelmann3 ¹ Centro de Bioplantas, UNICA, Car. a Morón km 9, CP-69450, Ciego de Avila, Cuba ² Centro Nacional de Investigaciones Científicas, Ave. 25 y 158, Apartado 6990, Cubanacán, Playa, La Habana, Cuba ³ IPGRI, 00145 Rome, Italy

Introduction Classical freezing protocols, which include slow controlled cooling (0.5– 1°C/min) to around –40°C followed by immersion of samples in liquid nitrogen, are usually employed for cell suspensions and embryogenic calluses (Kartha and Engelmann 1994). Storing embryogenic cell suspensions or calluses in liquid nitrogen preserves their regeneration capacities and limits the risks of somaclonal variation which increase with culture duration. In the case of sugarcane, cryopreservation protocols have been developed for various materials: apices of in vitro plantlets using the encapsulation-dehydration technique (González-Arnao et al. 1993a, 1993b; Paulet et al. 1993); cell suspensions (Finkle and Ulrich 1979) and embryogenic calluses using classical freezing protocols (Jian et al. 1987; Gnanapragasam and Vasil 1990; Eksomtramage et al. 1992). Simplified freezing protocols in which the expensive programmable freezers required for classical protocols are replaced with simpler freezing devices including alcohol baths and/or domestic freezers have been established for various materials (Sakai et al. 1991; Nishizawa et al. 1992; Engelmann et al. 1994, 1997). In this paper, we describe a simplified cryopreservation protocol and its application to embryogenic calluses of three commercial varieties of sugarcane. We also present data on the effect of extended cryopreserved storage duration on the survival and plantlet production of calluses of one sugarcane variety.

Materials and methods

In vitro culture Embryogenic calluses were initiated by culturing 3 to 5-mm-long immature inflorescence segments on a modified MS medium as described by Martínez- Montero et al. (1998). Calluses were cultured in the dark at 25±2°C, with monthly subcultures. For plantlet regeneration, calluses were transferred to a medium devoid of 2,4–D at 27±2°C under a 16-h light/8-h dark photoperiod, with a 40 mmol m-2 s-1 photon dose.

Cryopreservation For cryopreservation experiments, 15 to 25-d-old calluses, about 3–5 mm in diameter, were employed. About one-third of sterile 2-ml cryotubes were filled Cryopreservation techniques 111 with fragments of calluses which were pretreated in liquid medium with sucrose concentrations ranging between 0.3 and 0.75M for 1 h at 0°C. Dimethylsulfoxide (DMSO) was added progressively to the liquid medium over a period of 30 min until the desired final concentration (5, 10 or 15%, v/v) was reached. Freezing was performed using a home-made ethanol bath, consisting of a polypropylene container filled with 700 ml of ethanol precooled at 0°C. Cryotubes were inserted in holes pierced in a thin polypropylene plate floating on top of the ethanol, which allowed immersion of the cryotubes in the coolant. The ethanol bath was then placed in a –40°C freezer, thus allowing an average cooling rate of 0.4–0.6°C/min between 0°C and –40°C. Crystallization was induced manually in the cryoprotective medium at a temperature intermediate between the nucleation and the crystallization temperature of the cryoprotective medium, by briefly putting the base of the cryotubes in contact with liquid nitrogen. Once the temperature of –40°C was reached, the cryotubes were kept for 2 h at this temperature, then immersed rapidly in liquid nitrogen. Samples were kept for a minimum of 2 h at –196°C. Rapid thawing was carried out by plunging the cryotubes in a +40°C water-bath. Calluses were then transferred directly (without washing) to recovery medium. Three replicates, each consisting of six fragments of embryogenic calluses, were used for each experimental condition. The survival rate, evaluated 40–50 d after freezing, corresponded to the percentage of calluses which had increased in size during the recovery period. For the measurement of plantlet production, six calluses randomly chosen in each condition were transferred to standard medium without 2,4–D, and the number of plantlets produced was estimated after 80 d of culture. The survival rate and plantlet production of calluses of variety CP 5243 were evaluated after storage for 2 h, 4 and 14 months in liquid nitrogen.

Results Survival of calluses after pretreatment was comparable between the three varieties. It was high with 5% and 10% DMSO, and decreased slightly with 15% DMSO. After freezing in liquid nitrogen, higher survival was achieved with variety CP 5243 than with the other two varieties. With varieties CP 5243 and C 91-301, high survival rates were obtained for a large range of pretreatment conditions. In contrast, for variety C 1051-73, high survival was achieved after pretreatment with 5% DMSO and 0.3M sucrose only. Fully developed plants could be obtained from regenerating calluses of all three sugarcane varieties (Table 1). The number of plantlets produced from control calluses was higher than from cryopreserved ones and regeneration from calluses of variety CP 5243 was much higher than from the two other varieties. Regeneration of plantlets was obtained from a much broader range of experimental conditions than those which had ensured optimal survival. 112 Cryopreservation of Tropical Plant Germplasm

Table 1. Effect of DMSO and sucrose concentration in the cryoprotective solution on the number of plantlets produced from control (-LN) and cryopreserved (+LN) embryogenic calluses of sugarcane varieties CP 5243, C 91-301 and C 1051-73 (Reprinted from Martinez Montero et al. 1998, with permission) Plantlets produced DMSO Sucrose CP 5243 C 91-301 C 1051-73 (%) (M) -LN +LN -LN +LN -LN +LN 0.3 190 143 88 0 90 0 5 0.5 196 165 69 33 90 35 0.75 211 150 72 34 88 44 0.3 210 150 82 40 95 48 10 0.5 213 106 73 42 99 51 0.75 200 119 63 41 88 45 0.3 243 165 70 18 82 27 15 0.5 209 171 65 18 81 29 0.75 209 161 51 18 75 25

The effect of storage duration on the survival rate and plantlet regeneration of cryopreserved calluses of variety CP 5243 is presented in Table 2. Even though a slight decrease in survival between 2 h and 4 months in storage could be noted under some conditions, both the survival rate and the number of plantlets regenerated from cryopreserved calluses were not modified after 14 months under LN storage.

Table 2. Effect of extended storage duration on the survival rate and plantlet production of callus of variety CP 5243 (Reprinted from Martinez Montero et al. 1998, with permission) Survival (%) Plantlets produced DMSO (%) Sucrose (M) 2 h 4 mo 14 mo 2 h 4 mo 14 mo 0.3 81 75 77 150 113 95 10 0.5 90 81 78 106 124 115 0.75 94 73 77 119 99 100

Discussion/Conclusion The simplified freezing protocol developed in this study was efficient since it achieved results comparable to those obtained with the classical protocols employed by other authors to cryopreserve sugarcane calluses (Jian et al. 1987; Gnanapragasam and Vasil 1990; Eksomtramage et al. 1992). Differences in sensitivity to the different cryoprotective mixtures were noted among clones, as previously reported by Eksomtramage et al. (1992). Previous experiments (Jian et al. 1987; Eksomtramage et al. 1992) using a classical freezing procedure had indicated that sugarcane embryogenic calluses could be successfully frozen using a relatively wide range of cooling rates, between 0.1 and 1°C/min. This allowed us to obtain positive results after freezing the embryogenic calluses with a cooling rate ranging between 0.4 and 0.6°C/min. Cryopreservation techniques 113

In this work, the use of a simplified freezing protocol achieved good survival and numerous plantlets from regenerating calluses of all sugarcane varieties. The variety CP 5243 gave a much higher survival rate and production of plantlets than the other two varieties tested. However, it is important to mention that control calluses of these two varieties (C 91-301 and C 1051-73) had a much slower growth rate and released a large amount of phenolic compounds in the medium. This underlines the importance of the in vitro propagation procedure in the successful establishment of a cryopreservation protocol for any given material. Extending storage duration in liquid nitrogen to 14 months did not induce any modification in the survival rate and plantlet production of cryopreserved calluses, which confirms the applicability of cryopreservation for the long-term conservation of material produced in vitro. In conclusion, efficient and simple cryopreservation protocols have been developed for cell suspensions, embryogenic calluses and apices of sugarcane and successfully applied to a wide range of genotypes. Sugarcane could thus be one of the first tropical crops to which cryopreservation is applied routinely.

Acknowledgements The authors gratefully thank IPGRI for allowing F.E. to participate in this research project.

References Eksomtramage, T., F. Paulet, E. Guiderdoni, J.C. Glaszmann, and F. Engelmann. 1992. Development of a cryopreservation process for embryogenic calluses of a commercial hybrid of sugarcane (Sacharum sp.) and application to different varieties. Cryo-Letters 13: 239-252. Engelmann, F., D. Dambier and P. Ollitrault. 1994. Cryopreservation of embryogenic cell suspensions and calluses of citrus using a simplified freezing process. Cryo-Letters 15: 53-58. Engelmann, F., M. Lartaud, N. Chabrillange, M.P. Carron and H. Etienne. 1997. Cryopreservation of embryogenic calluses of two commercial clones of Hevea brasiliensis. Cryo-Letters 18: 107-116. Finkle, B.J. and J.M. Ulrich. 1979. Effect of cryoprotectants in combination on the survival of frozen sugarcane cells. Plant Physiology 63: 598-604. Gnanapragasam, S., and K. Vasil. 1990. Plant regeneration from a cryopreserved embryogenic cell suspension of a commercial sugarcane hybrid (Saccharum sp.). Plant Cell Reports 9: 419-423. González-Arnao, M.T., F. Engelmann, C. Huet and C. Urra. 1993a. Cryopreservation of encapsulated apices of sugarcane: Effect of freezing procedure and histology. Cryo- Letters 14, 303-308. González-Arnao, M.T., F. Engelmann, C. Urra and P. Lynch. 1993b. Crioconservación de meristemos apicales de plantas in vitro de caña de azúcar mediante el método de encapsulación-deshidratación. Biotecnología Aplicada 10: 225-228. Jian, L.C., D.L. Sun and L.H. Sun. 1987. Sugarcane callus cryopreservation. Pp. 323-337 in Plant Cold Hardiness, P.H. Li (ed). Alan R. Liss, Inc., New York. 114 Cryopreservation of Tropical Plant Germplasm

Kartha, K.K. and F. Engelmann. 1994. Cryopreservation and germplasm storage. Pp. 195- 230 in Plant Cell and Tissue Culture, I.K. Vasil and T.A. Thorpe (eds). Kluwer Academic Pub. Dordrecht, The Netherlands. Martinez-Montero, M.E., M.T. Gonzáles-Arnao, C. Borroto-Nordelo, C. Puentes-Díaz and F. Engelmann. 1998. Cryopreservation of sugarcane embryogenic callus using a simplified freezing process. Cryo-Letters 19: 171-176. Nishizawa, S., A. Sakai, Y. Amano and T. Matsuzawa. 1992. Cryopreservation of asparagus (Asparagus officinalis L.) embryogenic suspension cells and subsequent plant regeneration by a simple freezing method. Cryo-Letters 13: 379-388. Paulet, F., F. Engelmann and J.C. Glaszmann. 1993. Cryopreservation of apices of in vitro plantlets of sugarcane (Saccharum sp. hybrids) using encapsulation-dehydration. Plant Cell Reports 12: 525-529. Sakai, A., S. Kobayashi and I. Oyama. 1991. Cryopreservation of nucellar cells of navel orange (Citrus sinensis Osb.) by a simple freezing method. Plant Science 74: 243-248. Cryopreservation techniques 115

Cryopreservation of pollen Leigh E. Towill and Christina Walters USDA, ARS National Seed Storage Laboratory, 1111 S. Mason St., Fort Collins, CO 80521, USA

Introduction Interest in pollen biology is increasing due in part to its use in biotechnology, in selection at the gametophytic stage and in examining gene expression in gametophytic and sporophytic generations. A recent book summarizes current knowledge in pollen biology and future prospects (Shivanna and Sawhney 1997). Pollen banks, where pollen is cryopreserved, may make pollen easily and readily available for any use (Schoenike and Bey 1981; Bajaj 1987). Although pollen banks are conceptually useful, their establishment has been infrequent in germplasm systems, except in the forest industry for seed orchards and improvement prog- rammes (Jett et al. 1993; Mercier 1995). This review will examine pollen preser- vation, cryopreservation and procedures needed for developing pollen genebanks. Pollen is stored for facilitating crosses in breeding programmes, distributing and exchanging germplasm among locations, preserving nuclear genes of germplasm, studies in basic physiology, biochemistry and fertility, and studies for biotechnology involving gene expression, transformation and in vitro fertilization. Pollen preservation supplements seed or clone preservation for germplasm banks and is not intended to replace them. Pollen also has been stored for allergen studies, but viability in this case is of minor concern. Storage for use in haploid generation through pollen embryogenesis also has been advocated. Storage of mature pollen is probably of minor importance for this use since in most systems pollen at the uninucleate stage is more responsive to embryogenesis. This may herald a need for long-term storage of immature pollen, but such storage has not been addressed yet. For most purposes the goal when preserving pollen is to retain viability and functionality in a large percentage of stored grains. Longevities required by users differ. A breeder may need to retain viability for about a year so that wide crosses can be made; longer-term storage may obviate the need to continually grow out pollinator lines. Orchard growers may release stored pollen to enhance nut set when pollinator lines produce insufficient amounts of pollen. Pollen used for germplasm conservation should remain viable for many years, which usually requires cryogenic storage. This is preferred if suitable equipment and a reliable, inexpensive supply of cryogen are readily available. A pollen bank should have facilities to collect, preserve and distribute pollen. These are analogous to issues in germplasm banks for seed or vegetative propagules. Pollen is an exhaustible reserve; provision must be made for periodic replenishment of the pollen bank. In this context it is obvious why pollen preservation is supplemental: the seed or clone must be conserved to yield the pollen. Multiple generations introduce the risk of population genetic problems such as loss of alleles through random drift or splitting of adaptive complexes. 116 Cryopreservation of Tropical Plant Germplasm

Pollen collection Collection is a critical component for a pollen bank (Hoekstra 1995) and criteria need to be established to determine what samples are brought into the bank. Sufficient amounts of pollen must be gathered and processed for banking purposes (viability testing, storage and distribution). Pollen collection from species which shed copious amounts is easily accomplished, but extraction of pollen from many species is difficult and time-consuming. Flowers must be gathered just before anthesis, and the stamens or anthers excised. The pollen or anthers are then desiccated to the required moisture level, usually by equilibration over a known relative humidity, or if the ambient humidity is low, simply on a laboratory bench. Various drying units have been devised for bulk amounts of pollen extracted from catkins or cones of conifers. In some species, the pollen must be separated from the anther before desiccation; in other species drying of the whole anther followed by gentle crushing is sufficient for storage and use. Pollen that clumps is difficult to extract and manipulate. With some species there may not be enough pollen for moisture measurements and assurance that moisture equilibration at a given relative humidity had occurred. These species probably would not be utilized within a pollen bank. Across genotypes, the differences in flowering, time of shedding and amounts produced are very demanding for pollen-gathering and bank operation. Pollen yield, quality and viability depend upon environmental conditions during flowering and microgametophyte differentiation and development. It is well known that heat and drought stresses can reduce quality and quantity of pollen. Some compensation for stress can be made with annual plants, such as replanting, but no such option is easily available for perennial plants, other than to wait for the next flowering time. Once collected, the sample must be processed in a timely fashion. Careful handling is important to maintain whatever viability and vigour exist for the pollen sample (Connor and Towill 1993). Handling usually consists of quality and viability tests, moisture content adjustment and packaging. Contamination with pollen from other samples must be avoided.

Viability tests Pollen quality and viability are important for banks. Seed quality is an important factor of seed longevity and it is assumed the same principle applies to pollen. With seed storage, it is well understood that the greatest longevities occur with good-quality seed. Quality is difficult to define, but a number of seed vigour tests have been devised for this purpose (Hampton and TeKrony 1995). Pollen quality is similarly important, but adequate tests of the state are lacking. Rate of germination (Shivanna et al. 1991) or rate of pollen tube growth may be useful, but determining this for all samples and making comparisons with known standards is time-consuming. Quality estimates for tricellular pollen, many types of which lack simple germination tests, might be even more complex. Nevertheless, some characterization of the pollen lot with regard to initial quality will be useful in comparing the utility of different storage regimes and in predictions of longevity. Cryopreservation techniques 117

Measurement of viability is crucial for any storage study. Conceptually, pollen viability may be measured by metabolic activity (usually respiration rate), membrane semipermeability, germination and seed set. Metabolic activity is rarely used as a method, probably because it is difficult to quantify, requires a fair amount of pollen and is time-consuming. Evaluating ATP and ADP contents may provide a new and rapid method for evaluating pollen function; data in maize have correlated well with seed set (Roeckel-Drevet et al. 1995). Simple measures of membrane semipermeability are much more commonly applied. Potassium leakage into an osmotically adjusted medium is one test. Tetrazolium dyes can be used to determine the ability of the membrane to retain some reducing agent (NADH, NADPH) such that the tetrazolium becomes reduced upon permeation, usually forming an insoluble, coloured formazan. Another common test uses fluorescein diacetate (FDA), which is permeable and is cleaved within the cell by non-specific esterases to form fluorescein (Heslop-Harrison and Heslop-Harrison 1970; Shivanna and Heslop-Harrison 1981; Heslop- Harrison et al. 1984). Fluorescein is impermable and can be detected by cellular fluorescence. If the cell has lost membrane semipermeability, the cleavage is slow and the product is not concentrated. Flow cytometers can be used to rapidly quantify percentages of stained pollen. Both tetrazolium and FDA tests require an adequate osmoticum and are used with rehydrated pollen. A slow rehydration of pollen is beneficial, and often obligatory; this is accomplished by humidification over 95 or 100% relative humidity. In vitro germination is commonly used for desiccation-tolerant pollen after samples have been rehydrated. Various media have been used, but the Brewbaker and Kwack (1963) formulation still is widely applicable. Modifications of boron and sucrose levels are usually needed for optimization; adjustment of calcium and potassium levels also may be necessary. Inclusion of polyethylene glycol has been beneficial for improving germination percentages in several species (Hong-Qi and Croes 1982; Shivanna and Sawhney 1995). Adequate in vitro germination tests for many tricellular pollens are still lacking. Pollen germination on the stigma and growth in the style also are used as a viability test and can be observed either using staining techniques to detect callose (aniline blue) within the pollen tube or by direct light microscopy. Fertility is an absolute measurement of the ability of the pollen grain to germinate and to set a seed. Unfortunately, the quantitative determination of viability in a pollen sample is very difficult using seed set data. Greater detail and discussion of most tests are described in Stanley and Linskens (1974), Hoekstra (1995) and Stone et al. (1995). The FDA test is generally considered the most rapid and accurate staining test for viability, though correlations with germination are not always high. A pollen grain can be viable in the sense of being alive, retaining membrane semipermeability and metabolizing, but may have lost the ability to germinate, or the ability to fertilize an egg. A pollen genebank would have to use rapid methods for viability assessment, but should develop correlative data on true functionality of the sample. Monitoring the quality and viability of samples placed into the bank is 118 Cryopreservation of Tropical Plant Germplasm necessary to ensure that the sample is not deteriorating with time. The frequency of such tests decreases if lower storage temperatures are used.

Pollen preservation Numerous studies over about 100 years have reported pollen longevities under various storage conditions. These have been summarized in a number of reviews (Visser 1955; Singh et al. 1961; Towill 1985; Akahima and Omura 1986). Many reports are merely observations made after storing pollen under what may be considered logical conditions and do not explore maximum longevities or factors affecting longevity. Sometimes factors have been intertwined, such as storage over an anhydrous desiccant where moisture is reduced continually during storage. The use of different viability assays, suboptimal in vitro germination tests and inadequate rehydration of dried pollen by humidification makes it difficult to compare studies and define actual longevities. The cell number in a pollen grain (bicellular or tricellular) at anthesis has been described for taxonomic families (Brewbaker 1967). In many families all species possess the same pollen cell number. All species of the Rosaceae contain bicellular pollen and all species of the Compositae contain tricellular pollen. Some families contain genera with bicellular pollen and other genera with tricellular pollen. Most genera only contain a single type, but some rare exceptions occur. Sporophytic incompatibility is associated with the tricellular state, but the Gramineae are a notable exception, having tricellular pollen and gametophytic incompatibility. The knowledge of the cellular trait is useful because prediction of viability tests and storage characteristics is possible. Generally pollen which is bicellular at anthesis usually can be germinated in vitro, is shed at a lower moisture content, survives extensive desiccation, and is longer-lived. Thus, viability is more easily measured and longevity can be greatly extended by desiccation and lowering the storage temperature. Of course, there are exceptions. Pollen from the Cucurbitae is bicellular, but is large, shed at a high moisture content and very desiccation-sensitive (Digonett-Kerhoas et al. 1989). Tricellular pollen at anthesis usually has a high moisture content and survives only limited desiccation. The viability of this pollen is difficult to measure, often being done by in situ growth in styles or seed set. Systems must be set up to avoid incompatibility problems. Longevities of untreated or partially desiccated tricellular pollen at most non-cryogenic temperatures are very short. Again, there are exceptions. Sugarbeet pollen is tricellular, but survives considerable desiccation. Among the Gramineae, Pennisetum spp. pollen has been reported to be tolerant to at least about 3% moisture (Hanna 1990). The limits of desiccation tolerance of tricellular pollens are generally not well defined, so it is uncertain what physiological and biochemical similarities these tolerant, tricellular pollens share with the bicellular desiccation-tolerant class. Certainly the designation of two classes of desiccation behaviour is simplistic and various intermediate types exist, as has been recognized in seed (Hong and Ellis 1996). The desiccation responses described below illustrate the complexity. Cryopreservation techniques 119

Non-cryogenic storage Sufficient longevities can be obtained for some pollen preservation purposes using simple and available storage conditions. Thus, chest freezers at temperatures of –20°C may allow 1 to several years of storage. Desiccation- sensitive pollen has extremely short longevities at temperatures from about -20°C to ambient. Parenthetically we note that there are few storage data for temperatures between about –20°C and –80°C; obviously this relates to the lack of suitable temperature chambers for this range. The following observations are mainly for bicellular, desiccation-tolerant pollen. Storage characteristics are very similar to those observed for desiccation- tolerant seed. Longevity is increased by using lower temperatures and lower moisture contents. The general relationship between storage temperature and longevity is illustrated in Figure 1. The actual longevities at temperatures of -20°C and above differ considerably among species and genotypes, and probably harvest year.

Fig. 1. Survival of desiccation-tolerant pollen with time of storage at different temperatures. Scales for survival and time differ with species and genotype. 120 Cryopreservation of Tropical Plant Germplasm

The interaction between water content and longevity in bicellular pollens is probably complex. In seeds, maximum longevity is achieved by storage at relative humidities between 15 and 25% (Walters 1998b and references within). The relationship between water content and relative humidity varies among seeds according to their chemical compositions, and thus, if the relative humidity of storage is kept constant at say 20%, the water content giving maximum longevity varies among seed species. The discovery that the optimum relative humidity is fairly constant among seed species gives a thermodynamic rationale for the established recommendation for storing seeds at water contents between 3 and 7% depending on species (IBPGR 1985). Consistent with studies of seed longevity, studies of the storage behaviour of pollen from Typha latifolia demonstrate that a relative humidity of about 15% provides maximum longevity (Buitink et al. 1998b). If equilibration over a given relative humidity provides the best survival for pollen as it does for seeds, then we predict that the optimum water content for storing pollen depends on chemical composition. Experimental evidence as well as theoretical predictions suggest that the optimum water content for storage of a given seed or pollen sample increases with decreasing storage temperature (reviewed by Buitink et al. 1998a, 1998b; Walters 1998a, 1998b). Differences in optimum water content are relatively small over narrow temperature ranges, but they may have profound effects on longevity over a period of years. The observation of moisture optima implies that pollen can be ‘overdried’. Practical experience has shown this to be a problem in determining viability (Jensen 1970; Hoekstra 1995; Buitink et al. 1998b), although rehydration duration and extent somewhat confound the issue among other literature reports. Pollen grains are sensitive to imbibitional damage and without slow rehydration and/or warm imbibition temperatures, grains that are overly dry may be irreparably damaged by the rapid uptake of water (Crowe et al. 1989). Studies from Hoekstra’s and Crowes’ laboratories show compelling correlations between initial water content of pollen, phase behaviour of membrane lipids (detected using DSC or FTIR spectroscopy) and pollen viability following imbibition (Crowe et al. 1989). The presence of sugars appears to moderate imbibitional damage by keeping polar lipids fluid even when dried to low water contents (Crowe et al. 1998). Thus, unless proper rehydration procedures are used or viability decreases are progressive with time (multiple samplings), care must be used when interpreting the mechanism of damage when sharp reductions in viability in extremely dry pollen are noted. Injury may result from imbibitional damage, desiccation damage and/or aging. What is responsible for viability decline during storage at temperatures greater than –20°C? There is a catalogue of changes that occur in seeds and pollen during storage and it is not particularly clear which are causes and which are effects of aging (reviewed in Walters 1998a). Based on seed studies and the food science literature, the mechanism of aging is likely to differ at different moisture levels. Respiration becomes very low for pollen held at about 85% relative humidity (approx. 25–30% moisture content on a fresh weight basis) for Cryopreservation techniques 121 both desiccation-tolerant and -sensitive pollen (Hoekstra and Bruinsma 1975). If pollen is stored at moisture contents above this, depletion of reserves and, perhaps, build-up of fermentation products when samples are held within a confined space, probably account for deterioration. When respiration is limited by low moisture content, viability still declines and is associated with loss of membrane semipermeability (Hoekstra et al. 1992a, 1992b). The data of van Bilsen and Hoekstra (1993) and van Bilsen et al. (1994a, 1994b) provide strong evidence that de-esterification of phosholipids, leading to accumulation of lysophospholipids (LPL) and free fatty acids (FFA), is correlated with permeability loss and was due to free radical events. The content of LPL and FFA was greater with aging of a relatively short-lived pollen of Papaver rhoeas than for the longer-lived pollen of Narcissus poeticus. For both pollens, product formation was faster with a moisture content of 15% compared with 7–8%. The production of LPL and FFA lead to membrane defects during rehydration as membrane lipids undergo the transition from gel to liquid crystalline phase. The damaging events associated with the ‘overdried’ condition are still uncertain, but probably relate to both free radical associated lipid damage and phase changes during the kinetics of dehydration and rehydration. Water content influences the types of reactions that cause aging. It also affects the kinetics of those aging reactions. For example, formation of LPL and FFA in pollen from Papaver rhoeas and Narcissus poeticus was faster when grains were stored at 15% than with 7–8% water (van Bilsen et al. 1994a, 1994b). This is because the viscosity of the cytoplasm is profoundly affected by water content. The science of amorphous states explains the interactions between viscosity, water content and temperature and the interested reader is referred to various reviews on how these interactions affect the stability of dried biological systems (Roos 1995; Sun and Leopold 1997; Bernal-Lugo and Leopold 1998; Crowe et al. 1998; Walters 1998a). When cells are dried sufficiently, there may be an abrupt decrease in the viscosity in the cytoplasm which is described as a glass transition. When glasses form, aging supposedly slows down and the temperature dependency of aging decreases (greater temperature coefficient at temperatures above the glass transition). Changes in the kinetics of aging of Typha latifolia pollen were associated with glass transitions in the grains, but the changes were substantially less than predicted by glass theory (Buitink et al. 1998b). In addition to storage temperature and moisture content, storage atmosphere can greatly affect longevity. Freeze-dried and vacuum-dried pollen subsequently stored in a vacuum have shown greater longevities than storage in air (Jensen 1964). Storage of dried pollen in nitrogen also prolongs viability (Hoekstra 1992). Both vacuum and nitrogen atmosphere enhancement of longevity are more demonstrable at –5°C to ambient conditions and it is uncertain if any benefit occurs with cryogenic storage. Storage in light has been detrimental to seeds and is associated with free radical events (Khan et al. 1996), but there are only anecdotal reports for pollen. Both storage atmosphere and light effects are understandable if free radical damage from oxyradicals is a major contributor to deterioration in the dried state. 122 Cryopreservation of Tropical Plant Germplasm

Desiccation-sensitive pollens (those with tricellular structure) are most often associated with members of the Compositae, Cruciferae, Gramineae and Umbelliferae. Moisture levels of these pollens at anthesis are often quite high (approx. 40-60%) and the water contents to which they can be dried vary among species and laboratories. Within the Gramineae, several reports document that pollens from Pennisetum sp. are relatively desiccation-tolerant, surviving down to at least 3% moisture (Hanna 1990). Maize pollen can survive to moisture levels below 10% (Barnabas and Rajki 1976), though damage was initially observed at about 40% moisture content (Buitink et al. 1996). In many studies there appears to be an effect of genotype on sensitivity. The survival moisture limits for pollen from many other species, for example wheat and barley, are not well described owing to limitations in obtaining enough pollen for moisture tests, lack of useful in vitro germination tests, and generally short lifetimes. Tricellular pollen is metabolically more active than bicellular pollen at comparable moisture levels (Hoekstra and Bruinsma 1975). Thus, storage lifetimes generally are measured in minutes to hours, rather than the days to weeks characteristic of desiccation- tolerant pollen. A detailed understanding of the nature of desiccation sensitivity in some pollens is lacking. The predominance of desiccation sensitivity in tricellular pollens or grains with particularly large cells suggests that there may be an ultra- structural basis for the sensitivity. In maize, the greatest loss of viability occurred when pollen was dried to water contents below about 25%, a water content that corresponds to changes in the freezing properties of water (Kerhoas et al. 1987; Buitink et al. 1996). Based on comparisons between Pennisetum and maize, low sugar contents are also associated with the desiccation-sensitive condition (Hoekstra et al. 1989). Hydrogen bonding by sugars, especially sucrose, is believed to prevent polar lipids from undergoing phase transitions or fusions during drying, thereby protecting membrane structure during desiccation (Crowe et al. 1998). It also has been suggested that sugars help to form a glassy matrix at relatively high water contents (e.g. Leopold et al. 1994), and this protects cells from structural derangements upon desiccation. In spite of different sugar compositions, the glass transition properties of a desiccation-tolerant and intolerant pollen were similar and there were no apparent relationships between the state-phase behaviour of the cytoplasm and that of the polar membranes or of the incidence of desiccation or imbibitional damage (Buitink et al. 1996). While the link between desiccation tolerance and glass transitions is not clear (reviewed by Vertucci and Farrant 1995), stability in the desiccated state may be achieved by particular properties of the glasses, as described previously.

Cryogenic storage Certainly the preferred storage condition, if available and affordable, is at cryogenic temperatures, which for the purpose of this article are below about -130°C. This can be achieved by a mechanical refrigeration system, but is more commonly accomplished either in the vapour phase over liquid nitrogen (approx. -160°C) or within the liquid nitrogen (–196°C). The use of such low temperatures Cryopreservation techniques 123 should minimize the deterioration described above. Most reports consist of a one-time sampling demonstrating that pollen survived for a set period. From this information, we know that storage at –80°C usually extends survival times of desiccation-tolerant pollens compared with storage temperatures of –20°C and above, but there are few direct comparisons. Viability after several years of –80°C storage also has been reported for desiccation-sensitive pollen from maize (Barnabas 1994), sunflower (Frank et al. 1982) and pearl millet (Hanna 1990). The benefits of still lower temperatures are obvious if these results are extrapolated to storage at cryogenic temperatures. There are more numerous examples of pollen that has retained high levels of viability after being desiccated and then stored under cryogenic temperatures (Towill 1985; Hanna and Towill 1995). Most examples have been with desiccation-tolerant pollen, but several desiccation-sensitive pollens have been reported. A list of species where pollen has been successfully cryopreserved is presented in Hanna and Towill (1995). Pollen with high moisture levels does not survive immersion to low temperatures, probably because of intracellular ice formation. Although it is logical that injury is due to ice formation during LN exposure, there is little direct evidence for the event. Desiccation-tolerant pollen with moisture levels below where freezable water does not exist usually has a viability after cryopreservation approximating that of an uncooled sample. There are, however, instances where the viability of the cryopreserved sample is considerably lower than that of the uncooled sample. Reasons for this reduced viability are not apparent. In a practical sense, desiccation-sensitive pollen can be cryopreserved by partially desiccating pollen to moisture levels where no freezable water exists but above levels where desiccation injury is apparent. With such an approach, Barnabas and workers (reviewed in Barnabas 1994) showed that maize pollen with desiccation to 9-25% moisture (fresh weight basis) survived LN exposure and storage and set seed. The highest seed set occurred with samples of about 12- 20% moisture. Other researchers have observed similar results with desiccation- sensitive pollen from maize (Shi and Tian 1989a), rice (Hu and Gou 1996), rye (Shi and Tian 1989; Kovacs and Barnabas 1993), sugarcane (Tai 1995), triticale (Kovacs and Barnabas 1993) and wheat (Andreica et al. 1988). In rye the moisture range of cryopreserved pollen giving seed set similar to fresh pollen was 6-16% (Shi and Tian 1989b). It is not apparent if this strategy will be applicable to all desiccation-sensitive pollen. Sunflower pollen survived low-temperature storage with no prior desiccation (Frank et al. 1982), but no moisture levels were recorded. In most cases cooling and warming rate studies have not been detailed. Cooling is by immersion of the vial at the temperature of interest and warming is by placement at room temperature. In some cases more rapid cooling rates, in conjunction with rapid warming rates, may be beneficial. Biotechnology has spawned interest in manipulations of gametes for various studies, particularly for use with cereals. In vitro fertilizations using isolated sperm cells are feasible, but problems of developing useful storage systems for 124 Cryopreservation of Tropical Plant Germplasm desiccation-sensitive pollens have led some to examine cryopreservation of isolated sperm cells so that they are readily available for experiments. Some survival has been reported at –80°C (Roeckel and Dumas 1993), but lack of reported success at –196°C suggests that requirements are stringent. Storage of pollen is technically simpler than first isolating gametes and then storing them. The development of reliable methods for cereal pollens may obviate the need for male gamete storage. Recently it has been shown that freeze-dried sperm of mouse, which was not viable, still had a functional nucleus such that microinjection into an egg produced embryo development (Wakayama and Yanagimachi 1998). Although no comparable information is available for higher plants, nuclei from non-germinating and, perhaps, non-viable pollen grains might be useful for microinjection methods for in vitro fertilization. This, of course, would not be a goal of a pollen bank, but might allow use of samples which have very low viability.

Longevity under cryogenic storage The ready availability of cryogenic storage for biological systems is relatively recent and there are few data that describe longevity. This is understandable since it is argued that the use of low temperatures greatly retards most types of molecular reactions; hence lifetimes might be many hundred of years. Most literature reports with a variety of biological specimens (mainly microbes, blood cells, semen) cite retention of viability at levels approximating that of short-term exposure after about 1 to a few years in liquid nitrogen. For pollen, several reports chronicle survival after about 1 year of storage in liquid nitrogen, including Allium sp. (Ganeshan 1986; Kanazawa et al. 1992), Juglans nigra (Luza and Polito 1988) and Diospyros khaki (Wakisaka 1964). Jojoba (Lee et al. 1985) and hop (Haunold and Stanwood 1985) pollens retained viability over a 2-year test period in cryogenic storage. Pecan pollen stored for 3 years at –196°C retained high fertility levels (Yates and Sparks 1990). Vitis vinifera L. pollen showed no apparent decline in viability, as assessed by in vitro germination, during 5 years of storage (Ganeshan and Alexander 1990). Fertility rates were low with 5 year stored samples, but there were insufficent data to make comparisons with 1 year stored samples. Pollen from tomato and eggplant was fertile after 6 years of cryogenic storage (Alexander and Ganeshan 1989). Maize pollen stored for 10 years at –196°C set seed (Barnabas 1994), but percent seed set declined somewhat from that tested after 2 years of storage. Gladiolus pollen stored cryogenically for 10 years retained high levels of in vitro germination and seed set (Rajaskharan et al. 1994). Some data suggest that viability declines over a relatively short time. Examples exist for pollen from maize, Hevea and conifers. Hevea pollen with 7- 11% moisture exhibited a decline from 20% in vitro germination after 1 month to 2% after 5 months of storage in liquid nitrogen (Hamzah and Leene 1996). Some pine and spruce pollen stored in liquid nitrogen also showed a storage decline over a 24-month period, surprisingly at a rate similar to that occurring at –20°C (Lanteri et al. 1992). These observations are also surprising since good-quality conifer pollen stores very well at non-cryogenic temperatures. Sugarbeet pollen Cryopreservation techniques 125 stored at about –160°C also showed a drop in in vitro germination after 5 years of storage (Stanwood and Towill, unpubl.); this study was incomplete because of fairly low initial germination and limited replication. Maize and lily pollen were reported to decline rapidly while stored in liquid nitrogen (Nath and Anderson 1975). Andreica et al. 1988 also noted that wheat pollen viability declined during cryostorage. The reasons for the reported declines are not known. It is important to determine if longevities at cryogenic temperatures are truly less than anticipated or if some source of error exists in handling procedures. Experimental details in many publications are insufficient to describe exact manipulations of the sample and thus to determine if these contribute to the decline. One such issue is whether a given vial is cooled and warmed and then recooled for subsequent samples. In our hands fresh germination medium gave better survival than a medium stored at +4°C; stored media could contribute to declines observed over a short duration. Over a several-year period differences in personnel, media preparations and alterations, and manipulations may affect estimates of viability. Obviously, critical tests for acquiring long-term storage data are needed. The implication for pollen genebanks is that standardized procedures for processing samples and for measuring viability must be developed and that periodic testing of aliquots is needed even for storage at cryogenic temperatures.

Pollen genebanks The outline of the operation for a pollen genebank is fairly clear and the understanding of the processes is sufficient, at least for some crops, to warrant establishment. Methods exist for the cryopreservation of pollen from many crops. This is particularly true for temperate-zone fruit and nut species, but also for some subtropical species such as citrus (Rajaskharan et al. 1995) and papaya (Ganeshan 1986). Establishment for many herbaceous plants may not be as important, as it may be just as easy to grow the male parent needed for crossing. The main impediment for adoption, in our view, is that of cost. The uses of a pollen bank are usually supplemental to the established national or international bank’s role in supplying seed or vegetative propagules. Supplying pollen to the user community is viewed as an additional service and the ‘costs’ associated with the user harvesting the pollen from the plant are transferred to the genebank. Since genebanks operate on very limited budgets, it is difficult to incorporate new approaches, even if the overall efficiency of germplasm use is improved. Thus, pollen banks would appear to be more valuable in integrated systems or in crops with a high economic value. A number of private companies have developed in-house preservation systems for pollen storage for use in breeding and hybrid generation. Examples would include maize as an agronomic crop and petunia as a floral crop. The challenge for a pollen bank, then, is not so much in the development of pollen cryopreservation, but in the development of a system that utilizes the advances made in pollen biology and is able to fulfil the goal of providing pollen samples to the user community. 126 Cryopreservation of Tropical Plant Germplasm

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Hamzah, S. and C. Leene. 1986. Pollen storage of Hevea. Journal of Natural Rubber Research 11:115-204. Hanna, W.W. 1990. Long-term storage of Pennisetum glaucum (L.) R. Br. pollen. Theoretical and Applied Genetics 79:605-608. Hanna, W.W. and L.E. Towill. 1995. Long-term pollen storage. Pp. 179-207 in Plant Breeding Reviews, Vol. 13 (J. Janick, ed.). Wiley and Sons. New York. Haunold, A. and P.C. Stanwood. 1985. Long-term preservation of hop pollen in liquid nitrogen. Crop Science 25:194-196. Heslop-Harrison, J. and Y. Heslop-Harrison. 1970. Evaluation of pollen viability by enzymatically induced fluorescence: intracellular hydrolysis of fluorescein diacetate. Stain Technology 45:115-120. Heslop-Harrison, J., Y. Heslop-Harrison and K.R. Shivanna. 1984. The evaluation of pollen quality, and a further appraisal of the fluorochromatic (FCR) test procedure. Theoretical and Applied Genetics 67:367-375. Hoekstra, F.A. 1992. Stress effects on the male gametophyte. Pp. 193-201 in Sexual Plant Reproduction (M. Cresti and A. Tiezzi, eds.). Springer-Verlag, Berlin. Hoekstra, F.A., J.H. Crowe and L.M. Crowe. 1992a. Germination and ion leakage are linked with phase transitions of membrane lipids during imbibition of Typha latifolia pollen. Physiologia Plantarum 84:29-34. Hoekstra, F.A., J.H. Crowe, L.M. Crowe and D.G. van Bilsen. 1992b. Membrane behavior and stress tolerance in pollen. Pp. 177-186 in Angiosperm Pollen and Ovules (E. Ottaviano, D. L. Mulcahy, M. S. Gorla and G. B. Mulcahy, eds.). Springer-Verlag, Berlin. Hoekstra, F.A. 1995. Collecting pollen for genetic resources conservation. Pp. 527-550 in Collecting Plant Genetic Diversity. Technical Guidelines. (L. Guarino, V.R. Rao and R. Reid eds.) CAB International. Hoekstra, F.A. and J. Bruinsma. 1975. Respiration and vitality of binucleate and trinucleate pollen. Physiologia Plantarum 34:221-225. Hoekstra, F.A., L. M. Crowe and J.H. Crowe. 1989. Differential desiccation sensitivity of corn and Pennisetum pollen linked to their sucrose contents. Plant Cell and Environment 12:83-91. Hong, T.D. and R.H. Ellis. 1996. A protocol to determine seed storage behaviour. IPGRI technical Bulletin No. 1. International Plant Genetic Resources Institute, Rome. Hong-Qi, Z. and A.F. Croes. 1982. A new medium for pollen germination. Acta Botanica Neerlandica 31:113-119. Hu, J. and C. Gou. 1996. Studies on the cryopreservation (–196°C) of pollen of restoring line in hybrid rice. Acta Agronomica Sinica 22:72-77. IBPGR. 1985. International Board for Plant Genetic Resources Advisory Committee on Seed Storage: Report on the Third Meeting. IBPGR, Rome. Jensen, C.J. 1964. Pollen storage under vacuum. Royal Veterinary Agricultural College Yearbook: 133-146. Jensen, C.J. 1970. Some factors influencing survival of pollen on storage procedures. FAO/IUFRO Working group meeting on sexual reproduction of forest trees, Reprint #148, 18 pp. Jett, J.B., D.L. Bramlett, J.E. Webber and U. Ericksson. 1993. Pp. 41-46 in Advances in Pollen Management. (D.L. Bramlett, G.R. Askew, T.D. Blush, F.E. Bridgewater, and J.B. Jett eds) USDA-Forest Service Agricultural Handbook 698. Kanazawa, T., S. Kobayashi and T. Yakuwa. 1992. Flowering process, germination and storage of pollen in Allium victorialis L. ssp. platyphyllum Hult. Journal of the Japanese 128 Cryopreservation of Tropical Plant Germplasm

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Use of stored pollen for wide crosses in wheat haploid production Masanori Inagaki JIRCAS, Tsukuba, Ibaraki, 305-8686 Japan

Introduction Haploid production followed by chromosome doubling offers the quickest method for developing homozygous breeding lines. Doubled haploids derived from heterozygous materials show complete uniformity when used as recombinant inbred lines in selection procedures. The breeding method using doubled haploids thus has the advantage of shortening the period required for development of new varieties and increasing the efficiency of selection. Efficient production of doubled haploids complements conventional breeding programmes. The progress achieved in developing a method for haploid production of hexaploid wheat (Triticum aestivum L.) through wide crosses followed by chromosome elimination was reviewed by Inagaki (1997). Maize (Zea mays L.) is one of the Panicoides subfamily species that can successfully hybridize with wheat egg cells and produces hybrid zygotes (Laurie and Bennett 1986). The maize chromosomes were rapidly eliminated from hybrid zygotes, requiring artificial rescue of haploid embryos at early developmental stage (Laurie and Bennett 1988a). Maize pollination and subsequent 2,4–dichlorophenoxyacetic acid (2, 4–D) treatment of wheat resulted in the stable production of immature wheat embryos capable of regenerating haploid plants (Suenaga and Nakajima 1989), irrespective of wheat genotypes (Inagaki and Tahir 1990). Methodologies using wide crosses always require viable pollen at the time of crossing, that is, flowering of wheat and pollen donors must be synchronized. This method can be applied in seasons and places where both wheat and pollen donors grow. An adequate technique for long-term pollen storage is helpful for performing wide crosses without having to synchronize flowering times of both parents. This paper presents the technical development in use of stored pollen for wheat haploid production.

Pollen sources Wide crosses of wheat with members of the subfamily have been attempted in alien genetic transfer, which is still a major approach for incorporating desirable traits from alien species into cultivated species. Cytological evidence indicates successful fertilization and elimination of paternal chromosomes from hybrid zygotes in sorghum (Sorghum bicolor (L.) Moench) and pearl millet (Pennisetum glaucum (L.) R. Br.) crosses (Laurie and Bennett 1988b; Laurie 1989). Sorghum and pearl millet, in addition to maize, are potential pollen sources for wheat haploid production (Ahmad and Comeau 1990; Ohkawa et al. 1992; Inagaki and Bohorova 1995). Cryopreservation techniques 131

Table 1 shows the comparison of pollen donors on embryo formation frequencies in crosses of wheat with maize, pearl millet and sorghum. Wheat haploid embryos were obtained at high frequencies from all crosses with three pollen donors. However, sorghum crosses expressed a strong genotypic barrier of wheat to embryo formation. Haploid production through wide crosses with maize and pearl millet appears more stable than that with sorghum because of its less pronounced genotypic effect on haploid embryo formation (Inagaki and Mujeeb-Kazi 1995).

Table 1. Effect of pollen donors on embryo formation frequencies (%) in crosses of wheat with maize, pearl millet and sorghum Wheat genotype Pollen donor Chinese Spring Norin 61 Siete Cerros Maize Population-93A 26.0 22.4 12.6 Hybrid-33 18.4 26.8 13.1 Pearl millet NEC-7006 39.4 37.6 13.6 NEC-7268 23.3 15.6 0.8 Sorghum ICSR-LM-90166 22.1 42.1 0.0 Toluca-1 20.4 41.1 0.0 Source: Inagaki and Mujeeb-Kazi (1995).

Pollen viability of maize and pearl millet Maize and pearl millet pollen have been successfully preserved at ultra-low temperatures for long periods (Barnabás and Rajki 1981; Hanna 1990). From a range of experiments on maize pollen storage, Barnabás and Rajki (1981) summarized that 50% of the pollen grains could be kept viable for a year with seed-setting ability of 30% when pollen water content was reduced to 14%. Pearl millet is one species with relatively long-lived pollen grains. Hanna (1990) recently reported that pearl millet pollen stored for several years showed 100% seed set when pollen water content was reduced below 7.2% and stored. Understanding the effects of drying and freezing on pollen viability is essential for achieving successful long-term storage. Figure 1 shows water content and germination frequencies of maize and pearl millet pollen after drying at 35°C and freezing at –196°C. These data suggest that the optimum range of pollen water content for the pollen viability after drying and freezing is different for maize and pearl millet. Pearl millet pollen is relatively tolerant to drying and freezing, in contrast with maize pollen, which has a narrow water content range for maintaining viability during drying and freezing (Inagaki and Mujeeb-Kazi 1994, 1996). 132 Cryopreservation of Tropical Plant Germplasm

Fig. 1. Water content and germination frequencies of (a) maize and (b) pearl millet pollen after drying and freezing. Maize: F1 hybrid line (CML–246 × CML–242); pearl millet: inbred line NEC–7006. Source: Inagaki and Mujeeb–Kazi (1994, 1996).

Crosses of wheat with stored pollen Embryo-formation frequencies in crosses of wheat with pearl millet pollen stored at various temperatures are shown in Table 2. Even when there was a large variation in water content of pearl millet pollen at collection, pollen water content was reduced to a minimum value of 6%, and did not affect embryo formation frequency in crosses of wheat with stored pearl millet pollen. Storage temperature of –20°C reduced embryo formation frequencies but could be used only when storage period was not longer than 3 months. Ultra-low temperatures of –80°C and –196°C are required for long-term storage of pearl millet pollen. Cryopreservation techniques 133

Table 3 shows that satisfactory embryo formation was obtained in crosses of four wheat genotypes with pearl millet pollen stored for 12 months. In a study of detached-tiller culture, Table 4 confirms that pollen storage at ultra-low temperature did not affect haploid production frequency in pearl millet crosses, but greatly reduced the frequency in maize crosses. Therefore, stored pearl millet pollen can be used as an alternative medium for wheat haploid production when fresh pollen is not available. Development of pollen storage technology in wide crosses could thus complement the application of the haploid breeding method in wheat improvement programmes, which will accelerate the release of new varieties in developed countries, as well as in developing countries where rapid varietal development is critical for sustainable wheat production systems.

Table 2. Effect of pollen storage on embryo formation frequency (%) in crosses of wheat with stored pearl millet pollen† Pollen water content (%) Storage temp. Storage period (months) Fresh Dried (°C) 0 1 3 5 12 Embryo formation frequency (%) 36.8 – – 27.6 – – – – 54.7 6.6 –196 – 24.3 28.5 21.5 16.3 –80 – 21.1 26.0 17.5 16.4 –20 – 23.8 26.7 4.8 4.2 36.8 7.6 –196 – 25.4 29.1 28.9 28.8 –80 – 22.1 26.4 26.8 29.7 –20 – 17.4 14.2 3.2 5.1 22.0 5.7 –196 – 27.5 25.7 19.6 14.6 –80 – 22.4 20.5 16.9 18.1 –20 – 18.2 14.3 8.0 7.0 † Wheat: variety Norin 61; pearl millet: inbred line NEC-7006. Source: Inagaki and Mujeeb-Kazi (1996).

Table 3. Embryo formation frequencies (%) of four wheat genotypes crossed with 12- month stored pollen of pearl millet † Wheat genotype Pollen water Storage Siete content (%) temp. (°C) Norin 61 Haruhikari Cerros Attila Mean Embryo formation frequency (%) Fresh (48.7 - – 21.3 24.4 4.9 13.4 16.0 22.7) Stored (7.9) –196 12.9 22.1 4.1 14.6 13.4 –80 15.6 26.1 10.3 16.9 17.2 Stored (5.3) –196 20.3 31.9 9.4 21.7 20.8 –80 10.8 24.5 18.4 9.4 15.8 Stored (4.7) –196 18.1 27.8 3.8 11.1 15.2 –80 15.9 24.0 3.8 10.0 13.4 † Pearl millet; inbred line NEC-7006. Source: Inagaki and Mujeeb-Kazi (1996). 134 Cryopreservation of Tropical Plant Germplasm

Table 4. Effect of pollen storage on haploid production frequencies (%) in crosses of wheat with maize and pearl millet Pollen Tiller Embryo Plant Haploid production donor culture formation (a) regeneration (b) (axb) Maize Fresh On plant 20.4 67.0 13.7 Detached 19.4 42.5 8.3 Stored On plant 2.8 65.0 1.8 Detached 7.0 46.5 3.3 Pearl millet Fresh On plant 19.7 45.8 9.0 Detached 21.2 56.7 12.0 Stored On plant 20.4 44.3 9.0 Detached 27.7 54.5 15.0 † Wheat: variety Norin 61; maize: F1 hybrid line (CML-246 x CML-242); pearl millet: inbred line NEC-7006. Source: Inagaki et al. (1997).

Acknowledgements This paper is a contribution resulting from a collaborative research project (1993 - 1998) with the International Maize and Wheat Improvement Center (CIMMYT) on wheat haploid production.

References Ahmad, F. and A. Comeau. 1990. Wheat x pearl millet hybridization: consequence and potential. Euphytica 50: 181-190. Barnabás, B. and E. Rajki. 1981. Fertility of deep-frozen maize (Zea mays L.) pollen. Annals of Botany 48: 861-864. Hanna, W.W. 1990. Long-term storage of Pennisetum glaucum (L.) R. Br. pollen. Theoretical and Applied Genetics 79: 605-608. Inagaki, M.N. 1997. Technical advances in wheat haploid production using ultra wide crosses. JIRCAS Journal (Japan) 4: 51-62. Inagaki, M.N. and N. Bohorova. 1995. Factors affecting the frequencies of embryo formation and haploid plant regeneration in crosses of hexaploid wheat with pearl millet. Breeding Science 45: 21-24. Inagaki, M.N. and A. Mujeeb-Kazi. 1994. Storage of maize pollen for use in haploid production of hexaploid wheat. Breeding Science 44: 387-390. Inagaki, M.N. and A. Mujeeb-Kazi. 1995. Comparison of polyhaploid production frequencies in crosses of hexaploid wheat with maize, pearl millet and sorghum. Breeding Science 45: 157-161. Inagaki, M.N. and A. Mujeeb-Kazi. 1996. Production of polyhaploids of hexaploid wheat using stored pearl millet pollen. Pp. 319-325 in Proceedings of 5th International Wheat Conference, Ankara, Turkey. Inagaki, M.N., T. Nagamine and A. Mujeeb-Kazi. 1997. Use of pollen storage and detached-tiller culture in wheat polyhaploid production through wide crosses. Cereal Research Communications 25: 7-13. Inagaki, M.N. and M. Tahir. 1990. Comparison of haploid production frequencies in wheat varieties crossed with Hordeum bulbosum L. and maize. Japanese Journal of Cryopreservation techniques 135

Breeding 40: 209-216. Laurie, D.A. 1989. The frequency of fertilization in wheat x pearl millet crosses. Genome 32: 1063-1067. Laurie, D.A. and M.D. Bennett. 1986. Wheat x maize hybridization. Canadian Journal of Genetics and Cytology 28: 312-316. Laurie, D. A. and M.D. Bennett. 1988a. The production of haploid wheat plants from wheat x maize crosses. Theoretical and Applied Genetics 76: 393-397. Laurie, D. A. and M.D. Bennett. 1988b. Cytological evidence for fertilization in hexaploid wheat x sorghum crosses. Plant Breeding 100: 73-82. Ohkawa, Y., K. Suenaga and T. Ogawa. 1992. Production of haploid wheat plants through pollination of sorghum pollen. Japanese Journal of Breeding 42: 891-894. Suenaga, K. and K. Nakajima. 1989. Efficient production of haploid wheat (Triticum aestivum) through crosses between Japanese wheat and maize (Zea mays). Plant Cell Reports 8: 263-266. 136 Cryopreservation of Tropical Plant Germplasm

Storage of pollens for long-term conservation of yam genetic resources Nyat Quat Ng and I.O. Daniel International Institute of Tropical Agriculture (IITA), Ibadan, Nigeria

Introduction The storage of pollens has been for a long time a subject of interest to plant breeders involved in fruit tree and ornamental plant improvement. It allows circumvention of some of the problem faced by breeders, such as seasonal limitations in species that flower irregularly, physiological limitations in species with non-synchronization of flowering of male and female flowers and limitations due to geographical separation of plants growing in different parts of continents. Storage of pollens under low temperature, especially in liquid nitrogen, as a means for conservation of plant genetic resources has been widely recognized. Bajaj (1987) has reviewed methods for storage of pollens and suggested establishment of pollen genebanks. The application of pollen storage, as an integrated method for long-term conservation of yam genetic resources and for immediate use by yam breeders, holds great promises. The majority of yam varieties do not produce botanical seeds. Relatively few varieties produce female flowers. Those that produce flowers are generally dioecious, a small percentage of the flowering plants being monoecious. On the other hand, many varieties have male flowering plants. A survey of the flowering behaviour of about 700 accessions of Dioscorea rotundata and 80 accessions of Dioscorea bulbifera collected from Central and West Africa, growing under normal seasons in Ibadan, Nigeria indicated that more than 60% of D. rotundata and about 50% of D. bulbifera produce male flowers. If the viability of yam pollens could be maintained for an extended long period of time, it would be of immediate value for yam breeders and for the conservation of yam genetic resources. Reports by Akoroda (1981) and Akoroda et al. (1981) indicated that yam pollens stored under dry conditions at 5°C remained viable for over 6 months. The aim of the present study was to investigate the longevity of yam pollens stored under four different storage temperatures (15, 5, –20 and –80°C) over a 2-year period. An investigation also was conducted to examine the possibility of storing yam pollens in liquid nitrogen for long-term conservation of yam genetic resources.

Materials and methods Male flowering plants of D. rotundata accessions TDr 1766, TDr 2385, TDr 3577, TDr 3303, TDr 3370 and TDr 3605, growing in Ibadan during the planting seasons between 1993 and 1998, were used as sources of pollen for the experiments. The ambient relative humidity surrounding the fields, during pollen collection, was about 70-80%. A D. rotundata breeding line (TDr 93-1) and a germplasm accession (TDr 3099) were used as female plants for pollination experiments. Male flower buds were collected from the fields just prior to anthesis. Cryopreservation techniques 137

Air-dried storage Freshly excised anthers or male flower buds of TDr 3577 were collected in vials. Each vial contained at least 15 anthers. The vials containing the anthers were left uncapped and placed for 2 days in a cold room at 5°C with a relative humidity of about 35%. Pollen was then packed and sealed in an aluminium envelope containing 1 g of silica gel. The sealed envelopes were stored at –80°C, –20°C, 5°C and 15°C. Pollen samples were taken out from each storage treatment for the assessment of their viability at every 10-day interval, for the first 100 days, and at every 100 days thereafter.

Wet-cold storage Freshly excised anthers or whole male flower buds of TDr 3577, from newly collected male spikes, were tightly closed in vials and sealed with film tape. They were then stored at the four experimental storage temperatures stated above. The viability of the stored pollen samples was tested at regular intervals as stated above, over a period of 2 years.

Freeze-drying and storage in liquid nitrogen Freshly sampled male flower buds of D. rotundata accessions TDr 3577, TDr 3370 and TDr 1766 were freeze-dried in a Ohausä lyophilizer set at –60°C and 50 mm Hg vacuum for 24 h. The viability of the pollens was evaluated immediately after freeze-drying. Male flower buds collected in cryovials were plunged directly into liquid nitrogen or the vapour-phase nitrogen for 1 h. Viability of the pollen was assessed immediately after the vials were rewarmed to room temperature.

Pollen viability and fertility assessments Pollen viability was assessed by staining test in 2% acetocarmine solution and germination in in vitro, using Brewbaker and Kwack (1963) medium (BK). Anthers excised from flower buds were placed on glass slides. One to two drops of the acetocarmine solution were applied on the anthers, which were teased out to release pollens. Stained and unstained pollens observed in three fields, under a compound microscopes, were counted. For the in vitro germination test, whole anthers containing pollens were cultured on the surface of the solidified BK medium. The culture was kept in laboratory room conditions. After 3 h, cultured anthers were picked from the medium, teased onto a glass slide, stained with acetocarmine solution and observed under a compound light microscope. Pollen germination counts were made in three microscope fields. In field fertility trials, female spikes with unopened flowers were bagged with thrips-proof nylon bags. At pistil maturity, pollens collected from fresh or stored flower buds were placed on the stigmas of the previously bagged female flowers. The hand-pollinated flowers were re-bagged and observed for seed setting after 8–10 days. Swelling of fruits indicated successful fertilization. 138 Cryopreservation of Tropical Plant Germplasm

Results and discussion The results of the air-dry storage are summarized in Table 1, wet-cold storage in Table 2, and field and laboratory assessments of fertility of stored and fresh pollens in Table 3. The study showed that the pollens, after air-drying in a 5°C cold room with 35% RH for 2 days and then stored over silica gel, had lost their germinability within 30 days of storage. The pollens maintained good stainability (49% with accession TDr 3577 and 19% with TDr 2385) after 400 days of storage at 5°C. Studies by Akoroda (1981) and Akoroda et al. (1981), on the other hand, showed that yam pollens stored in a vial placed over sulphuric acid in a desiccator with 5% relative humidity, and kept in a refrigerator at about 5°C, retained their germinability for over 6 months. Akoroda’s study also suggested that pollens stored better under drier conditions (over sulphuric acid) than under humid conditions (without predrying). Our study did not show such a trend. In our study, fresh pollen collected from the field (without predrying) and stored at subzero temperatures gave very encouraging results. Pollen of TDr 3577 retained a germination rate of 28% and 40%, after 2 years of storage at –20°C and –80°C, respectively (Table 3). The fresh pollen also could be stored viable at 15°C, with a germination rate of 10% after 100 days of storage. The differences in results may be due to differences in the procedures adopted for drying and storing pollens.

Table 1. Effect of dry-air storage (over silical gel) on pollen viability of D. rotundata Storage period (days) 0 30 400 Accession germ.† stain. germ. stain. germ. stain. TDr 3577 59 50.9 0 50.3 0 48.9 TDr 2385 - 73.0 0 59.7 0 18 † Germ. = germination (in %); stain. = stainability (in %).

Table 2. Percentage germination of pollen of D. rotundata (TDr 3577) after storage at various cold temperatures Storage temperature (°C) Storage period Initial % germination –80 –20 5 15 100 days 70 62 66 40 10 2 years 70 40 28 0 0

The procedure adopted for freeze-drying of pollens in our experiment might have a detrimental effect on pollen germinabilty. After 24 h of freeze-drying, only 2% of the pollens of accession TDr 1766 had germinated. All others did not germinate, even though more than 50% of the pollens were positively stained with the acetocarmine solution. Similarly, no germination was recorded from pollen after they had been stored for 1 h in the vapour phase or liquid nitrogen, although about 100% of the pollen still remained positively stained by acetocarmine. Improvement of procedures for storing yam pollen in liquid nitrogen may give better results. Cryopreservation techniques 139

Experiments conducted in our laboratory and field assessment of pollen fertility of fresh and stored pollen indicated that percentage in vitro germination and percentage fruit set by hand-pollination were correlated (Table 3). The results indicated that the pollen of TDr 3577 maintained a germination rate of 40%, at the end of 2 years of storage at –80°C. Artificial pollination of female flowers with the stored pollen gave 50% fruit set. The percentage fruit set resulting from pollination with frozen pollen was comparable to that from fresh pollen. This suggests that storage of yam pollen at low temperature, –80°C, could maintain the fertility of yam pollen for a long time. Storage under this and other favourable conditions could be explored for long-term conservation of yam genetic resources.

Table 3. Fruit set in D. rotundata (TDr 3577) using fresh pollen and pollen frozen at -80°C for 2 years Pollen parent % germination % fruit set Frozen pollen TDr 3577 † 40 50 Fresh pollen TDr 3577 † 73 68 TDr 3303 ‡ 60 85 TDr 3605 ‡ 68 50 † Female parent TDr 3099. ‡ Female parent breeding line TDr 93-1.

Conclusion Manipulation of storage conditions of yam pollen as reported in the present study indicates the potential for establishment of a pollen genebank for the conservation and utilization of yam genetic resources. The results showed that yam pollen remained viable after being frozen at –80°C for 2 years. Further experiments on procedures of drying and storing of pollen, particularly in liquid nitrogen, should be investigated.

References Akoroda, M.O. 1981. Studies on the genetics and floral biology of yams (D. rotundata and D. cayenensis). PhD thesis, Department of Agronomy, University of Ibadan, Ibadan, Nigeria. Akoroda, M.O., J.E. Wilson and H.R. Chheda. 1981. Artificial pollination, pollen viability, and storage in white yam. Pp. 189-194 in Tropical root crops: Research strategies for the 1980s. IDRC-163c. Ottawa, Canada. Bajaj, Y.P.S. 1987. Cryopreservation of pollen and pollen embryos, and the establishment of pollen banks. Pp. 297-420 in International Review of Cytology, Vol. 107, (Gikks, K.L. and J. Prakash, eds). Academic Press. Brewbaker, J.L. and B.H. Kwack. 1963. The essential role of calcium ion in pollen germination and pollen tube growth. American Journal of Botany 50:859–865. 140 Cryopreservation of Tropical Plant Germplasm

Cryopreservation of recalcitrant zygotic embryos Patricia Berjak¹, Marieanne Walker¹, D.J. Mycock³, J. Wesley-Smith¹,², Paula Watt¹ and N.W. Pammenter¹ ¹ School of Life and Environmental Sciences, University of Natal, Durban, 4041 South Africa ² Electron Microscope Unit, University of Natal, Durban, 4041 South Africa ³ Department of Botany, University of the Witwatersrand, Johannesburg, P.O. Wits, 2050 South Africa

Introduction From our present perspective, it seems that cryopreservation is the only way in which to conserve the germplasm of species producing recalcitrant seeds. There are several different contemporary approaches to achieving this goal (Engelmann 1997a, 1997b), all of which depend on reducing the water content of the material to levels that will allow its freezing without lethal ice formation. These are: · Encapsulation of meristematic shoot apices in alginate followed by partial dehydration achieved by their maintenance on a high-sucrose medium. This is called encapsulation-dehydration. · Vitrification – achieved by freezing explants in very concentrated cryopro- tective solutions, which has been used for shoot apices and somatic embryos. · Encapsulation-vitrification, which is a self-explanatory combination of the first two methods. · Rapid dehydration, achieved by placing explants in an airstream, or by the used of an efficient desiccant such as silica gel. · A procedure termed pregrowth, which involves culturing the material – generally on a high-sucrose medium – prior to rapid dehydration. This is, of course, also applicable to encapsulated material.

The method chosen for cryopreservation of the germplasm of species producing recalcitrant seeds, is usually that of rapid – or relatively rapid – dehydration of zygotic embryonic axes prior to their introduction into the cryogen. Sometimes, certain pretreatments may be used. However, only very limited success has been achieved (reviewed by Berjak et al. 1996; Normah and Marzalina 1996; Engelmann 1997a), and the present contribution explores some of the reasons for this. Most recalcitrant seeds are relatively large and are lethally damaged by dehydration. However, the bulk of such seeds is constituted by the cotyledons (or endosperm) with the embryonic axis usually making up only a tiny fraction of the entire volume. So, generally the embryonic axes of such seeds are used as explants, as they most often are sufficiently small to be rapidly dehydrated and, theoretically, to be successfully cryopreserved. Use of intact embryonic axes has the advantage that, if cryopreservation procedures are successful, it should merely be a matter of manipulating post-freezing conditions to produce vigorous plantlets. However, real success, measured as a high percentage of vigorous plantlets produced from cryopreserved axes from seeds that have been unequivocally established to be Cryopreservation techniques 141 recalcitrant, often proves to be elusive! In a few cases, variably successful plantlet development has been reported following cryopreservation [e.g. Hevea brasiliensis (Normah et al. 1986); Euphoria longan (Fu et al. 1990); Cocos nucifera (Assy-Bah and Engelmann 1992); Calamus manan (Krishnapillay et al. 1992); Artocarpus heterophyllus (Chandel et al. 1995); Camellia japonica (Janeiro et al. 1996)]. However, much of the reporting on the outcome of cryopreservation does not allow one to conclude whether axes that showed regrowth in vitro would have developed into plantlets; and, in other instances, greening and/or root development only is reported as indicating survival. Our own experience with zygotic axes from a range of recalcitrant seed species indicates that hypocotyl/root elongation, generally accompanied by greening, frequently occurs after cryopreservation. Shoot development, however, often just does not take place and close examination shows that the apical meristems have become necrotic (see later), thus precluding plantlet formation, no matter how vigorously the root may have grown. In other cases, callus alone has been developed from zygotic axes after cryopreservation, as reported for Castanea sativa and Quercus spp. by Pence (1990). The present contribution examines, and attempts to account for, the responses of the root and shoot apices of axes of the temperate Quercus robur L. (Fagaceae) to cryopreservation. The effects of three different protocols are compared, two of which yield little or no success, while the third [protocol N (Natal) developed in our laboratory] affords good survival levels when root growth is assessed. The problem of the negative response of the shoot apices to cryopreservation (using the otherwise successful N protocol) is also examined, with a promising prognosis for its alleviation.

Procedures, results and discussion Quercus robur, the pedunculate oak, is a valuable temperate tree producing recalcitrant seeds, as do several species of this genus. Several authors reported that little success could be achieved in attempts to cryopreserve zygotic axes of Quercus spp. (Pence 1990; Poulsen 1992; Chmielarz 1997). However, the preliminary studies of one of us (Walker) had shown that the use of fast flash-drying (to about 0.21 g/g) and very rapid cooling (freezing) would give repeatable survival levels of around 40%, for relatively newly harvested Q. robur seeds, which improved to 66% when seeds stored for about 10 weeks were used. (This improvement itself is interesting, as it is known that the physiological status of hydrated, recalcitrant seeds changes during storage, as development continues and/or the processes of germination are entrained, e.g. Berjak et al. 1989). We thus decided to compare survival and examine the tissues and cells of both root and shoot apices of axes cryopreserved by this protocol (termed the N protocol) at each of the critical stages during their manipulation, with those obtained using the protocols of Poulsen (1992) and Chmielarz (1997) (termed the P and C protocols, respectively). Assessment was done following surface-sterilization, dehydration and cryopreservation, each carried out according to the N, P and C protocols. The microscopical results reported here are for root and shoot apices. For clarity, details of the procedures used for each of the critical manipulations according to the N, P and C protocols 142 Cryopreservation of Tropical Plant Germplasm immediately precede analysis of the results of that manipulation. Details of the control (newly excised) material are, however, presented before the consideration of any of the manipulations. It should be noted that all microscopical results are illustrated after the axes were afforded a 6-d 'recovery' period in culture (excepting those of the control material, which was newly excised). Thus persisting effects of various manipulations should be appreciated as indicating serious damage.

The control material The root apex of a Q. robur axis is protected by a substantial root cap, consisting of compact cells that are not highly vacuolated (Fig. 1). In a growing root, the cap is continuously regenerated distally from the meristem, while the most peripheral cells are sloughed: in this way the cap affords constant protection to the vital meristematic region as the root encounters sharp and abrasive soil particles. Similarly, if the integrity of the root cap is preserved, then the root apical meristem will be partially protected from the effects of the manipulations involved in axis cryopreservation. It is suggested that this tissue plays a significant role in the successful cryopreservation of these axes, as is revealed below, when the consequences of the various manipulations are considered. Whether constituting the cap or root proper, cells of the apex were characterized by large nuclei with prominent nucleoli, and contained many, predominantly spherical, mitochondria, starch-filled plastids, profiles of rough endoplasmic reticulum (rER), polysomes and Golgi bodies (Fig. 2). All these intracellular features are indicative of the actively metabolic state of recalcitrant embryonic axes when the seeds are shed. The shoot apex of a newly excised zygotic axis is Q. robur is shown in Figure 3. Aside from the incidence of associated fungi (discussed below), it can be seen that the shoot apical meristem is entirely superficial, and not protected by any equivalent of the root cap. The outermost two meristematic cell layers constitute the tunica, and the cone of meristematic corpus cells lies immediately below. The meristematic cells (Fig. 4) were dominated by prominent nuclei, and contained plastids with little starch, frequent aggregations of rER profiles, and, particularly in the corpus cells, exceptionally well-developed mitochondria and many polysomes.

Surface-sterilization of axes Surface-sterilization is a vital procedure for any axes that are to be cultured in vitro, as it seems inevitable that fungal propagules, or even active hyphae, are closely associated (Fig. 3). The problem is exacerbated the longer the seeds have been in storage before axes are excised. The procedures commonly used range from relatively gentle to harsh. Surface-sterilization according to the C protocol was very gentle yet effective in removing contaminants, consisting of immersion of the axes in 0.1% mercuric chloride for 2.5 min, followed by thorough rinsing in sterile, distilled water (Chmielarz 1997). No immediate adverse effects of this procedure were seen in any of the cells of the cap, or root proper, the ultrastructure (not illustrated) remaining similar in all respects to that of the control material. The N protocol utilized 1% Cryopreservation techniques 143 sodium hypochlorite immersion of the excised axes, for 10 min, which proved to be a highly effective mode of surface-sterilization. Only the most peripheral cap cells, which were poised to slough naturally (see above), were adversely affected by this surface-sterilization procedure. The only other noticeable effect was the development of large vacuoles containing granular debris in the relatively superficial cap cells underlying the most peripheral layer (Fig. 5). In contrast, the P protocol incorporated many steps involving considerable handling, and is rated as relatively harsh. The procedure of Poulsen (1992) was followed, during which whole embryos (i.e. after pericarp removal only) were swabbed with 70% ethanol, followed by immersion in 6% calcium hypochlorite for 15 min. After the embryos were washed (three times) in sterile, distilled water, axes were excised, and immediately immersed into a filter-sterilized solution containing 10 and 5 mg/L ascorbic acid and citric acid, respectively. The axes were then placed in 5% sodium hypochlorite for 3 min, washed (as after the calcium hypochlorite step) and finally immersed in the antioxidant solution (as above) for 30 min. Perhaps not surprisingly, the procedures involved in this aspect of the P protocol resulted in marked deterioration of root cap cells, even deep within this protective tissue (Fig. 6). While some of these cells were completely deteriorated, many others showed diffuse clearing of both the nucleo- and cytomatrix, membranes became ill-defined and there was marked wall abnormality. The unprotected shoot meristems were affected by all the surface-sterilization procedures used, with least damage being manifested after short immersion in mercuric chloride (Fig. 7; C protocol). While treatment for 10 min with 1% sodium hypochlorite (N, Fig. 8) did produce some small, localized areas of necrosis, many more such pockets of necrotic cells occurred following the harsher surface-sterilization of the P protocol. Among the surviving cells, buckling of the thinner cell walls occurred, which was accompanied by plasmalemma withdrawal and vesiculation (Fig. 9). Although this phenomenon was most often observed and appeared most severe after application of the P protocol, it was not restricted to axes so treated. It is possible that the activity of fungi, which were most prevalent among the leaf bases around the shoot apex (Fig. 3), had predisposed the cell walls to damage during subsequent processing. Considering that surface-sterilization of the axes by all the methods described proved to be highly effective in removing fungi, it is obvious that, at least in the case of Q. robur, over-rigorous procedures are not only unnecessary, but actually deleterious. However, the harsh surface-sterilization procedure of the P protocol alone did not affect viability adversely, as all axes germinated whether this treatment was applied, or that of the C or N protocols. But it is important to realize that axes were assessed ultrastructurally after a 6-d recovery period: in the actual cryopreservation protocol, no such remission is facilitated, and the immediate consequences of a damaging procedure become exacerbated as the axes are further manipulated. 144 Cryopreservation of Tropical Plant Germplasm

Axis dehydration The three protocols differed markedly in the drying procedures used, that of the N approach being the most rapid – in fact, half the time used by the P protocol while the C-protocol methodology was very complex and took over five times as long. Although axes remained 100% viable irrespective of the drying protocol, it is significant that those which dehydrated most rapidly (N) germinated more vigorously than those subjected to twice the period under dehydrating conditions (following the harsh surface-sterilization, P), while germination of axes subjected to the prolonged procedure of the C protocol was very slow, indicating that axis vigour had been adversely affected. According to the N protocol, dehydration was by fast flash-drying over a period of 4 h, after which axis water content was reduced from the original 0.88 g/g (after 10 weeks of cold storage) to an average of 0.27 g/g. Aside from a peripheral band of necrosis in the root cap, the integrity of this structure as a protective organ was retained. Although they had become more vacuolated, the cap cells appeared ultrastructurally normal and relevantly (see later) the nuclei had retained their normal, spherical morphology (Fig. 10). Cells of the apical meristem were in exceptionally good condition and appeared generally active following the 6-d recovery period (Fig. 11). Axes were dried over an 8-h period in a laminar airflow reaching an average water content of 0.32 g/g, using the P approach. During the 6-d recovery period used here (but not afforded when axes are to be cryopreserved), root cap regeneration occurred, with the highly damaged cells seen after surface-sterilization having been sloughed. However, there were signs of persisting abnormality in the cap cells, particularly instances where nuclear shape was not spherical (Fig. 12). Another phenomenon indicating persisting damage, but the ability for repair, was manifested by the frequency of intensive vacuolation and autophagy (not illustrated), which is evidence of the removal and lysis of damaged intracellular structures. While the root apical meristem cells were in essentially good condition, there were occasional pockets of necrosis in the parenchyma (Fig. 13). The C dehydration protocol was complex, involved and protracted, drying, per se, taking over 21 h (Chmielarz 1997). It involved initial cryoprotection over 24 h by axis immersion in sucrose solutions of increasing molarity, and finally the use of 1M glycerol. Individual axes were subsequently encapsulated with calcium alginate.

Figs. 1–7. Figs. 1–4 illustrate aspects of newly excised axes. er, rough endoplasmic reticulum; M, mitochondrion; Me, meristem; N, nucleus; Nu, nucleolus; P, plastid; s, starch; V, vacuole; W, wall. Fig. 1. The substantial root cap, consisting of many ranks of compact cells, the greater proportion of which are not highly vacuolated, is illustrated. x130. Fig. 2. The ultrastructure typical of apical root cells is characterized by large, starch-filled plastids, mitochondria that are not highly differentiated, profiles of ER and many polysomes (conferring considerable cytomatrical [cytoplasmic] density), among other organelles that are not shown here. x15 000. Fig. 3. The shoot apical meristem typically is composed of the tunica and underlying corpus. All the axes presently used Cryopreservation techniques 145

harboured fungal hyphae between the leaf bases and shoot apex, as can be seen in this illustration. x130. Fig. 4. Shoot apical meristem cells presented a highly active appearance, with well-developed mitochondria, ranks of parallel ER and many polysomes. Each large nucleus had a prominent nucleolus (not illustrated) and patches of heterochromatin were always apparent. x10 000. Fig. 5. Following sodium hypochlorite surface-sterilization (N protocol), the relatively superficial root cap cells developed large vacuoles, containing a granular debris. x6000. Fig. 6. The very rigorous surface- sterilization procedure of the P protocol resulted in marked cap cell deterioration, even in the deep-lying portions of the tissue. x20 000. Fig. 7. After brief surface-sterilization with mercuric chloride (C protocol), very little damage was seen in shoot apical meristem cells. x20 000. 146 Cryopreservation of Tropical Plant Germplasm

Figs. 8–15. Fig. 8. Some damage to the shoot apex, resulting in small patches of necrotic cells, followed immersion of the axes in sodium hypochlorite (N protocol). The cells seen here were adjacent to a small necrotic zone, and typically for such neighbours, had become markedly more vacuolate. x7200. Fig. 9. Pockets of necrosis occurred relatively frequently, following application of the multi-step surface-sterilization of the P protocol. Some surviving cells were characterized by buckled cell walls from which plasmalemma withdrawal was evident, as illustrated here. x16 000. Figs. 10 & 11. Dehydration by fast flash-drying (N protocol) resulted only in the contents of the root cap cells (10, x8000) becoming dense and compacted. The ultrastructure of both cap and apical meristem (11, x16 000) was indicative of the minimal dehydration injury occurring on very rapid water removal. Note particularly the retention of the spherical shape of the nucleus. Figs. 12 & 13. Slower dehydration, as used in the P protocol, resulted in marked changes in nuclear morphology in some of the Cryopreservation techniques 147

Following this, the encapsulated axes were dehydrated for 1 h in a laminar flow airstream, after which they were maintained above dry silica gel for 20 h, reaching an average water content of 0.19 g/g in the present experiments, which is a little higher than the 0.15 g/g reported by Chmielarz (1997) as 24% (wmb). This treatment resulted in extensive degeneration of the root cap. Not only were many ranks of cells, from the outermost in, completely deteriorated, but virtually all the cells of the central cap region were damaged and wall collapse had occurred (Fig. 14). There were also groups of deteriorated cells in both the root apical meristem and parenchyma, and even in seemingly undamaged cells there were signs of abnormality, including localized distentions of the nuclear envelope and rER profiles, and damaged plastids and mitochondria (Fig. 15). Following fast flash-drying (4 h, N protocol) and a 6-d recovery period, the shoot apical meristem cells were in excellent and very active condition. Figure 16 shows that nuclei had retained a normal, spherical morphology, mitochondria appeared active and, most significantly, there was evidence of endomembrane activity in terms of the production of rER and particularly Golgi bodies. Application of protocol P involving more extended, slower dehydration, resulting in relatively extensive areas of necrosis in the shoot apices, which is considered to be an exacerbation of the situation seen after the harsh surface-sterilization. However, the proportion of apical meristem cells surviving was adequate to ensure shoot devel- opment when the axes were set to germinate in culture. Such cells, when viewed after the 6-d recovery period, showed two major effects which are interpreted as the outcome of the cumulative stresses of the manipulations to which they had been subjected. Atypically large vacuoles had developed and, as was seen in the root apex, the nuclear profile tended to be irregular (Fig. 17), both features also seen in root apical cells of this material. Loss of the spherical conformation of the nucleus suggests that some abnormality of the nuclear lamina and matrix, which provide an essential framework for this body (Moreno Díaz de la Espina 1995), had persisted during the recovery period presently afforded the axes after dehydration. The extended dehydration period of the C protocol was associated with poorly differentiated organelles and an extreme degree of vacuolation, especially of the tunica cells (Fig. 18), while the corpus was composed of a mosaic of reasonably well-organized cells, contiguous with those that were deteriorating or had deteriorated (Fig. 19). There was a marked build-up of starch in the plastids of the corpus cells, which is correlated with the provision of sucrose during the dehydration procedure, and its non-utilization during the 6-d recovery period. cap cells (12, x12 800) as well as occasional pockets of necrosis in the parenchyma of the root apex (13, x6400). Figs. 14 & 15. Prolonged retention of axes under dehydrating conditions (C protocol) caused serious and widespread degeneration from the exterior to the deep-lying cells in the centre of the cap (14, x12 800) as well as centres of deterioration in the apical meristem itself. Some surviving meristem cells (15, x16 000) showed damage in the form of distentions of the nuclear envelope and ER, as illustrated here. Fig. 16. Not only had most shoot apical meristem cells not been damaged by fast flash-drying of the N protocol, but these were in a highly active condition following the 6-d recovery period, as indicated particularly by the frequent occurrence of Golgi bodies. x17 000. 148 Cryopreservation of Tropical Plant Germplasm

Figs. 17–19. Fig. 17. After 8-h laminar flow drying, following the rigorous surface- sterilization of the P protocol, surviving shoot meristem cells became highly vacuolated and the nuclei were no longer entirely spherical, as illustrated here. x9600. Fig. 18. The stress imposed by the prolonged dehydration of the C protocol resulted in marked vacuolation of the tunica cells, accompanied by extreme compaction of the cytomatrix and poor resolution of membranes. x9000. Fig. 19. Dehydration by the C protocol included pretreatment with sucrose, the results of which are seen in the large starch grains accumulated in the corpus cells. The two uppermost cells illustrated border on a necrotic area, the signs of which are apparent in the lower cell. x9000. Cryopreservation techniques 149

This comparative study of dehydration according to the N, P and C protocols clearly shows the deleterious effects of longer drying periods. Remembering that during normal handling of axes for cryopreservation, no time elapses between dehydration and freezing, no repair or compensatory processes can be entrained. Thus the axes – which still show signs of dehydration damage after the 6-d recovery period – are in a stressed condition, which increases in severity with increasing drying time, when introduced into the cryogen.

The critical cooling (freezing) step Despite the damage that accrued, or its comparative severity, after dehydration 100% germination was obtained irrespective of the protocol used, although vigour was compromised in proportion to the length of the dehydration period (see above). Several different approaches to freezing the axes dehydrated by the three protocols, were then used. Axes prepared by the N, P and C protocols were: 1. Enclosed in batches of five in cryotubes which were plunged into liquid nitrogen. None survived. 2. Fast-frozen (according to a procedure developed by Wesley-Smith in this laboratory) by plunging 'naked' axes individually into isopentane held in a liquid nitrogen reservoir, which were then placed in liquid nitrogen. Only axes prepared according to the N protocol survived (see details below). 3. Frozen by cooling at 1°C/min to –38°C, and placed in this state in liquid nitrogen (Poulsen 1992) or cooling at 2°C/min to 0°C, then at 1°C/min to –20°C, and introduced into liquid nitrogen at this point (Chmielarz 1997). Irrespective of the prefreezing protocol, none survived.

Ultrastructural data are presented only for material that had been fast-frozen (2, above), this final step of the N protocol alone emerging as successful for the cryopreservation of axes of Q. robur . When axes were prepared by the P protocol and then fast-frozen, the root cap was extensively degraded (Fig. 20). This tissue had already shown distinct evidence of the effects of the stresses imposed by surface-sterilization and relatively prolonged dehydration, and is suggested not to have been able to function as a protective structure for the root proper during freezing. There were aggregates of deteriorated cells in both the root meristem and parenchyma, while surviving cells showed distinct abnormalities (Fig. 21). The most significant of these were changes in nuclear morphology, as shown in Figure 21, and clustering of organelles in the perinuclear area (not illustrated). Despite the fact that some obviously living cells survived fast freezing, no root development occurred, and it was not ascertained in these experiments whether or not these cells would produce callus. In axes that were fast-frozen after being prepared by the C protocol, all the cells of the cap and the root apex were completely destroyed (Fig. 22). Following rapid freezing of axes that had been fast flash-dried (the N protocol), necrosis in the root cap was confined to essentially the same peripheral ranks of cells so affected by dehydration, indicating the retention of a substantial volume of living (cap) tissue protecting the root apical meristem and parenchyma. Most of the 150 Cryopreservation of Tropical Plant Germplasm cells of the root meristem were in very good condition: nuclear morphology was normal, as was the disposition of the organelles, and there was evidence of active protein synthesis in terms of the abundance of polysomes in the material sampled after a 6-d recovery period (Fig. 23). The parenchymatous derivatives of the meristem bore evidence of a high degree of metabolic activity, as evidenced by the highly organized mitochondria and plastids (Fig. 24). It is obvious that the cooling/freezing step is critical, and that two factors come into play. The first pertains to cooling rate: only rapid freezing facilitated survival. Even axes that had been prepared to this point by the least-damaging N protocol did not survive freezing by either of the P or C procedures, both of which employed slow, stepwise cooling. The cooling rate achieved when axes in cryotubes are plunged into liquid nitrogen, although far more rapid, is nevertheless relatively slow, and no axes survived this freezing procedure. The second vital consideration is that the quantitative and qualitative aspects of the stresses imposed before axes are frozen, and the nature and degree of the resultant damage, have profound consequences. Using the fast-freezing method of the N protocol, axes prepared to this stage by the P and C protocols did not survive. In contrast, 66% of axes prepared according to the N protocol and fast-frozen produced vigorous roots when cultured after retrieval from cryogenic storage. Regarding root survival in particular, it is suggested that if a substantial root cap, composed of cells that are not significantly vacuolated, is present, then if surface- sterilization and dehydration cause damage to the peripheral layers only, the root is likely to survive rapid freezing. In contrast, where the preparative methods cause the cap tissue to deteriorate substantially, as in the P, and especially the C, protocols, then the vital meristematic region of the root apex and its surrounding parenchyma are lethally damaged by the additional stresses imposed by freezing, even under the best possible conditions. In cases where the root cap is a relatively small structure, and especially if the component cells are highly vacuolated, as has been demonstrated for Trichilia dregeana (Berjak et al. 1999), then it is unlikely that any regime achieving a low enough water content for successful freezing will not have lethally damaged the entire root cap beforehand.

Figs. 20–28 illustrate material prepared by each of the three protocols, but all rapidly cooled (frozen), after the 6-d recovery period. Figs. 20–24 are of root tissues / cells: 20 & 21, P; 22, C and 23 & 24 N protocols, resp. Shoot cells are shown in Figs. 25–28, with 25 & 26, P; 27, C and 28, the N protocol, resp. Fig. 20. The extent of degradation of the cap is shown following the P protocol. x150. Fig. 21. Abnormal nuclear morphology and complete degradation (right) in root cells after freezing are shown, consequent upon the P protocol. x7500. Fig. 22. The complete destruction of all the root apical cells after axes prepared by the C protocol are frozen, is apparent even at the light microscope level. x130. Figs. 23 & 24. Most of the cap cells and those of the root proper showed undamaged ultrastructure and a high degree of activity following axis manipulation by the N protocol. Evidence of this is implicit in the appearance of the nucleus and nucleolus, the proliferation of ER and the marked cytomatrical density that is a function of a great number of polysomes (arrowheads; Cryopreservation techniques 151

23, x16 000), while the degree of development of the mitochondria and plastids attests to a high level of metabolic activity (24, x40 000). Figs. 25 & 26. After freezing of axes prepared by the P protocol, occasional groups of surviving shoot apical meristem cells showed activity evidenced especially by polysome formation (arrowhead, 25, x30 000), but most of the cells were extensively deteriorated (26, x9000). Fig. 27. Shoot apex cells were extensively degraded following freezing after manipulation by the C protocol. x12 000. Fig. 28. The typical appearance of shoot apical meristem cells from axes prepared by the N protocol and then rapidly frozen, is shown. Nuclear morphology is not normal (cf. Fig. 4) and, while organelles abound, there is a lack of normal spatial organization. x12 000. 152 Cryopreservation of Tropical Plant Germplasm

The case of the shoot apex (ref. Fig. 3) of the embryonic axis is different, and poses a far greater problem, because there is no tissue equivalent to the root cap, as an integral part of the structure. The shoot apical meristem is, in fact, entirely superficial, with the outermost one or two layers constituting the tunica and the underlying cone of tissue, the corpus. These two components together constitute the shoot apical meristem, and both are presumably vital to normal shoot growth. It is possible, however, that the leaf pairs, which closely surround the apical meristem (ref. Fig. 3) might afford some measure of protection against dehydration-imposed stress and direct freezing damage, although it is unlikely to be as effective as that suggested to be afforded by the root cap. Aside from isolated groups of surviving shoot apical meristem cells (which showed an evidence of a surprising degree of activity, Fig. 25), when axes prepared by the P protocol were rapidly frozen, most of the tissue was extensively degraded (Fig. 26). In the material which had undergone the 24-h dehydration followed in the C protocol, there were no surviving cells, all having undergone extensive degradation (Fig. 27) when the stress of freezing was applied. Although 66% of the axes of Q. robur cryopreserved by the N protocol survived and produced vigorous roots after retrieval from liquid nitrogen, the shoots of none developed. As the shoot apices remained whitish and did not look necrotic to the naked eye, the problem was initially seen as a developmental stasis rather than one of degeneration. (It is worthwhile mentioning that we had found this situation to be common to the axes of many species after retrieval from cryostorage.) This appearance was misleading as, when the shoot apices of these Q. robur axes were examined after about 30 d in culture, they were found to be completely necrotic. However, on ultrastructural examination of the shoot apices after the 6-d recovery period following axis retrieval from cryostorage, we found few obvious changes that could be interpreted as potentially necrotic (Fig. 28). In fact, the cells appeared relatively active, but this activity obviously did not lead to organized further development. Examination of Figure 28 does reveal some features that suggest that intracellular organization might be deficient, among which is the appearance of the nuclei. Not only were these bodies not spherical, but there was an absence of visible heterochromatin and nucleoli were infrequent, all of which are features common to shoot apical cells of control material (Fig. 4). Plant cells have a well-developed nucleoskeleton that has been convincingly demonstrated, and is very prominent underlying the nucleolus (Moreno Díaz de la Espina 1995): this structural framework not only maintains nuclear shape, but it imposes organization on the nucleus by supporting and localising the chromatin in discrete domains (Spector 1993). Thus we tentatively have concluded that the persistent damage to the shoot apical meristem cells following cryopreservation by the N protocol might well be an inability for the nucleoskeleton to be reconstituted. The better-known structural framework is the cytoskeleton, which ramifies throughout the cytomatrix (cytoplasm) and is vital in imposing intracellular spatial order. It is a feature of all eukaryotic cells, those of plants being no exception (Lloyd 1989; Staiger and Lloyd 1991; Lambert 1993; Shibaoka and Nagai 1994; Olyslaegers and Verbelen 1998). If Cryopreservation techniques 153 the nucleoskeleton is not reconstituted, then the cytoskeleton too is assumed to be similarly affected, as the assembly of the structural elements of both are basically controlled by similar factors, particularly the appropriate concentrations of divalent cations (e.g. Wolfe 1995). Failure of the nucleoskeleton (and of the cytoskeleton) to reassemble in an organized manner could well not only underlie the nuclear features seen after cryopreservation (N protocol) in the shoot apices of Q. robur , but would also result in uncontrolled and chaotic intracellular events. To this point, axes had been retrieved from cryostorage and thawed in either liquid medium or distilled water. Thus, with the last remnants of the seeds we still had in storage, we repeated the experiments using the N protocol, but this time retrieved the axes from cryostorage into a solution containing Ca 2+ and Mg2+ in appropriate concentrations [based on work by one of us (Mycock) on cryopreserved somatic embryos] to promote re- assembly of nucleo- and cytoskeletal elements. The difference in response of the axes was dramatic: not only did recovery assessed as root development improve from 66 to 100%, but within a short time 70% of the axes had initiated organized shoot development. This was the situation in October 1998, when these results were reported at the JIRCAS Workshop. Because the seeds used had been stored for several months, and harboured such severe fungal infections which could not be eliminated by conventional surface-sterilization, we have only later (at the time of going to press) been in a position to repeat these investigations with sizeable samples of axes from fresh seeds of Q. robur. The current results entirely substantiate those reported above, for material prepared by the N protocol and thawed in the Ca2+/Mg2+ solution. We are presently assessing the ultrastructure and attempting examination of the cyto- and nuceo-skeletal situations following retrieval of these axes, compared with those thawed in distilled water (when severe ion loss must occur). From the work presented here, it is apparent that major factors that determine the success or failure of cryopreservation protocols depend very much on the parameters used in the protocols. As a generalization from the work on Q. robur, the most gentle, yet effective, surface-sterilization procedure must be used; dehydration should be carried out as quickly as possible (and not below the water content that will just facilitate freezing without untoward injury), and the cooling/freezing step should also be rapid. Optimization of these factors, particularly the parameters involved in the cooling/freezing step for a variety of species, is currently the subject of intense investigation by one of us (Wesley-Smith), who has reported some of his findings elsewhere (Wesley-Smith et al. 1999). Work reported on axes of T. dregeana (Berjak et al. 1999), on the other hand, showed clearly that the tissue composition and organ and cell structure of the axes are of prime importance. If these properties render an axis 'unsuitable' for cryopreservation, then no matter how efficiently the experimental parameters are optimized, success will not be obtained. In such cases, the use of other explant material (e.g. buds) must be investigated, and it might eventuate that only somatic embryos will afford success. 154 Cryopreservation of Tropical Plant Germplasm

Acknowledgements The authors are pleased to acknowledge the skilled technical assistance, enthusiasm and willingness to go to extraordinary lengths, of Priscilla Maartens and Nthabiseng Motete, and financial support from the International Plant Genetic Resources Institute and the South African FRD and THRIP Programmes. We also thank W.E. Finch-Savage (Horticulture International, UK) and P. Vorster (University of Stellenbosch, SA), without whose assistance in collecting seeds the work on Q. robur could not have been done.

References Assy-Bah, B. and F. Engelmann. 1992. Cryopreservation of mature embryos of coconut (Cocos nucifera L.) and subsequent regeneration of plantlets. Cryo-Letters 13, 117-126. Berjak, P., D.J. Mycock, J. Wesley-Smith, D. Dumet and P. Watt. 1996. Strategies for in vitro conservation of hydrated germplasm. Pp. 19-52 in Normah, M.N., Narimah, M.K. and Clyde, M.M. (Eds) In vitro Conservation of Plant Genetic Resources. Percetakan Watan Sdn.Bhd., Kuala Lumpur, Malaysia. Berjak, P., J.I. Kioko, M. Walker, D.J. Mycock, J. Wesley-Smith, P. Watt and N.W. Pammenter. 1999. Cryopreservation - an elusive goal? in Proceedings of the IUFRO Symposium, "Recalcitrant Seeds" , Kuala Lumpur (in press). Berjak, P., J.M. Farrant and N.W. Pammenter. 1989. The basis of recalcitrant seed behaviour. Pp. 89-108 in Taylorson, R.B. (Ed.) Recent Advances in the Development and Germination of Seeds. Plenum Press, New York. Chandel, K.P.S., R. Chaudhury, J. Radhamani and S.K. Malik. 1995. Desiccation and freezing sensitivity in recalcitrant seeds of tea, cocoa and jackfruit. Annals of Botany 76, 443-450. Chmielarz, P. 1997. Preservation of Quercus robur L. embryonic axes in liquid nitrogen. Pp. 765-769 in Ellis, R.M., Black, M., Murdoch, A.J. and Hong, T.D. (Eds) Basic and Applied Aspects of Seed Biology. Kluwer Academic Publishers, Dordrecht. Engelmann, F. 1997a. Importance of desiccation for the cryopreservation of recalcitrant seeds and vegetatively propagated species. Plant Genetic Resources Newsletter 112, 9-18. Engelmann, F. 1997b. In vitro conservation methods. Pp. 119–162 in B.V. Ford–Lloyd, J.H. Newbury and J.A. Callow (eds.), Biotechnology and Plant Genetic Resources: Conservation and Use. CABI, Wellingford. Fu, J.R., B.Z. Zhang, X.P. Wang, Y.Z. Qiao and X.L. Huang. 1990. Physiological studies on desiccation, wet storage and cryopreservation of recalcitrant seeds of three fruit species and their excised embryonic axes. Seed Science and Technology 18, 743-754. Janeiro, L.V., A.M. Vieitez and A. Ballester. 1996. Cryopreservation of somatic embryos and embryonic axes of Camellia japonica L. Plant Cell Reports 15, 699-703. Krishnapillay, B., M. Marzalina and M.Y. Aziah. 1992. Cryopreservation of excised embryos of rattan manau. Pp. 325-330 in Proceedings of the 3rd National Biotechnological Research Conference of National Universities of Malaysia, Kuala Lumpur, Malaysia. Lambert, A.M. 1993. Microtubule-organizing centers in higher plants. Current Opinion in Cell Biology 5, 116-122. Lloyd, C.W. 1989. The plant cytoskeleton. Current Opinion in Cell Biology 1, 30-35. Moreno Díaz de la Espina, S. 1995. Nuclear matrix isolated from plant cells. International Review of Cytology 162B, 75-139. Normah, N.M. and M. Marzalina. 1996. Achievements and prospects of in vitro conservation for tree germplasm. Pp. 253-261 in Normah, M.N., Narimah, M.K. and Clyde, M.M. (Eds) In vitro Conservation of Plant Genetic Resources. Percetakan Watan Sdn.Bhd., Kuala Cryopreservation techniques 155

Lumpur, Malaysia. Normah, N.M., H.F. Chin and Y.L. Hor. 1986. Desiccation and cryopreservation of embryonic axes of Hevea brasiliensis Muell.-Arg. Pertanika 9, 299-303. Olyslaegers, G. and J.-P. Verbelen. 1998. Improved staining of F-actin and co-localization of mitochondria in plant cells. Journal of Microscopy 192, 73-77. Pence, V.C. 1990. Cryostorage of embryo axes of several large-seeded temperate tree species. Cryobiology 27, 212-218. Poulsen, K.M. 1992. Sensitivity to desiccation and low temperatures (–196°C) of embryo axes from acorns of the pedunculate oak (Quercus robur L.). Cryo-Letters 13, 75-82. Shibaoka, H. and R. Nagai. 1994. The plant cytoskeleton. Current Opinion in Cell Biology 6, 10- 15. Spector, D.L. 1993. Macromolecular domains within the cell nucleus. Annual Review of Cell Biology 9, 265-315. Staiger, C.J. and C.W. Lloyd. 1991. The plant cytoskeleton. Current Opinion in Cell Biology 3, 33-42. Wesley-Smith, J., C. Walters, P. Berjak and N.W. Pammenter. 1999. A method for the cryopreservation of embryonic axes at ultra-rapid cooling rates. Proceedings of IUFRO Symposium, "Recalcitrant Seeds" , Kuala Lumpur (in press) Wolfe, S.L. 1995. Introduction to Cell and Molecular Biology. Wadsworth, California. 156 Cryopreservation of Tropical Plant Germplasm

Cryopreservation of zygotic embryos of tropical fruit trees – a study on Lansium domesticum and Baccaurea species M.N. Normah, G. Mainah and R. Saraswathy Department of Botany, Faculty of Life Sciences, Universiti Kebangsaan Malaysia, 43600 UKM, Bangi, Selangor, Malaysia

Introduction There is a vast diversity of tropical fruit species. Lansium domesticum Corr. and Baccaurea species, fruit trees of the family Meliaceae and Euphorbiaceae respectively, are considered as being less utilized and promise to add to both nutrition and economic development. Nevertheless, conservation is necessary prior to wider utilization of them. Questions, however, have risen about the best way to preserve genetic diversity of these species. Seeds of Lansium domesticum and B. motleyana are recalcitrant and those of B. polyneura were found to be minimally recalcitrant (but not orthodox) (Normah et al. 1997). These seeds cannot survive drying to the low moisture content required for long-term storage. An alternative method to store the germplasm of these tropical fruit species is through in vitro techniques and cryopreservation. The cryopreservation system is based on the reduction and subsequent detention of the metabolic functions, including the cellular division of the explants. This is accomplished when material is brought to the temperature of liquid nitrogen (–196°C). Zygotic embryos/embryonic axes have been used quite widely in attempts at conserving germplasm of plants with recalcitrant seeds or those with seeds in the intermediate catagory. Examples of these include rubber (Normah et al. 1986), coffee (Normah and Vengadasalam 1992), Corylus sp. (Normah et al. 1994), Camellia sinensis and Artocarpus heterophylus (Chandel et al. 1995). However, there also have been reports of failures or unsuccessful attempts of cryopreserving embryos of the tropical tree species (Hor et al. 1990; Normah et al. 1996, 1997). Preliminary attempts on cryopreserving embryonic axes of Lansium domesticum , Baccaurea motleyana and B. polyneura are described and discussed.

Lansium domesticum Four cryopreservation methods were employed, namely: desiccation, encapsulation-dehydration, vitrification and slow freezing. Details of the investigation were reported by Normah et al. (1996). Desiccation involves dehydrating the axes under the air stream of a laminar flow hood for 0-3 h followed by direct immersion in liquid nitrogen. Thawing was carried out in a water-bath (40±2°C). It was found that the moisture content of freshly excised axes was 57.98% and decreased with desiccation to 18.27% after 3 h. At 38.68% moisture content, 70% viability was obtained but at a lower moisture content, the axes failed to survive. At this high level of moisture, it was expected the cryopreserved axes would not have survived. Cryopreservation techniques 157

For encapsulation-dehydration, the effect of sucrose pretreatment was investigated. Sucrose concentrations of 0.1M, 0.5M and 1.0M were used and it was shown that 0.1M sucrose gave better survival than with 0.5M and 1.0M. Further desiccation of the encapsulated axes in the laminar flow hood reduced viability to 49-58%. No axes survived cryopreservation. The effect of preculture medium was studied for the vitrification method. Embryonic axes were precultured on Murashige and Skoog (1962) medium supplemented with 5% DMSO and 5% glucose, 1.2M sorbitol, 1.0M sorbitol or 0.6M sorbitol followed by the standard procedure by Yamada et al. (1991). The preculture medium, however, did not have any effects on the viability of the non- cryopreserved axes. No viable axes were found after cryopreservation. With slow freezing, the embryonic axes were precultured on MS medium with 5% DMSO for 2 days at room temperature. The samples were transferred to 0.25 ml liquid MS medium in 2-ml cryotubes and cryoprotectant PGD or PVS2 was added over 30 min. A 30-min equilibration at 4°C was followed by cooling at 0.3°C/min to –35°C and plunging into liquid nitrogen. Samples were thawed in a 40°C water-bath, rinsed in MS liquid medium and cultured on recovery medium. Embryonic axes exposed to both PGD and PVS2 died even before the exposure to liquid nitrogen.

Baccaurea species Baccaurea motleyana and B. polyneura were the two species studied. The desiccation method was used for both but encapsulation-dehydration and vitrification were investigated only for B. motleyana . Embryonic axes were desiccated under the laminar airflow for 0, 15, 30, 45, 60 and 75 min for B. motleyana; 0, 30, 60, 90 and 120 min for B. polyneura followed by direct immersion in liquid nitrogen. Baccaurea motleyana axes were very sensitive to desiccation: a fall in moisture content from about 51% to about 36% reduced viability by 85%. The viable axes, however, formed callus. None of the axes survived cryopreservation (Normah et al. 1997). Immature embryos were tested for the same cryopreservation method, and the results showed that at 39.86% moisture content, the viability was 50% and the viability was lost at a lower moisture content (Mainah 1995). The axes for B. polyneura are slightly more tolerant to desiccation and cryopreservation. At 27-30% moisture content, the axes survived desiccation with various viability rates. There was an indication of survival after cryopreservation when some axes formed callus in culture (Normah et al. 1997). For the encapsulation-dehydration method, after excision, the embryonic axes were encased in alginate beads for 72–h pretreatment in liquid MS medium with 0.35, 0.50 or 0.65M sucrose. The beads were then air-dried in the laminar flow hood for 0, 1, 2 or 4 h, placed in cryotubes and plunged into liquid nitrogen. Thawing was carried out at room temperature. All axes pretreated with different concentrations of sucrose and consequently dehydrated gave 100% survival. However, after liquid nitrogen exposure, none survived (Normah et al. 1996). 158 Cryopreservation of Tropical Plant Germplasm

As for vitrification, embryonic axes were precultured on MS medium supplemented with 5% DMSO and 5% sucrose, 1.2M sorbitol, 1.0M sorbitol or 0.5M sorbitol. The effect of different concentrations of PVS2 (0, 40, 50, 60, 70, 80 and 100%) was also investigated. In general, viability of axes declined with the increase in PVS2 concentration. None of the axes survived liquid nitrogen exposure. However, for axes precultured on medium with 5% DMSO and 5% sucrose and cryopreserved with 60% PVS2 concentration, the radicles remained green for 2 days after culture. The axes showed some positive tetrazolium reaction (Normah and Mainah 1996).

Discussion The results of the above studies have shown that none of the standard procedures for cryopreservation is applicable to embryonic axes of Lansium domesticum and the Baccaurea species. Modification of these procedures is necessary taking into consideration the different factors involved in obtaining successful cryopreservation results. For the desiccation technique, flash-drying (fast-drying) probably will give more positive results. It has been shown that excised embryonic axes of recalcitrant seeds can be dehydrated to low water contents, providing dehydration is rapid (Berjak et al. 1989). For the encapsulation-dehydration method, the effects of pretreatment media could be studied further. The role of sugars for the acquisition of tolerance to desiccation and cryopreservation could be investigated. The induction of dehydration (concentration of sucrose or glycero), and duration of induction such as 16-18 h as suggested by Sakai (pers. comm.) may give positive results too. The encapsulation-vitrification method has been investigated on wasabi meristems by Matsumoto et al. (1995) and offers another alternative for conservation of these recalcitrant species. Concentration of PVS2, loading and unloading solutions, and pretreatment media are among the many factors involved that can be investigated further for the vitrification procedure. Loading temperature against length of exposure to PVS2 also may play a role. The importance of sucrose in cryopreservation was shown by Dumet et al. (1993) with somatic embryos of oil-palm and as for the encapsulation and dehydration method, the role of sugars is worth investigating. A key to successful cryopreservation in the vitrification method is to induce a high level of dehydration tolerance. A high level of sugars or sorbitol during preculture has been reported to be important in improving survival (Dereuddre et al. 1988; Uragami et al. 1990). A question which arose from the present study is what factors allow some recalcitrant species to survive cryopreservation and some others not. The ratio of sucrosyl-oligosaccharide to sucrose in seed tissue is generally a good indicator of seed storage category (Steadman et al. 1996) where the ratio for orthodox seed is 1:7, and for recalcitrant seed, 1:12. This indicator could probably be applied to the different categories of the axes too. Hor et al. (1990) suggested that unsuccessful attempts at cryopreservation of embryos of recalcitrant seeds might Cryopreservation techniques 159 be due to the absence of a safe window between high critical moisture content and threshold moisture. This is an important factor that one needs to consider when storing embryonic axes of recalcitrant seeds in liquid nitrogen. Moisture content of embryonic axes is much higher (6-20%) than for the whole seeds for tea, cacao and jackfruit (Chandel et al. 1995). However, embryonic axes can be desiccated to a lower moisture content than for whole seeds. Nevertheless, for cacao, axes at all maturity stages failed to survive cryopreservation. When comparing the size of the axes, cacao has large embryonic axes. So does the axis for Lansium domesticum. It is known that the smaller and less complex the specimens, the better will be the freezing rate achieved. Thus, for L. domesticum the large axes size together with the existence of coarse brownish hairs covering them probably play an important role in desiccation sensitivity. The embryonic axes of Baccaurea species are relatively smaller and in fact they are about the same size as rubber axes which have been successfully cryopreserved. Other factors such as the amount of freezable water probably play a role here. For rubber, the freezable water was probably absent when the naked embryos were partially dried to 17% moisture content, as no exotherm activities were detected (Yap 1998). Successful cryopreservation of plant material should be achievable by the appropriate balance between tissue water content and freezing rate making the use of cryoprotectants a secondary consideration. Thus, flash-drying and very fast freezing rate could be a solution to achieving successful cryopreservation method as suggested by Berjak et al. (1999).

Conclusion Existing cryopreservation procedures developed for temperate or non-woody tropical species are not readily adaptable to woody tropical species. The development of cryopreservation procedures thus needs to consider the unique characteristics of each species. There is a range of parameters that must be empirically determined at a species (perhaps organ) level for successful cryopreservation of tropical recalcitrant seed species.

Acknowledgement This work was supported by the Malaysian National IRPA grant.

References Berjak, P., J.M. Farrant, D.J. Mycock and N.W. Pammenter. 1989. Recalcitrant (homoiohydrous) seeds: the enigma of their desiccation-sensitivity. Seed Science and Technology 18: 297-310. Berjak, P., N.W. Pammenter, M. Norris, J.I. Kioko, J. Wesley-Smith, J.M. Farrant and D.J. Mycock. 1998. Cryopreservation – An elusive goal? IUFRO Seed Symposium 1998, Recalcitrant Seeds, Programme and Abstracts, 12-15 Oct. 1998, Kuala Lumpur. Chandel, K.P.S., R. Chaudhury, J. Radhamani and S.K. Malik. 1995. Desiccation and freezing sensitivity in recalcitrant seeds of tea, cocoa and jackfruit. Annals of Botany 76: 443-450. 160 Cryopreservation of Tropical Plant Germplasm

Dereuddre, J., J. Fabre and C. Bassaglia. 1988. Resistance to freezing in liquid nitrogen of carnation (Dianthus caryophyllus L. var. Kolo) apical and axillary shoot tips excised from different aged in vitro plantlets. Plant Cell Reports 7: 170-173. Dumet, D., F. Engelmann, N. Chabrillange, Y. Duval and J. Dereuddre. 1993. Importance of sucrose for the acquisition of tolerance to desiccation and cryopreservation of oil palm somatic embryos. Cryo-Letters 14: 243-250. Hor, Y.L., P.C. Stanwood and H.F. Chin. 1990. Effects of dehydration on freezing characteristics and survival in liquid nitrogen of three recalcitrant seeds. Pertanika 13: 309-314. Mainah, G. 1995. Viability Studies on Rambai (Baccaurea motleyana) Seeds. BSc.Thesis, Department of Botany, Universiti Kebangsaan Malaysia. Matsumuto, T., A. Sakai, C. Takahashi and K. Yamada. 1995. Cryopreservation of in vitro- grown apical meristems of wasabi (Wasabia japonica) by encapsulation-vitrification method. Cryo-Letters 16: 189-196. Murashige, T. and F. Skoog. 1962. A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiologia Plantarum 15: 473-497. Normah, M.N. and G. Mainah. 1996. Cryopreservation of rambai using encapsulation- dehydration and vitrification of embryonic axes. Pp. 88-90 in Proceedings of the 4th Symposium of Applied Biology, 28-29 May 1996, Universiti Kebangsaan. Malaysia. S.C. Quah and M.K. Vidyadaran (eds.). Malaysian Society of Applied Biology. Normah, M.N. and M. Vengadasalam. 1992. Effects of moisture content on cryopreservation of Coffea and Vigna seeds and embryos. Cryo-Letters 13: 199-208. Normah, M.N., H.F. Chin and Y.L. Hor. 1986. Desiccation and cryopreservation of embryonic axes of Hevea brasiliensis Muel.-Arg. Pertanika 9: 299-303. Normah, M.N., M.S. Jamilah and M.N. Siti Dewi Serimala. 1996. Viability studies on seeds and embryonic axes of Lansium domesticum Corr. Malaysian Applied Biology 25: 39-43. Normah, M.N., B.M. Reed and X. Yu. 1994. Seed storage and cryoexposure behavior in hazelnut (Corylus avellana L. cv. Barcelona). Cryo-Letters 15: 315-322. Normah, M.N., D.R. Saraswathy and G. Mainah. 1997. Desiccation sensitivity of recalcitrant seeds – a study on tropical fruit species. Seed Science Research 7:179-183. Steadman, K.J., H.W. Pritchard and P.M. Dey. 1996. Tissue-specific soluble sugars in seeds as indicators of storage category. Annals of Botany 77: 667-674. Uragami, A., A. Sakai and M. Nagai. 1990. Cryopreservation of dried axillary buds from plantlets of Asparagus officinalis L. grown in vitro. Plant Cell Reports 9: 328-331. Yamada, T., A. Sakai, T. Matsumura and S. Higuchi. 1991. Cryopreservation of apical meristems of white clover (Trifolium repens L.) by vitrification. Plant Science 78: 81-87. Yap, L.V. 1998. Desiccation and Preculture Effects on Survival of Encapsulated Zygotic Embryos of Rubber Following Liquid Nitrogen Exposure. Master of Agricultural Science Thesis, Universiti Putra Malaysia. Cryopreservation techniques 161

Cryopreservation of coffee (Coffea arabica L.) seeds: toward a simplified protocol for routine use in coffee genebanks S. Dussert¹, Nathalie Chabrillange¹, Florent Engelmann², F. Anthony³ and S. Hamon¹ ¹ ORSTOM, 34032 Montpellier Cedex 1, France ² IPGRI, 00145 Rome, Italy ³ Centro Agronómico Tropical de Investigación y Enseñanza, Apartado 59, 7170 Turrialba, Costa Rica

Introduction

Though Coffea arabica seeds can withstand desiccation down to 0.06-0.08 g H2O/g dw (Becwar et al. 1983; Ellis et al. 1990), they cannot be considered orthodox because they remain cold-sensitive and desiccation does not improve their longevity (Van der Vossen 1977; Ellis et al. 1990). Coffea arabica seeds are also characterized by their very short lifespan in the hydrated state (Couturon 1980). Whatever their water content, C. arabica seeds do not withstand direct immersion in liquid nitrogen (Becwar et al. 1983). However, successful cryopreservation of zygotic embryos extracted from mature seeds has been achieved with C. liberica (Normah and Vengadasalam 1992), C. arabica (Abdelnour-Esquivel et al. 1992; Florin et al. 1993), C. canephora and the interspecific hybrid arabusta (Abdelnour-Esquivel et al. 1992). With all species tested, partial dehydration of excised embryos to 0.2 g H2O/g dw was sufficient to obtain high survival rates after their direct immersion in liquid nitrogen. Even if cryopreservation of excised zygotic embryos represents an interesting alternative strategy for the long-term preservation of C. arabica genetic resources, this technique presents some drawbacks for routine use in coffee genebanks: (i) in the case of coffee seeds, embryo extraction is very time-consuming and labour- intensive, (ii) low reproducibility was observed when desiccation of zygotic embryos used classical desiccation methods (airflow or silica gel), and (iii) all stages of the cryopreservation procedure have to be performed under aseptic conditions, which does not allow avoiding the use of in vitro culture techniques. Cryopreservation of whole seeds, instead of zygotic embryos, would allow elimination of these drawbacks. With this aim, the effects of several parameters of the cryopreservation protocol (desiccation, cooling, thawing and post-treatment) were investigated with C. arabica seeds to define conditions which ensure survival of both the endosperm and the embryo (Dussert et al. 1997, 1998). It was shown that: (i) the optimal water content for cryopreserving whole coffee seeds was 0.2 g H2O/g dw, (ii) a two-step freezing procedure including precooling at 1°C/min to –50°C was imperatively required to recover normal seedlings after cryopreservation, and (iii) there was no effect of the thawing rate on survival of cryopreserved seeds. Under these conditions, the maximal percentage of normal seedlings produced after 162 Cryopreservation of Tropical Plant Germplasm cryopreservation was about 30% (Dussert et al. 1997). In addition, it was observed that, whatever the cooling process, the survival rate of zygotic embryos extracted from cryopreserved seeds after thawing was always very high (80-90%). In this study, the reproducibility of seedling recovery was investigated by carrying out several cryopreservation experiments using the same optimal conditions. In addition, the effect of various post-thawing treatments was studied in order to improve the production of normal seedlings after cryopreservation of whole seeds.

Materials and methods

Plant material, desiccation and cryopreservation Fresh mature seeds of C. arabica var. Typica were provided from CATIE, Costa

Rica. After the testa was removed, seeds were desiccated to 0.2 g H2O/g dw by equilibration for 3 weeks under 78% RH obtained using a saturated NH4Cl solution (Dussert et al. 1997). The different cryopreservation procedures (rapid cooling, two-step cooling, slow and rapid rewarming) were carried out following the protocols described by Dussert et al. (1997, 1998).

Osmopriming After thawing, some seeds were primed for 2 weeks at 27°C in the dark using PEG solutions. Osmopriming was carried out by placing batches of 10 seeds in Petri dishes sealed with Parafilm Ribbon, on a thin layer of cotton wool imbibed with 20 ml of aqueous PEG 6000 solution. PEG concentrations were calculated to achieve osmotic potentials of –1, –2 and –4 MPa at 27°C using the equation given by Michel and Kaufmann (1973).

Culture conditions After thawing and/or osmopriming, seeds were cultured in vitro for survival assessment. Disinfection and in vitro culture were performed as described by Dussert et al. (1997). Extraction of zygotic embryos and in vitro culture were carried out according to the method of Bertrand-Desbrunais and Charrier (1989).

Survival assessment Both germination sensu stricto and development of normal seedlings were used to assess seed survival. Emergence of the hypocotyl and radicle was used as the criterion for estimating the germination rate. Seedlings which stood upright on the medium were considered normal. Excised embryos were considered viable when they stood upright on the culture medium and when their first pair of leaves was developed. The time to reach half of the final proportion (Pf) of normal seedlings, T50, was estimated using the least square regression and the following model where P is the proportion of normal seedlings, T the time in days and A a treatment-dependent variable describing the synchronization of seedling development: P = Pf / {1 + exp[A (T - T50)]}. Cryopreservation techniques 163

Results

Effects of slow and rapid cooling When cooled rapidly (200°C/min) by direct immersion in LN, none of the cryopreserved seeds produced normal seedlings. By contrast, viability of zygotic embryos extracted from rapidly cooled seeds was always very high. If seeds were precooled to –50°C at 1°C/min before immersion in LN (slow cooling), over seven repeats, a mean value of 17% of cryopreserved seeds developed into normal seedlings. Under these conditions, some normal seedlings were always recovered after germination of whole seeds, but high variability in the final survival rate was observed (6–29%) (Table 1).

Table 1. Number of experiments (n) and percentage (mean, minimal and maximal values) of normal seedlings recovered from whole seeds or extracted zygotic embryos after desiccation of seeds to 0.2 g H2 O/g dw, followed by rapid cooling (direct immersion in LN; 200°C/min) or slow cooling (1°C/min to –50°C prior to immersion in LN) Whole seeds Zygotic embryos n Seedlings (%) [min-max] n Seedlings (%) [min-max] Rapid cooling 4 0 3 91 [83-98] Slow cooling 7 17 [6-29] 1 70

Effect of osmopriming of seeds after rewarming Osmopriming of seeds after rewarming improved the final proportion of normal seedlings recovered from cryopreserved seeds but the gain in survival decreased in line with decreasing osmotic potential of the PEG solution used for the osmopriming treatment (Fig. 1). Production of normal seedlings under optimal priming conditions (–1 MPa) was 3-fold higher than that of unprimed cryopreserved seeds. Post- thawing seed osmopriming drastically reduced T50, the time to reach half of the final proportion of seedlings: when cryopreserved seeds were placed under germination conditions directly after thawing, T50 value was 36 d, while with osmoprimed seeds, it was about 12-13 d, independently of the osmotic potential of the priming solution.

Discussion

When seeds of C. arabica at 0.2 g H2O/g dw were cooled rapidly (200°C/min), none of them developed into normal seedlings. This result is consistent with those of Becwar et al. (1983) who showed that C. arabica seeds did not survive after immersion in LN, even if all freezable water had been removed from the seeds. By contrast, when seeds were slowly precooled to –50°C at 1°C/min before immersion in LN, an average value of 17% (over seven repeats) of cryopreserved seeds developed into normal seedlings. It is thus clear that slow precooling of C. arabica seeds had a dramatic effect on their survival and their capacity to develop normally (Dussert et al. 1998). However, in view of the high variability observed for the survival rate over seven experiments, improvement to the method appeared necessary before routine use and various post-thawing treatments were investigated. 164 Cryopreservation of Tropical Plant Germplasm

50

40

30

20

10 Normal seedlings (%)

0 0 20 40 60 80 Culture period (d)

Fig. 1. Evolution with time in culture under germination conditions of the percentage of normal seedlings recovered from cryopreserved seeds after a 2-week osmopriming treatment on PEG solutions at –1 (s), –2 (l) and –4 (n) MPa or without osmopriming treatment (¡).

It was shown for the first time that seed osmopriming (osmoconditioning) carried out after thawing had a dramatic beneficial effect on the proportion of normal seedlings recovered after cryopreservation and on seedling growth rate: under optimal osmoconditioning conditions (–1 MPa for 2 weeks), the percentage of cryopreserved coffee seeds which developed into normal seedlings was 3-fold that of unprimed cryopreserved seeds (39% vs. 13%) and the time to reach half of final percentage of normal seedlings (T50 ) was about 3-fold lower (13 vs. 36 d). To our knowledge, the effect of seed osmopriming after cryopreservation has been investigated previously in one study on celery seeds only (Gonzales-Benito et al. 1995). In this study, no effect of osmopriming could be found since cryopreservation did not affect germination rates and T50 values. Osmopriming was first employed to improve the rate and uniformity of germination (Heydecker et al. 1975). Since then, a beneficial effect of osmopriming after seed ageing has been observed both on germination percentage and on germination rate with numerous species (Bewley and Black 1994; Bray 1995). Thus, even if the percentage of seeds which developed into normal seedlings remained relatively low in comparison with that obtained from excised zygotic embryos, a combination of slow cooling and osmopriming treatment could represent a simple and efficient complementary option to field conservation for genebanks which cannot afford in vitro culture facilities. Moreover, this method might be simplified by using a –80°C freezer for precooling seeds to –50°C and could thus become more easily employed routinely in a larger number of genebanks maintaining coffee genetic resources. However, additional research should be undertaken to optimize osmopriming conditions and to carry out direct germination tests under greenhouse or nursery conditions. Cryopreservation techniques 165

In cases where very high survival rates are required for routine use, we propose a second alternative approach based on the extraction of zygotic embryos after rewarming of seeds, which would avoid most of the drawbacks of cryopreservation protocols developed for zygotic embryos. Equilibrating coffee seeds under 78% RH allowed seeds to reach optimal water content for cryopreservation in a very easy and reproducible manner. This method also allows the processing of large amounts of seeds at the same time. Moreover, aseptic culture conditions are required only after thawing. In conclusion, depending on the genebank's facilities, one of the two protocols proposed in this study could be easily applied for the establishment of C. arabica germplasm cryobanks.

References Abdelnour–Esquivel, A., V. Villalobos and F. Engelmann. 1992. Cryopreservation of zygotic embryos of Coffea spp. Cryo–Letters 13: 297–302. Becwar, M.R., P.C. Stanwood and K.W. Lehonardt. 1983. Dehydration effects on freezing characteristics and survival in liquid nitrogen of desiccation-tolerant and desiccation- sensitive seeds. Journal of the American Society of Horticultural Science 108: 613–618. Bertrand–Desbrunais, A. and A. Charrier. 1989. Conservation des ressources génétiques caféières en vitrothèque. Pp. 438–447 in Proceedings of the 13th ASIC, Paipa, Colombia. Bewley, J.D. and M. Black (eds.). 1994. Seeds. Physiology of Development and Germination. 2 nd Edition. Plenum Press, New York. Bray, C. 1995. Biochemical processes during the osmopriming of seeds. Pp. 767–780 in Seed development and germination (J. Kigel and G. Galili, eds.). Marcel Dekker Inc., New York. Couturon, E. 1980. Le maintien de la viabilité des graines de caféiers par le contrôle de leur teneur en eau et de la température de stockage. Café Cacao Thé 1: 27–32. Dussert, S., N. Chabrillange, F. Engelmann, F. Anthony and S. Hamon. 1997. Cryopreservation of coffee (Coffea arabica L.) seeds: importance of the precooling temperature. Cryo–Letters 18: 269–276. Dussert, S., N. Chabrillange, F. Engelmann, F. Anthony, J. Louarn and S. Hamon. 1998. Cryopreservation of seeds of four coffee species (Coffea arabica, C. costatifructa, C. racemosa and C. sessiliflora): importance of water content and cooling rate. Seed Science Research 8: 9–15. Ellis, R.H., T.D. Hong and E.H. Roberts. 1990. An intermediate category of seed storage behaviour ? I. Coffee. Journal of Experimental Botany 41: 1167–1174. Florin, B., H. Tessereau and V. Pétiard. 1993. Conservation à long terme des ressources génétiques de caféier par cryoconservation d'embryons zygotiques et somatiques et de cultures embryogènes. Pp. 106–113 in Proceedings of the 15th ASIC, Montpellier, France. Gonzales–Benito, M.E., J.M. Iriondo, J.M. Pita and F. Perez–Garcia. 1995. Effect of seed cryopreservation and priming on germination in several cultivars of Apium graveolens. Annals of Botany 75: 1–4. Heydecker, W., J. Higgins and Y.J. Turner. 1975. Invigoration of seeds. Seed Science & Technology 3: 881–888. Michel, B.E. and M.R. Kaufmann. 1973. The osmotic potential of polyethylene glycol 6000. Plant Physiology 51: 914–916. 166 Cryopreservation of Tropical Plant Germplasm

Normah, M.N. and M. Vengadasalam. 1992. Effects of moisture content on cryopreservation of Coffea and Vigna seeds and embryos. Cryo–Letters 13:199–208. Van der Vossen, H.A.M. 1977. Methods of preserving the viability of coffee seed in storage. Kenya Coffee 45: 31–35. Cryopreservation techniques 167

Cryopreservation of melon somatic embryos by desiccation method Kei Shimonishi¹, Masaya Ishikawa², Seiichi Suzuki³ and Katsuji Oosawa²* ¹ Kagoshima Biotechnology Institute, Kushira, Kagoshima, 893-1601 Japan ² National Institute of Agrobiological Resources, Tsukuba, Ibaraki, 305-8602 Japan (* present address: Hokkaido National Agricultural Experiment Station, Sapporo, Hokkaido, 062-0045 Japan) ³ Miyagi Agricultural Research Center, Natori, Miyagi, 981-1243 Japan

Introduction Cryopreservation has become a promising tool for long-term conservation of plant genetic resources, and remarkable progress has been made in the past few years. For successful cryopreservation, the key is to apply an appropriate pretreatment to the plant material prior to storage in liquid nitrogen (LN) to avoid freezing injury. Current procedures applied for cryopreservation include pretreatment of samples with permeating hydrogen-bond-rich compounds followed by slow , dehydrative freezing with a programmable freezer. In addition, the vitrification method is now widely applied for cryopreservation of plant tissues, especially shoot apices, and has been successful for a wide range of species. However, a precise treatment duration is required to achieve survival because a prolonged treatment with the vitrification solution could be toxic to the specimens. Therefore, it is difficult to handle large numbers of specimens at a time. Desiccation, which does not involve complicated treatments both before and after immersion in LN, is also one of the effective methods which avoid intracellular freezing because the cells would sustain little damage upon exposure to LN if we could eliminate most of the free water in embryos. However, somatic embryos, which were used as the materials in this study, are considered to be less resistant to desiccation than seeds because the former contain much water. Desiccation of somatic embryos has been attempted for conservation of synthetic seeds, but the viability achieved was low. Some reports indicated that preculture with ABA was effective in increasing the desiccation tolerance of celery somatic embryos (Kim and Janick 1989) and in increasing freezing tolerance in bromegrass suspension cultures (Ishikawa 1989). Our preliminary experiments showed that ABA preculture increased the viability of desiccated melon somatic embryos. Therefore, in the current study, we attempted to identify optimal desiccation conditions which would allow embryos to be directly immersed in LN. Here we report on the cryopreservation of melon somatic embryos by using a controlled desiccation method which does not involve the use of expensive facilities or the application of any cryoprotectants. 168 Cryopreservation of Tropical Plant Germplasm

Material and methods

Induction of somatic embryos and culture conditions Hypocotyls of mature seeds of melon (cv. Earls favorite) were excised and placed on Murashige and Skoog (1962) medium containing 3% sucrose (MS medium) solidified by 0.2% gellan gum. To induce embryos, 1 mg/L 2,4– dichlorophenoxyacetic acid (2,4–D), 1–4 mg/L naphthaleneacetic acid (ANA) and 0.1 mg/L 6–benzyladenine (BA) were added as growth regulators. All the cultures in this study were incubated at 25°C and exposed to a 16-h light/8-h dark photoperiod with cool white fluorescent illumination.

Preculture and desiccation procedure Induced embryos were precultured on MS gellan gum medium with 10 mg/L abscisic acid (ABA) for 3 d to enhance their desiccation tolerance. Following preculture, the embryos were classified into three groups according to their size (S, M, L) and desiccated in 200-ml culture pots made of polycarbonate, with two holes on the lid especially designed for desiccation. The holes were covered with membrane filters (Milliseal, Millipore Co. Ltd), which allow ventilation under aseptic conditions. The embryos were placed inside the pots on a filter paper which was wetted with a few drops of MS liquid medium containing 10 mg/L ABA. The culture pots with embryos inside were put in a chamber in which the RH was kept constant by the addition of a sulphuric acid solution, and desiccated slowly at 25°C. The RHs in this experiment were 50, 60 and 65% because preliminary experiments showed that somatic embryos desiccated at a RH over 80% were not dehydrated but germinated. RH of the chambers was monitored with a digital hygrometer (ACE Scientific Laboratory, Model AD-2) when necessary. Water content of embryos was determined as follows. Total fresh weight of about 70 somatic embryos collected from the desiccation pots was measured every day during the desiccation period. On the final day of desiccation, the embryos were oven-dried at 75°C for 48 h to determine the dry weight. Embryo water content on each date was calculated from these values and expressed as the percentage of water weight versus total fresh weight.

Immersion in liquid nitrogen, thawing and reculture When the embryos were sufficiently desiccated (visual observation), half of them were picked up and transferred to 10-ml glass vials. They were immersed into LN directly from room temperature, stored for 1 d, then thawed rapidly in a 40°C water-bath. The thawed embryos were directly placed on regeneration medium (½ MS gellan gum medium). Viability was determined after 2–3 weeks of incubation, based on the presence of green colour, callus formation or germination of recultured embryos. The other half of the desiccated embryos was directly placed on regeneration medium without cryopreservation to evaluate the effects of desiccation. Cryopreservation techniques 169

Results and discussion The desiccated embryos were shrunken and smaller in size. However, when they were recultured, they rehydrated quickly, rapidly regained their initial size and became as large as before desiccation. The colour of the desiccated embryos was white to yellow when they were placed on the medium, but viable embryos turned yellow to green within about 1 week. Then they germinated within about 2 weeks. Callus formation was not observed in germinated embryos and no secondary embryos were produced. However, the regeneration rate was lower than that of untreated embryos (data not shown). The viability of desiccated embryos and embryos cryopreserved after desiccation (cryopreserved embryos) is indicated in Figure 1. Embryos of medium (M) and large (L) sizes showed a higher survival rate than those with a small (S) size for both desiccated embryos and cryopreserved embryos, irrespective of the RH values (50–65%). This indicates that the smaller the size of the embryos, the more severe the physiological damage caused by desiccation. The observation by Gray et al. (1987) with orchardgrass that the larger or more developed embryos showed a higher survival rate when desiccated at a RH of 70% is similar to ours. The survival rate was 55% (M) and 65% (L) when desiccated at a RH of 60%, and 45% (M) and 57% (L) at a RH of 65% (Fig. 1). These values are not apparently lower than the survival rate of 47–80% (Shimonishi et al. 1990) or 65.7% under optimum conditions (Niwata et al. 1991) when cryopreserved by conventional slow prefreezing method, which suggests that desiccation could be an alternative in melon cryopreservation.

100 Desiccated Cryopreserved 80

60

40 Viability(%) 20

0

S/50 M/50 S/60 M/60 L/60 S/65 M/65 L/65 Size/RH%

Fig. 1. Effect of embryo size (S, M, L) and relative humidities on the viability after controlled desiccation and subsequent cryopreservation. 170 Cryopreservation of Tropical Plant Germplasm

There was no clear difference in the viability rate between desiccated embryos and cryopreserved embryos, regardless of the size classes (Fig. 1). This result indicates that the water content of embryos decreased to a suitably low level which enabled them to survive direct immersion in LN after desiccation at RHs in the range of 50–65%. Water content of the embryos decreased rapidly in the first 2–3 days of the desiccation period. Then it decreased slowly and reached an equilibrium within about 7 days. The equilibrium of the water content of the desiccated embryos at RHs 60% and 65% after 7 days was 11.8% and 13.9%, respectively (Fig. 2). These values are similar to those recorded with desiccated embryos of orchardgrass (Gray et al. 1987).

100 90 80 RH60% 70 RH65% 60 50 40 30 20 13.81 Water content (%) 10 11.81 0 0 1 2 3 4 5 6 7 Days of desiccation

Fig. 2. Changes in water content during desiccation under different relative humidities (RHs). Water content was expressed as the percentage of the fresh weight.

The RH in desiccation chamber became higher than the initially set RH when fresh embryos were inserted (data not shown). Within about 3 days, the rapid decline of the RH, which became close to the set level, coincided with the rapid decline in the water content of the embryos. This indicates that monitoring of RH could be used as a method for estimating the extent of embryo desiccation and the timing of immersion into LN. In the current study, we successfully achieved cryopreservation of melon somatic embryos by subjecting the materials to “predesiccation” followed by immersion into LN. This method allows for low-cost cryopreservation of cultured tissues because no sophisticated facilities are necessary except for a chamber with controlled desiccation conditions. Further studies should be carried out to achieve a more efficient regeneration of plants from cryopreserved embryos and to apply this method to a wide range of materials including tissues Cryopreservation techniques 171 of species more sensitive to desiccation. Cryopreservation of sweetpotato somatic embryos reported by Yoshinaga and Yamakawa (1994) is one of the cases showing the applicability of this method, in which 95.0% of embryos survived and 33.0% regenerated into plantlets, and these values were higher in larger embryos than in the smaller ones as reported here with melon. In recent years, encapsulation-dehydration has often been adopted for cryopreservation by the desiccation method as in the case of lily (Matsumoto and Sakai 1995). The slow desiccation under controlled relative humidities reported here takes longer to reach an equilibrium compared with rapid desiccation. However, this method allows easy control of water content of the specimen unlike with other desiccation methods. This is one of the advantages of this method, because the desiccated specimens can be exposed to LN at any time after reaching equilibrium, not at a specific time of desiccation. Also, this slow desiccation protocol could be applicable to materials with poor tolerance to rapid desiccation.

References Gray, D.J., B.V. Conger, and D.D. Songstad. 1987. Desiccated quiescent somatic embryos of orchardgrass for use as synthetic seeds. In Vitro 23:29–33. Ishikawa, M. 1989. Induction of freezing tolerance in plant cells. Japanese Journal of Freeze- Drying 35:118–124. Kim, Y.H. and J. Janick. 1989. ABA and polyox-encapsulation or high humidity increases survival of desiccated somatic embryos of celery. HortScience 24:674–676. Matsumoto, T. and A. Sakai. 1995. An approach to enhance dehydration tolerance of alginate-coated dried meristem cooled to –196°C. Cryo–Letters 16: 299–306. Murashige, T. and F. Skoog. 1962. A revised medium for rapid growth and bioassays with tobacco tissue culture. Physiologia Plantarum 15:473–497. Niwata, E., R. Ogawa, M. Ishikawa and K. Oosawa. 1991. Cryopreservation by prefreezing method for somatic embryos of melon. Japanese Journal of Breeding 41 (suppl. 1): 190–191. Shimonishi, K., M. Ishikawa, S. Suzuki and K. Oosawa. 1990. Survial of melon somatic embryos exposed to liquid nitrogen after slow prefreezing and after controlled desiccation. Japanese Journal of Breeding 40 (Suppl. 1): 122–223. Yoshinaga, M. and O. Yamakawa. 1994. Cryopreservation of sweet potato somatic embryos involving a desiccation step. Japanese Journal of Breeding 44 (Suppl. 2): 290. 172 Cryopreservation of Tropical Plant Germplasm

Cryopreservation of oil-palm polyembryonic cultures Dominique Dumet¹, Florent Engelmann², Nathalie Chabrillange³, S. Dussert³ and Y. Duval³ ¹ Plant Conservation Biotechnology Group, Division of Molecular and Life Sciences, School of Sciences and Engineering, University of Abertay-Dundee, Dundee DD1 1HG, Scotland ² IPGRI, 00145 Rome, Italy ³ ORSTOM, 34032 Montpellier Cédex 01, France

Introduction An oil-palm in vitro propagation process was set up by the ORSTOM/CIRAD research group in the early 1980s (Pannetier et al. 1981). In this process, the collections are maintained at the polyembryonic culture stage. To facilitate management of the in vitro collection so as to limit the risk of both somaclonal variation and contamination of cultures, a cryopreservation protocol has been developed. The original cryopreservation method was set up for a particular type of embryo: shiny white, finger-shaped, 2–4 mm long, so-called “fusiform embryos”. It was shown that these embryos presented high survival rates after cryopreservation when they were first pretreated for 1 week on a high-sucrose medium (0.75M) (Engelmann 1986). However, even if the appearance of fusiform embryos in the standard polyembryonic cultures were enhanced by subculturing them on a sucrose-enriched medium (0.3M), their production remained a limiting factor in the development of the cryopreservation process. In this paper we describe the different steps of a desiccation-based cryopreservation protocol set up for standard polyembryonic cultures and we discuss its efficiency. High sucrose pretreatment plays a key role in the success of this protocol; sucrose improves both desiccation and cryopreservation tolerance of the polyembryonic cultures. When provided at high concentration it can either act via the osmotic pressure it develops on the tissue (non-specific action), or via its structural characteristics and potential metabolization (specific action). In a second part of this paper we investigate the mode of action of sucrose by replacing it with other saccharides and polyols. We then study the effect of a high sucrose treatment on the sugar contents of embryo clumps.

Materials and methods Embryo clumps weighing 250–300 mg were dissected from standard polyembryonic cultures. They were then subcultured for 1 week on a 0.75M sucrose solid MS-based medium (Pannetier et al. 1981) before being desiccated for 16 h over silica gel (5 clumps per airtight box containing 40 g silica gel). Desiccated clumps were introduced in cryotubes which were plunged directly into liquid nitrogen. Thawing was performed quickly by plunging cryovials in a 40°C water-bath for 2 min. Embryo recovery was optimized by progressively decreasing sucrose concentration and by adding 0.2 mg/L of 2,4– Cryopreservation techniques 173 dichlorophenoxyacetic acid (2,4–D) in the recovery medium for 3 weeks. The osmotic effect of sucrose was studied by comparing the tolerance of embryos to desiccation and cryopreservation after pretreatment on a medium containing sucrose and/or mannitol; mannitol was chosen because of its limited absorption by plant tissue (Cram 1984). Sucrose specificity was approached by replacing it with the following compounds: ribose, fructose, galactose, glucose, maltose, raffinose, mannitol and sorbitol. The osmotic pressure of a 0.75M sucrose medium measured with an osmometer is 1090 mOsm. Consequently, the concentrations of the compounds used in association or as a substitute for sucrose were such that the final osmotic pressure of different media was close to 1000 mOsm. Owing to its dilution limit, the maximal osmotic pressure tested with raffinose was 804 mOsm. For each pretreatment, survival after 16 h of desiccation and/or cryopreservation was recorded. Sugar analysis was performed by ion chromatography HPLC/Dionex (BioLC unit, AG6 precolumn, ASG separation column) after extraction with 78% ethanol. Starch concentration was measured enzymatically using Boeringher detection test kits.

Application of the cryopreservation protocol The cryopreservation protocol described above has been tested with oil-palm polyembryonic cultures of clones maintained in vitro either in France (ORSTOM/CIRAD) or in Côte d’Ivoire (IDEFOR). Depending on the test the average age of the polyembryonic culture, i.e. time since their last subculture, varied from 4 to 12 weeks. Very high survival rates, ranging from 50 to 100% (Table 1), were obtained in the first test performed with 4-week-old cultures. The clones used in this case were those initially chosen to determine the optimal conditions of the cryopreservation process. An average of 27 and 31% survival was obtained in test nos. 2 and 4 performed with 4 and 5-week-old cultures, respectively. The 12- week-old cultures of test no. 3 showed lower tolerance to cryopreservation; indeed, their survival rate varied from as low as 2% up to 25% with an average value of only 11%. Polyembryonic cultures of all the 39 clones tested withstood freezing in liquid nitrogen. However, their survival rate was greatly dependent on the age of the culture that embryos were excised from. Embryo clumps are highly heterogeneous as regards their histological structure. They consist of meristem areas organized in adventitious embryos at different developmental stages. In order to keep their embryogenic proliferation property, i.e. high density of meristematic areas, it is necessary to subculture them regularly every 4 weeks. In the case of oil-palm somatic embryos, it seems that the more meristematic the clump is, the higher its tolerance to desiccation and cryopreservation. Consequently, it is necessary to maintain or reactivate the proliferation potential of the polyembryonic cultures prior to freezing. 174 Cryopreservation of Tropical Plant Germplasm

Table 1. Survival rate after cryopreservation of different polyembryonic cultures of the ORSTOM/CIRAD (France) and IDEFOR (Côte d’Ivoire) clone collections Test No. of clones Culture age Location of Survival rate (%) no. tested (weeks) the test Average Lowest Highest 1 3 4 France 73 50 100 2 7 4 France 27 13 53 3 19 12 France 11 2 25 4 10 5 Côte d’Ivoire 31 10 54

Osmotic effect and specificity of sucrose in desiccation and/or cryopreservation tolerance Only 20% of the non-pretreated polyembryonic cultures (control) withstood the 16-h desiccation period (Table 2). When the pretreatment medium contained a high concentration of either sucrose or various monosaccharides such as fructose, galactose and glucose, desiccation tolerance increased considerably; indeed after such treatment 77–100% of the embryos withstood the 16-h desiccation period. Lower tolerance was obtained in the presence of the other compounds tested, ranging from 56% (maltose) to 18% (lactose). In the case of mannitol, which was used as a “strict” osmoticum, 40% survival was recorded after desiccation. This value dropped down to 0% when mannitol was combined with sucrose. The presence of ribose in the culture medium was lethal for the polyembryonic cultures.

Table 2. Survival rate (%) after 16-h desiccation and/or cryopreservation of polyembryonic cultures pretreated for 7 days in presence of various sugars and polyols at different concentrations Survival rate (%) after Sugars/polyols Pretreatment + concentration during Pretreatment Pretreatment + Desiccation + pretreatment + Desiccation Cryopreservation Cryopreservation Sucrose 0.10M 20 0 0 Sucrose 0.75M 100 40 93 Mannitol 0.90 42 0 0 Sucrose 0.10M + Mannitol 0.75M 0 33 0 Sucrose 0.75M 77 20 57 Ribose 0.93M 0 0 0 Fructose 0.90M 100 0 53 Galactose 0.90M 100 0 47 Glucose 0.90M 92 0 12 Lactose 0.70M 18 0 0 Maltose 0.80M 56 0 0 Raffinose 0.24M 38 0 21 Sorbitol 0.90M 32 0 12 Cryopreservation techniques 175

Survival after cryopreservation of the non-desiccated embryo clumps was recorded only when the pretreatment medium contained sucrose, associated or not with mannitol, with 20–40% and 33% survival, respectively. By contrast, when embryo clumps were frozen after desiccation, various compounds such as fructose, galactose and to a lesser extent, glucose, raffinose and sorbitol, allowed clumps to survive after cryopreservation, with survival rates ranging from 12 to 53%. High sucrose pretreatment improved desiccation tolerance of the polyembryonic cultures and allowed them to survive after cryopreservation. The high osmotic pressure developed by such a medium is likely partly responsible for the improvement of their desiccation tolerance. Indeed, cultures maintained on a standard medium withstand less dehydration than those maintained in the presence of a high osmoticum concentration such as mannitol. Sucrose specificity was very low as regards desiccation tolerance as well as cryopreservation tolerance at low water content, as several compounds could mimic its effect. However, sucrose acted in a very specific way when embryo clumps were not desiccated prior to freezing. In that case, only a high osmotic pressure medium containing sucrose allowed embryonic tissues to withstand cryopreservation.

Changes in sugar profiles induced by high sucrose pretreatment Four different sugars – sucrose, glucose, fructose and starch – were detected initially in the polyembryonic clumps (Table 3). A 7–day high sucrose pretreatment resulted in a drastic increase in sucrose and starch concentrations which were multiplied by 10- and 20-fold respectively. No significant modifications were noticed in glucose and fructose concentrations (data not shown). The only qualitative change recorded was the appearance of arabinose, whose concentration remained very low (around 2 mg/g dry weight [DW]) throughout the pretreatment. The acquisition of high tolerance to desiccation and cryopreservation of the polyembryonic clumps was associated with an increase in their sucrose and starch concentrations.

Table 3. Sugar profile of polyembryonic cultures before and after high sucrose pretreatment Sugar concentration (mg/g DW) Sugars Before After Sucrose 64 679 Glucose 54 30 Fructose 43 35 Arabinose 0 2 Starch 2 45 176 Cryopreservation of Tropical Plant Germplasm

Discussion The cryopreservation protocol described in this paper is now used for oil-palm germplasm banking. In order to obtain satisfactory survival rates after cryopreservation it is essential to freeze only regularly subcultured polyembryonic cultures. High sucrose pretreatment played an important role in the success of this protocol. One of its consequences was a drastic increase in the saccharose concentration of the polyembryonic cultures. Whether this uptake is apoplastic or symplastic remains unclear; however, starch accumulation at the end of the pretreatment reflects that, probably, a large quantity of sucrose was absorbed and metabolized. According to Sagishima et al. (1989), sucrose is first hydrolyzed into glucose and fructose before being incorporated into the cell via invertase activity. However, for Stranzel et al. (1988), sucrose incorporation depends on its concentration in the medium; invertase activity would be necessary at low concentration while sugar absorption would be passive through hydrophilic domains at high concentration. Sucrose is well known for its implication in desiccation tolerance of plant tissues (Koster and Leopold 1988; Leopold 1990). Sucrose could act either by replacing the water molecules involved in the strutural maintenance of macromolecules, or by inducing vitrification of the intracellular medium (Crowe et al. 1988; Williams and Leopold 1989). In the case of oil-palm somatic embryos, other sugars such as fructose, galactose and glucose were able to mimic a sucrose effect as regards desiccation tolerance. Their efficiency could depend on their ability to be both absorbed into the symplastic compartment and metabolized into sucrose. Other studies have shown that as the crystallisable water is removed from the high sucrose pretreated polyembryonic culture their survival after cryopreservation increased (Dumet et al. 1993). It is likely that a similar effect occurred with sugars other than sucrose, allowing desiccated embryo clumps to withstand cryopreservation. By contrast, only sucrose provided cryoprotection when polyembryonic culture water content was relatively high. Our hypothesis is that the presence of sucrose in the apoplast compartment prevents lethal extra-cellular ice formation which may occur in relatively highly hydrated embryo clumps.

References Cram, W.J. 1984. Mannitol transport suitability as an osmoticum in root cells. Physiologia Plantarum 61:396–404. Crowe, J.H, L.M. Crowe, J.F. Carpenter, A.S. Rudolph, C.A. Wistrom, B.J. Spargo and T.J. Anchordoguy. 1988. Interactions of sugars with membranes. Biochimical and Biophysical Acta 947:367–384. Dumet, D., F. Engelmann, N. Chabrillange, Y. Duval and J. Dereuddre. 1993. Importance of sucrose for the acquisition of tolerance to desiccation and cryopreservation of oil palm somatic embryos. Cryo–Letters 14:243–250. Engelmann, F. 1986. Cryoconservation des embryons somatiques de palmier à huile (Elaeis guineensis Jacq.) – Mise au point des conditions de survie et de reprise. Thèse d’Université, Paris 6, 228p. Cryopreservation techniques 177

Koster, K.L. and A.C. Leopold. 1988. Sugars and desiccation tolerance in seeds. Plant Physiology 88:829–832. Leopold, A. C. 1990. Coping with desiccation. Pp. 37–56 in Stress response in Plant: Adaptation and acclimation mechanisms. (R.G. Alscher and J.R. Cuming eds.). Wiley– Liss. Inc. Pannetier, C., P. Arthuis and D. Liévoux. 1981. Néoformations de jeunes plantes d’Elaeis guineensis à partir de cals primaires obtenus sur fragments foliaires cultivés in vitro. Oléagineux 36:119–122. Sagishima, K., K. Kubota and H. Ashihara. 1989. Uptake and metabolism of sugars by suspension-cultured Catharanthus roseus cells. Annals of Botany 64: 185–193. Stanzel, M., R.D. Sjolund and E. Komor. 1988. Transport of glucose, fructose and sucrose by Streptanthus tortuosus suspensions cells. II: Uptake at low concentration. Planta 174:201–209. Williams, R.J. and A.C. Leopold. 1989. The glassy state in corn embryos. Plant Physiology 89:977–981. 178 Cryopreservation of Tropical Plant Germplasm

Recent developments in cryopreservation of shoot apices of tropical species Hiroko Takagi JIRCAS, Tsukuba, Ibaraki 305-8686, Japan

Introduction It is well known that conservation of plant genetic resources is an issue of common global concern owing to the rapid erosion of plant genetic diversity and the enormous potential value of genetic resources. With the rapid increase of germplasm accessions maintained in genebanks, the development of useful and efficient schemes and techniques for conservation is now being given priority in plant genetic resources activities. Especially, long-term conservation of recalcitrant seed and vegetatively propagated species is considered an important research area to overcome the problems and difficulties associated with clonal maintenance of plant germplasm (Engelmann 1997). For long-term conservation of problem species, it is generally accepted that cryopreservation is the only current method that could provide ideal conditions. Since the pioneer research in cryopreservation of cold-hardy plant tissues was reported, tremendous efforts have been made toward the development and application of techniques for cryopreservation of plant species. Liquid nitrogen storage is now available for different materials such as cells, callus, pollen, embryos (somatic and zygotic) and shoot apices, and the number of cryopreserved species is rapidly increasing. Although recent remarkable progress in cryobiological studies of plant materials has made cryopreservation a realistic tool for long-term storage, cryopreservation of non cold-hardy plants, especially tropical species, which are not intrinsically tolerant to low temperature and desiccation, has been less extensively investigated. There are growing interests and needs for cryopreserv- ation of crops in the tropics where species that require clonal maintenance are prevalent. In an increasing number of research programmes in the tropics, research activities in this field have been actively initiated during recent years. This paper reviews the current status of cryopreservation of tropical species with special emphasis on shoot apices (meristems), which are ideal explants for in vitro clonal conservation.

Recent progress in cryopreservation At the beginning of the 1990s, it was reported that cryopreservation was applied to more than 100 species as cell suspensions, callus, protoplasts, embryos and meristems. Engelmann (1991) reported that 44 species of tropical origin (87 reports) were cryopreserved. Out of those, 48 reports were on undifferentiated tissues (cells, callus and protoplasts), 7 on pollen, 19 on embryos (somatic and zygotic) and 13 on shoot apices/meristems. Classical cryopreservation techniques, which involve freeze-induced dehydration, were still the mainstream of the protocols Cryopreservation techniques 179 employed. The techniques have been successfully applied to undifferentiated culture systems such as cell suspensions and callus (Engelmann 1997). In the case of differentiated structures, these techniques also could be employed for freezing apices of cold-tolerant species (Reed and Chang 1997), but their utilization with high survival for tropical species was exceptional. Recent noteworthy progress in cryopreservation research was the development of simplified but effective procedures such as vitrification (Langis et al. 1990; Sakai et al. 1990; Towill 1990), encapsulation/dehydration (Dereuddre et al. 1990; Fabre and Dereuddre et al. 1990) and air-drying (Uragami et al. 1990). These new techniques are referred to as “vitrification-based techniques” in which cell dehydration is performed prior to direct plunging in to LN by exposure of samples to concentrated cryoprotective media or air desiccation to induce glass transitions during cooling and rewarming (Engelmann 1997). By precluding ice formation in the system, the vitrification-based procedures simplify the cryogenic procedure and eliminate concerns for the potentially damaging effects of intra- and extra- cellular crystallization (Sakai 1997). They are more appropriate for complex organs (embryos and shoot apices) that contain a variety of cell types, each with unique requirements under conditions of classical cryopreservation techniques (Withers 1979). The new techniques have produced high levels of post-thaw regrowth, and have greater potential for broad applicability since only minor modifications for different cell types are required (Engelmann 1997; Sakai 1997).

Recent advances in cryopreservation techniques of shoot apices The number of species which can be cryopreserved has rapidly increased over the last several years because of the new techniques and progress of cryopreservation research. Recent reports of successful cryopreservation of shoot apices are presented in Tables 1, 2 and 3 by the techniques applied with the detailed key points of the protocols. Although a few cryopreserved collections of temperate fruit, nuts and woody plants of economic importance have been established (Reed and Chang 1997), they are not included in the tables. The lists are mainly focused on herbaceous plants with tropical origin and some temperate plants for comparison. In comparison with the cryopreservation of shoot apices by classical techniques, the recent progress of cryopreservation techniques has considerably contributed to the improvement of post-thaw recovery in the last few years. Only post-thaw shoot recovery is considered as a criterion for survival (not the partial survival of tissues) because shoot recovery without intermediate callus formation is essential for clonal conservation of germplasm. Although some of the figures show less than 40% post-thaw shoot recovery rates, they are relatively high and compare to other reports on each plant species. Among the reports of successful cryopreservation presented here, the vitrification, encapsulation-dehydration and other methods are employed 16, 11 and 4 times, respectively. The key technical factors for successful cryopreservation of shoot apices will be discussed in detail based on those examples. 180 Cryopreservation of Tropical Plant Germplasm

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Basic protocols of the techniques

Vitrification method The principle of the vitrification method is to dehydrate a specimen by exposing it to a vitrification solution containing high osmoticum. The method basically involves the following steps: 1. Prepare and select appropriate samples 2. Preculture dissected shoot apices with osmoticum (e.g. 0.3–0.6M sucrose) 3. Treat with loading solutions 4. Dehydrate by exposing to vitrification solution at 0/25°C 5. Immerge directly into LN and store 6. Rewarm rapidly 7. Remove vitrification solution 8. Place apices under appropriate conditions for recovery.

This procedure has been reported to be successful with complex tissues such as somatic embryos and shoot apices rather than cell suspensions. Encapsulation- vitrification is a modified method where explants are encapsulated in alginate beads before being subjected to preculture and dehydration with highly concentrated solutions. Table 1 presents the list of the recent reports on successful cryopreservation of shoot apices by vitrification.

Encapsulation-dehydration method This method is based on the fact that encapsulation protects the explants, and preculture in medium enriched with osmoticum makes them tolerant to air- drying. The method basically consists of the following steps: 1. Prepare and select appropriate samples 2. Encapsulate dissected shoot apices in alginate beads 3. Preculture with osmoticum (e.g. sucrose, sorbitol, glycerol) 4. Desiccate in a laminar flow chamber or with silica gel 5. Immerge rapidly into LN and store 6. Rewarm rapidly 7. Place apices under appropriate conditions for recovery.

This method has been applied to cell suspensions, somatic embryos and apices of numerous species of temperate and tropical origins. The recent reports with successful results on shoot apices by encapsulation-dehydration are summarized in Table 2.

Other methods Successful cryopreservation by pregrowth-desiccation, pregrowth and droplet methods are cited in Table 3. These methods are operationally simple but have been applied to only a limited number of cases for shoot apices. Cryopreservation techniques 185

A typical procedure of pregrowth-desiccation is used for Asparagus (Uragami et al. 1990), i.e. dissected shoot apices are: (1) precultured with osmoticum, (2) dehydrated in laminar flow, (3) directly immersed into LN, (4) rapidly rewarmed, and (5) cultured under appropriate recovery conditions. The optimized procedure by Escobar et al. (1997) for cassava shoot apices is more complicated: a combination of different osmoticum and slow freezing. Panis et al. (1996) developed successful protocols of the pregrowth method for proliferating meristematic clumps of banana by means of sucrose pregrowth followed by a direct plunge into LN. A similar procedure was reported for axillary buds of asparagus, but with only 13.2% post-thaw shoot recovery (Uragami et al. 1991). The droplet method is unique to potato shoot apices (Schäfer–Menuhr 1996). After being cryoprotected with 10% DMSO, apices are placed into small droplets of cryoprotectant distributed on a small piece of aluminium foil and directly immersed in LN.

Technical key issues for cryopreservation of shoot apices

Plant materials

Proper set-up of in vitro culture systems Apices derived from shoot cultures maintained in vitro are the specimens commonly used for cryopreservation of vegetatively propagated species. These materials have the advantage of being available in large numbers, thus providing clonal replicates and permitting more satisfactory experimentation and duplication of samples in storage. A point of critical importance for successful cryopreservation is closely linked with tissue culture protocols. Adequate management of whole culture systems is essential to induce favourable physiological conditions for micropropagation of shoot-tips from donor plants, effective preculturing of dissected shoot apices and vigorous recovery of cryopreserved shoot apices without intermediate callus formation.

Selection of size and developmental stage of shoot apices It has been generally accepted that an appropriate unit specimen for cryopreservation consists of the apical dome plus a couple of leaf primordia, measuring 0.5–2 mm in length, depending upon the species. However, it has been reported that the proper selection of size and developmental stage of the specimen was one of the essential factors to achieve high post-thaw shoot recovery rate. For example, taro shoot-tips within the range 0.5–0.8 mm long, containing the apical dome and 1–2 leaf primordia, were effectively dehydrated within shorter periods of exposure to PVS2 and showed higher post-thaw recovery compared with those of larger size (Takagi et al. 1997). In the case of banana (Musa spp.), the shoot apices with the apical dome partially covered by leaf primordia, but not fully covered or exposed, showed the 186 Cryopreservation of Tropical Plant Germplasm best post-thaw survival in the vitrification method (Thinh 1997). The selection of this type of shoot apices was also successfully applied to other tropical monocotyl- edonous plants such as Cymbidium , pineapple and wild rice (Thinh 1997). Escobar et al. (1997) reported that viability and shoot growth increased significantly when small shoot apices (1–2 mm in length) were used as explants for cryopreservation by the desiccation method in comparison with larger shoot apices (3–4 mm in length).

Conditioning of donor plants The post-thaw survival of cold-hardy plants has always been improved through cold-hardening of donor plants. The treatment tends to induce an intrinsic tolerance to low temperature and desiccation by triggering genes responsible for cold stress. For the temperate herbaceous plants which are minimally cold-hardy, low-temperature treatments (0–5°C), either to the donor plants or to the dissected meristems themselves, were effective in obtain ing high and stable post-thaw recovery when the protocols were well optimized (Tables 1 and 2). One of the most important issues of cryopreservation of tropical species could be how to induce the optimal physiological state to induce tolerance to dehydration and cryogenic procedures. It has been reported that post-thaw survival of taro [Colocasia esculenta (L.) Schott.] shoot-tips was significantly increased and stabilized when donor plants were preconditioned on medium with high sucrose concentration (120 g/L) for 1 month (see Table 1; Takagi et al. 1997; Thinh 1997). The chemical analysis of these plants showed that the plants preconditioned with 120 g/L sucrose had significantly higher amount of soluble sugars, proline and starch than those grown on medium with 30 g/L sucrose. However, on the other hand, the preconditioning with elevated concentration of sucrose was not effective, and even decreased the post-thaw survival of tannia (Xanthosoma) which belongs to the same family, Araceae (Thinh 1997). Although information on the effects of conditioning culture with high sucrose is still limited, it definitely showed the possibility of using sucrose as a means of inducing tolerance to cryopreservation in tropical species.

Technical key factors of the vitrification method

Dehydration by vitrification solution Since vitrification solutions contain high concentrations of cryoprotectants such as glycerol, ethylene glycol or dimethylsulfoxide (DMSO), it is an essential step for successful vitrification to identify the minimum exposure time to the solutions to avoid the toxic effects but to obtain sufficient dehydration (Yamada et al. 1991). Sensitivity to dehydration by a vitrification solution varies among species (or even cultivars). For example, when shoot-tips of four species were exposed to PVS2 solution at different time intervals, significant difference among the species tested was observed in their sensitivity. Taro appeared to be quite tolerant, since Cryopreservation techniques 187 even after 60 min of exposure, 60% of treated shoot-tips resumed normal growth. In contrast, banana and sweet potato were very sensitive and exposure durations of 10–15 min were enough to kill all the specimens (Fig. 1). Understanding the degree of sensitivity to dehydration by a vitrification solution is the first step in accomplishing an optimized protocol.

100 90 Taro 80 (Colocasia esculenta) 70 White yam (Dioscorea rotundata) 60 50 40 30 Sweet potato (Ipomoea batatas) Shoot recovery (%) 20 Banana 10 (Musa spp.) 0 0 5 10 15 20 30 40 60 Exposure time to PVS2 (min)

Fig. 1. Survival rate of shoot-tips of some tropical crops after exposure to PVS2 at different time intervals. Dissected shoot-tips were exposed to PVS2 [30% (w/v) glycerol, 15% (w/v) ethylene glycol, 15%(w/v) DMSO and 0.4M sucrose; Sakai et al. 1990] and recultured under favourable culture conditions for each material (cited from Takagi et al. 1998).

Improvement of dehydration tolerance The key for successful cryopreservation by the vitrification method is how to overcome the sensitivity to the treatment with vitrification solutions; in other words, how to induce dehydration tolerance to a highly concentrated vitrification solution (Yamada et al. 1991; Matsumoto et al. 1994; Touchell and Dixon 1996; Sakai 1997). Recent dramatic progress in the vitrification method is much indebted to the development of additional approaches from this aspect.

Preculture of dissected shoot apices With the vitrification method, preculture of shoot apices with sucrose (or sorbitol)-enriched media prior to dehydration has been reported to be effective to improve post-thaw survival of some temperate species (Yamada et al. 1991; Kohmura et al. 1994; Matsumoto et al. 1994, 1995a, 1995b; Niwata 1995; Hirai et al. 1998). It is understood that the accumulation of sugars increases the stability of 188 Cryopreservation of Tropical Plant Germplasm membranes under conditions of severe dehydration (Crowe et al. 1989). It is revealed that for the tropical species (Table 1), preculture with sucrose (or sorbitol) was also an indispensable procedure, although preculture may not always lead to substantial increases in post-thaw recovery.

Loading Direct exposure of shoot apices to vitrification solutions often leads to detrimental effects. Exposure to cryoprotectants with lower concentrations than vitrification solutions (so-called “loading solution” or “LD solution”) has been reported to be useful to minimize the adverse effects of vitrification solutions, especially when applied to shoot apices of some crops relatively sensitive to PVS2 (Yamada et al. 1991; Matsumoto et al. 1994, 1995a, 1995b; Takagi et al. 1997; González–Arnao et al. 1998a; Thinh et al. 1999). Interestingly, loading did not influence post-thaw survival of shoot-tips of white yam (Kyesmu et al. 1997). For sweet potato it played an effective role only when certain circumstances of preconditioning, preculture and type of vitrification solution were properly combined (Takagi, unpubl.). During the brief incubation with a LD solution, cells are plasmolyzed by considerable dehydration, but little permeation of glycerol into the cytosol is observed. Although the action mechanism of the treatment is not well understood yet, it is considered that the protective effects of LD solution treatment may be a result of the concentration of cytosolic cryoprotectants accumulated during the preculture with sucrose and the protective effects of plasmolysis. Also, the presence of LD solution in the periprotoplasmic space of plasmolyzed cells may mitigate mechanical stress caused by severe dehydration by a concentrated vitrification solution (Matsumoto et al. 1998).

Lowering the temperature during dehydration The positive effect of using a lower temperature, 0°C, during the dehydration by a vitrification solution was first reported in white clover (Yamada et al. 1991). It was also very effective with other species, especially the tropical species which are relatively sensitive to the dehydration procedure such as pineapple (González–Arnao et al. 1998b), Grevillea scapigera (Touchell and Dixon 1996), sweet potato (Plessis and Steponkus 1996) and banana (Thinh et al. 1999). In the case of greater yam (Dioscorea alata, cv. UM680), the post-thaw shoot recovery was drastically improved from 47 to 91% by lowering the temperature from 25 to 0°C during the dehydration by PVS2 (Kyesmu 1998; Table 1).

Composition of vitrification solutions The most popular and effective vitrification solution so far is PVS2 (30% glycerol, 15% ethylene glycol and DMSO in 0.4M sucrose). Sakai and his group reported that PVS2 does not permeate into the cytosol during dehydration. However, the use of toxic chemicals such as DMSO still could be one of the issues for further investigation, not only for their physiological effects but also for their mutagenic effects on plant materials. More recently, Sakai et al. (unpubl.) found that a new Cryopreservation techniques 189 vitrification solution, PVS4, which contains 35% glycerol and 20% ethylene glycol in basal medium containing 0.6M sucrose produced nearly the same recovery growth as PVS2. If using toxic chemicals such as DMSO is still an issue of concern, the further development of less toxic vitrification solutions could promote the wider application of this method.

Technical key factors of the encapsulation-dehydration method The encapsulation-dehydration technique (Dereuddre et al. 1990; Fabre and Dereuddre 1990) is easy to handle and appears to be a practical method for cryopreservation of meristems and somatic embryos. In the encapsulation- dehydration technique, gradual extraction of water from encapsulated meristems is performed during the preculture in sucrose-enriched medium (usually around 0.8M sucrose). The sucrose molarity in the beads is further increased by the additional air-desiccation, and reached/exceeded the saturation point of the sucrose solution resulting in a glass transition during cooling to –196°C (Dereuddre et al. 1990). With this technique, the induction of desiccation tolerance, through the subsequent increase of sucrose concentration during preculture of encapsulated shoot apices, is the key for the successful cryopreservation.

Pregrowth and preculture Preculture of encapsulated shoot apices is the main procedure to induce desiccation tolerance in the encapsulation-dehydration technique. The technique is relatively simple, and the concentrations of sucrose and treatment duration are the only variables to be optimized for this step. A progressive increase in sucrose concentration during the preculture was used for coffee (Mari et al. 1995) and sugarbeet (Vandenbussche and de Proft 1996). Higher concentration of sucrose (0.4M sucrose) in beads was applied to wasabi (Matsumoto et al. 1995b), strawberry (Hirai et al. 1998) and Japanese pink lily (Matsumoto and Sakai 1995). It is an approach to simplify the procedure but results benefited from a progressive increase of sucrose concentration. Matsumoto et al. (1995a) reported that encapsulated lily meristems precultured with a mixture of 0.8M sucrose plus 1.0M glycerol produced considerably high post-thaw shoot recovery of 90% while preculture only with 0.8M sucrose gave 57%. However, a mixture of 0.8M sucrose in combination with ethylene glycol and DMSO produced toxic effects during the dehydration process. It is considered that glycerol contributes to minimizing the injurious membrane changes resulting from severe dehydration. This revised procedure was also applied to strawberry (Hirai et al. 1998) and potato (Hirai and Sakai, this vol. p. 205). Although it is not included in Table 2, culture of apices on standard medium after dissection was reported as an important procedure leading to the reactivation of metabolism through starch accumulation in all cells, and resulted in good survival for coffee, date palm and sugarcane (Engelmann 1997). This culture period may also reduce the physiological heterogeneity of apices by synchronizing their metabolism. 190 Cryopreservation of Tropical Plant Germplasm

Technical key factors of other methods As mentioned previously, the methods listed in Table 3 have been applied to shoot apices of a limited number of species so far. Technical details may not be applicable to a wider range of plant materials with less tolerance to dehydration and low temperature. The basic procedure for those methods is a combination of sucrose pretreatment, dehydration and rapid freezing without encapsulation and has proved successful for cold-hardened in vitro-grown shoot-tips of apple (Malus domestica) and winter buds of mulberry (Marus bombysis), etc. Banana shoot-tips are extremely sensitive to high sucrose concentrations and air-desiccation and encapsulation-dehydration led to very low survival. However, selection of the proliferating meristematic clumps as specimens and addition of sucrose to the pregrowth medium enabled the development of a very simple method with potential application to a wide range of banana germplasm (Panis et al. 1996).

Comparison of different methods The encapsulation-dehydration method has advantages: (i) the cryogenic procedure is relatively simple, (ii) only sucrose is used as osmoticum to induce desiccation tolerance, and (iii) handling and storage after desiccation are easy. However, for most of the species listed in the tables, the vitrification method (with or without encapsulation) produced much higher levels of post-thaw shoot recovery under well-optimized conditions than the encapsulation-dehydration method. Comparative studies of the different methods on wasabi (Matsumoto et al. 1995b), strawberry (Hirai et al. 1998) and Grevillea scapigera (Touchell and Dixon 1996) clearly showed the higher post-thaw recovery rate of vitrification method and also pointed out that recovery growth was much faster with the vitrification method. Besides the high post-thaw shoot recovery, the method has advantages in that it does not require expensive equipment, and the operation is economical and requires less time than other methods. In the vitrification procedure, different approaches and the combination of these means to increase tolerance of samples are available and this makes the applicability of the technique wider for different species and cultivars with different genetic and physiological backgrounds. Although the technique has been improved to be operationally more simple, a set of interacting variables still must be simultaneously optimized to accomplish successful cryopreservation for each species and/or cultivar. Although it still requires a series of tedious investigations, the practical guideline based on the past experiences could be helpful for systematic approach to optimize protocol for new materials (Thinh 1997). Cryopreservation techniques 191

Conclusion Considerable progress has been made in the cryopreservation of shoot apices. Since the newly developed protocols are simple in operation and do not require expensive equipment, the techniques will become practical tools in the near future for germplasm conservation activities in tropical regions which are rich in plant genetic diversity and prominent with vegetatively propagated species. Although cryopreservation techniques are fairly well developed, more research is needed to make the techniques more practical and reliable. There are few analyses of regenerants from cryopreserved materials. It is desirable to have more information on genetic stability of the materials kept in LN to check that cryogenic and culture protocols are effective in maintaining clonal integrity. Basic research on physiological mechanisms of the successful cryopreservation, especially the role of sucrose and glycerol, could contribute to improvement and stabilization of post-thaw shoot recovery, and to development of protocols for materials which are extremely sensitive to desiccation and cryogenic procedures. As mentioned above, technical developments in the last 10 years have made the cryopreservation of shoot apices realistic. To facilitate the application of these techniques, more information and considerations on practical issues are also needed to set up cryogenic systems in genebanks, such as required levels of viability to ensure safe conservation, required size of samples for each accession, guidelines for periodic testing of viability and genetic integrity, etc. Research must also expand to identify and develop the systems and technologies required from this aspect.

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Plessis, P. and P.L. Steponkus. 1996. Cryopreservation of sweet potato shoot-tips by vitrification. Cryobiology 33: 655–656. Reed, B.M. and Y. Chang. 1997. Medium- and long-term storage of in vitro cultures of temperate fruit and nut crops. Pp. 67–105 in Conservation of Plant Genetic Resources In Vitro. Volume 1: General Aspects. M.K. Razdan and E.C. Cocking (eds.). Science Publishers Inc., Enfield, USA. Sakai, A., S. Kobayashi and I. Oiyama. 1990. Cryopreservation of nucellar cells of navel orange (Citrus sinensis Osb. var. brasiliensis Tanak) by vitrification. Plant Cell Reports 9: 30–33. Sakai, A. 1997. Potentially valuable cryogenic procedures for cryopreservation of cultured plant meristems. Pp. 53–66 in Conservation of Plant Genetic Resources In Vitro. Volume 1: General Aspects. M.K. Razdan and E.C. Cocking (eds.). Science Publishers Inc., Enfield, USA. Schäfer–Menuhr, A. 1996. Refinement of cryopreservation techniques for potato. Final Report for the period 1 Sept., 1991–31 Aug., 1996. International Plant Genetic Resources Institute, Rome. Takagi H., N.T. Thinh, O.M. Islam, T. Senboku and A. Sakai. 1997. Cryopreservation of in vitro grown shoot tips of taro (Colocasia esculenta (L.) Schott) by vitrification. 1. Investigation of basic conditions of the vitrification procedure. Plant Cell Reports 16:594–599. Takagi, H., N.T. Thinh and P.M. Kyesmu. 1998. Cryopreservation of vegetatively propagated tropical crops by vitrification. Acta Horticulturae 461: 485–494. Thinh, N.T. 1997. Cryopreservation of germplasm of vegetatively propagated tropical monocots by vitrification. Doctoral Papers of Kobe University, Department of Agronomy, Japan. Thinh, N.T., H. Takagi and S. Yashima. 1999. Cryopreservation of in vitro-grown shoot tips of banana (Musa spp.) by vitrification method. Cryo–Letters 20 (in press). Touchell, D.H. and K.W. Dixon. 1996. Cryopreservation for conservation of Australian endangered plants. Pp. 169–180 in In Vitro Conservation of Plant Genetic Resources. (M.N. Normah, M.K. Narimah and M.M. Clyde, eds). Plant Biotech. Lab., Faculty of Life Sci., Univ. Kebangsaan Malaysia. Towill, L.E. 1990. Cryopreservation of isolated mint shoot tips by vitrification. Plant Cell Reports 9: 178–180. Uragami, A., A. Sakai and M. Nagai. 1990. Cryopreservation of dried axillary buds from plantlets of Asparagus officinalis L. grown in vitro. Plant Cell Reports 9: 328–331. Uragami, A., A. Sakai and M. Nagai. 1991. Cryopreservation of asparagus (Asparagus officinalis L. ) cultured in vitro. Japan Agricultural Research Quarterly 27:112–115. Withers, L.A. 1979. Freeze preservation of somatic embryos and clonal plantles of carrot (Daucus carota ). Plant Physiology 63: 460–467. Yamada, T., A. Sakai, T. Matsumura and S. Higuchi. 1991. Cryopreservation of apical meristems of white clover (Trifolium repens L.) by vitrification. Plant Science 78: 81–87. Vandenbussche, B. and M.P. de Proft. 1996. Cryopreservation of in vitro sugar beet shoot- tips using the encapsulation-dehydration technique: Development of a basic protocol. Cryo–Letters 17:137–140. 194 Cryopreservation of Tropical Plant Germplasm

Cryopreservation of vegetatively propagated species (mainly mulberry) Takao Niino¹, Ivette Seguel² and Toru Murayama¹ ¹ Department of Upland Farming, Tohoku National Agricultural Experiment Station, Arai, Fukushima, Fukushima 960-2156, Japan ² Experimental Center in Carillanca, Institute of Investigation on Agriculture and Livestock, Casilla 58-D, Temuco, Chile

Introduction The importance of plant genetic resources is well known and it is recognized that plant genetic diversity is a limited natural resource, which provides useful genes for breeding. However, the long-term conservation of crops which are vegetatively propagated, like roots and tubers, fruit trees and other woody plants, including tea and mulberry, of recalcitrant seed species is facing practical problems. In Japan, the conservation of clonal germplasm amounts to about 40 000 accessions. Fruit trees, tea and mulberry trees account for more than 16 000 accessions. These plants are being maintained as living plants in the field. However, there are several serious problems with field genebanks. They are not only time-consuming and cumbersome to maintain , requiring both space and labour, but the material is also exposed to pests, pathogens and environmental stress and there is always the risk of losing germplasm. Moreover, as yet, no method is known by which the germplasm can be stored. Cryopreservation procedures have been developed over the last decade using materials such as tissues, organs, meristems, pollen, embryos and cells of many plants. With many species, cryopreservation has been successfully achieved with the regeneration of entire plants from cryopreserved materials. These results have demonstrated that we have been able to apply the cryopreservation technique to many other materials such as vegetatively propagated crops, recalcitrant seeds, rare germplasm, in vitro cultures, pollen, transgenic cultures, valuable cell lines, virus- free properties, etc. Cryopreservation of plant materials has been proven to be a potentially ideal method for preserving plant germplasm for the long term using a minimum of space, labour, medium and maintenance. In this paper, we describe three aspects of the practical cryopreservation methods developed over the last 10 years.

Cryopreservation using mulberry winter buds In woody plants, using dormant winter buds for materials is one way to achieve successful cryopreservation. The protocol of cryopreservation for mulberry winter buds is the following (Niino et al. 1995). First, the branches of woody plants were collected in the winter season, when the buds were still in a state of quiescence. The axillary buds with about 10 mm of vascular tissue were removed from the branches. The winter buds were then put into a polyethylene bag or a polyethylene cryotube. They were prefrozen, with the temperature being Cryopreservation techniques 195 decreased by 10°C a day to –30°C and then transferred to a deep-freezer maintained below –135°C or immersed in a LN container. After storage, the buds were rapidly thawed in a water-bath at 37°C. After sterilization, shoot-tips consisting of the meristem and five to eight leaf primordia were dissected. The shoot-tips were then cultured on MS medium. If it is necessary to recover flowering plants more quickly, micrografting of cryopreserved winter buds is feasible. Excised buds with vascular tissue were directly grafted onto 1-year-old seedlings. After grafting, the top of the buds turned green in a few days, and the buds were unfolded in 2 weeks, and formed shoots in 2 months. It is very important that cryopreserved shoot-tips are capable of maintaining viability during storage. Figure 1 show s the changes in shoot formation rates of mulberry winter buds stored at –135°C for 1–8 years. No significant changes were observed after 8 years' storage. Another point of successful cryopreservation is that this method can be adapted for many varieties of mulberries. The shoot formation rate of 376 varieties cryopreserved for 5 years in a deep-freezer at –135°C was tested. Out of this total number, 279 (about 74%) achieved more than 50% shoot formation rates. Only 24 varieties (about 6%) gave low rates of less than 30% shoot formation. In the case of the varieties showing low survival, we obtained at least one regenerated shoot (Niino et al. 1995). These results clearly show that cryopreservation using winter buds is a reliable and safe method for long-term storage. Up to now , these methods have been successfully applied to winter buds of deciduous woody trees, such as apple (Tyler and Stushnoff 1998a, 1998b), pear (Suzuki et al. 1997), blueberry and raspberry (Niino et al. 1990).

Cryopreservation of in vitro-grown mulberry shoot-tips by vitrification For in vitro cultures, the non-hardy plants, the low-propagation plants and the plants that can not use winter bud, another cryopreservation method was required. The cryopreservation protocol of in vitro-grown mulberry shoot-tips by vitrification was established by Niino et al. (1992a). The procedure consists of cold- hardening of the shoot-tips, preculture, treatment by vitrification solution (PVS2: Sakai et al. 1990) and immersion in LN. For plant recovery, vitrified shoot-tips were rapidly rewarmed in water, washed, plated on the medium and recultured. An important parameter to determine is the optimum duration of treatment with PVS2. In mulberry, the shoot formation rate of vitrified shoot-tips gradually increased with increasing time of exposure to PVS2 and reached a maximum at about 90 min exposure. With this procedure, cold-hardening and preculture are crucial to obtain high recovery rates of mulberry shoot-tips cryopreserved using vitrification. Also, the effect of storage duration on survival of the in vitro-grown shoot-tips cooled to LN by vitrification was tested. There was no decrease in the shoot-formation rate after 3 years of storage in LN. 196 Cryopreservation of Tropical Plant Germplasm

Fig. 1. Shoot-formation rate of mulberry winter buds (variety Kenmochi) stored for 1–8 years in a deep-freezer at –135°C. Approximately 20 winter buds were tested for each of two replicates.

The successfully vitrified shoot-tips of mulberry resumed growth in 1 week after reculture and started to develop shoots within 3–4 weeks without intermediary callus formation. After growing and rooting the shoots, plantlets were transferred to pots and the field. There were no morphological abnormalities in the plants developed from the cryopreserved shoot-tips. A total of 11 mulberry cultivars (Niino et al. 1992a), 6 apple cultivars (Niino et al. 1992b), 8 pear cultivars (Niino et al. 1992b) and 8 Prunus cultivars (Niino et al. 1997a) were successfully cryopreserved by this vitrification method. Notably, seven non-hardy mulberry cultivars from Southeast Asia showed moderate survival rates (Niino et al. 1992a). Also, in vitro shoot-tips of yacon and Jerusalem artichoke (Niino et al. 1997b) which had tuberous roots and tubers, and multiple bud bodies of three cultivars of mulberry (Oka and Niino 1997) were successfully cryopreserved by vitrification. However, average survival rates are around 65% and therefore we should improve the technique to obtain higher survival rates. The incubation period in PVS2 appears to be species-specific. The duration of incubation in PVS2 might necessarily increase in line with the size of the shoot-tips. We usually use shoot- tips of 2–3 mm in length, because we believe that relatively large shoot-tips cooled to –196°C are able to survive and regrow more easily and faster than smaller ones. Also, we can use shoot-tips regardless of the age or stage of shoots after the last subculture.

Cryopreservation of in vitro-grown shoot-tips of chrysanthemum It is important to use uniform materials for cryopreservation to obtain a higher survival rate. In vitro-grown shoots of chrysanthemum were used in this study. The procedure for cryopreservation of in vitro-grown shoot-tips of Cryopreservation techniques 197 chrysanthemum is the same procedure as for the mulberry except for an additional treatment with a loading solution consisting of liquid MS medium supplemented with 2M glycerol and 0.4M sucrose (Matsumoto et al. 1994). After 40 days of cold-hardening at 5°C, apical shoot-tips were cryopreserved according to the procedure. The survival rate was about 60%. So we tried to use shoots of the same size and the same stage shoots. For preparing shoots at similar stages of development, immature lateral buds were used. Shoots 3–5 mm long with one bud were cut and cultured on MS medium containing 0.1M sucrose at 5°C for up to 42 days followed by 3 days incubation at 25°C. When these immature lateral buds were cultured at 25°C for 3 days, a small bud came out at the base of a former leaf. After incubation at 5°C for 24 days, the first leaf grew and unfolded. The 2nd, 3rd and 4th leaves also unfolded after 30, 36 and 42 days incubation, respectively. Elongation of the shoot started just after the second leaf unfolded. The effect of the growth stages of shoot on survival rates is shown in Table 1. The highest survival rate of cryopreserved shoot-tips was obtained for shoots with two unfolded leaves. In the case of shoots with more than three unfolded leaves, survival rates abruptly decreased. This result shows that with chrysanthemum, the use of shoots at the same growing stage, especially at the two-unfolded-leaf stage, is necessary for obtaining a high survival rate.

Table 1. Effect of growth stage on the survival (% ±SD) of chrysanthemum shoot-tips cooled to –196°C by vitrification, following preconditioning at 5°C for various durations Duration of Growth preconditioning at Survival stage no. Growth stage of shoots 5°C (d)† (% ±SE)‡ S Early bud 0 2.5±1.3 0 Swollen bud 12 58.8±3.7 1 One unfolded leaf 24 82.5±2.8 2 Two unfolded leaves 30 95.0±1.8 3 Three unfolded leaves and 36 65.0±5.6 shoot elongation 4 More than four unfolded 42 38.8±2.1 leaves and shoot elongation † Lateral buds were cut and put on MS medium containing 0.1M sucrose and cultured at 25°C for 3 d before transfer at 5°C for conditioning. ‡ After conditioning for various durations, shoot-tips were precultured on MS medium containing 0.3M sucrose for 3 d before cryopreservation. Twenty shoot-tips were tested for each of the four replicates. Shoot-tips were treated with the loading solution for 20 min at 25°C, then with PVS2 vitrification solution for 20 min at 25°C.

However, 5°C preconditioning is time-consuming, so preconditioning at other temperatures was tested. Table 2 shows the effect of preconditioning temperatures and growth stage of shoots on survival rate. The survival rate of cryopreserved shoot-tips reached 98% when shoot-tips at the two unfolded leaf stage and preconditioning at 10°C were used. The incubation duration at 10°C from 198 Cryopreservation of Tropical Plant Germplasm

Table 2. Effect of preconditioning temperature and growth stage on the survival (% ±SD) of chrysanthemum shoot-tips cooled to –196°C by vitrification Preconditioning temperature (°C) Growth 10 15 25 stage Duration Survival Duration Survival Duration Survival no. (d) (%) (d) (%) (d) (%) 1 16 85.0±2.4 11 71.7±3.6 5 50.0±12.5 2 21 98.3±1.4 14 80.0±2.4 7 63.3±5.4 3 26 76.7±3.6 17 65.0±2.4 9 33.3±9.8 4 30 68.3±3.6 21 25.0±8.5 12 20.0±4.7 Shoot-tips were precultured on MS medium containing 0.3M sucrose for 3 d. Twenty shoot-tips were tested for each of the three replicates. Shoot-tips were treated with the loading solution for 20 min at 25°C, then with PVS2 vitrification solution for 20 min at 25°C. the start of shoot preconditioning to the stage of the two unfolded leaves was 21 days. For the other preconditioning temperatures tested (15°C and 25°C), the same growing stage displayed high survival rates compared with the other growing stages. These results indicate that shoot-tips at the two unfolded leaf stage are suitable materials for cryopreservation, regardless of the preconditioning temperature. Even shoot-tips at 15 or 25°C, which were growing vigorously, displayed a moderate survival rate at the two-unfolded-leaf stage. This meant that successful cryopreservation does not always depend on the preconditioning temperature. The reason for this result is not clear yet, and we need more detailed examinations from many aspects. We have developed a successful cryopreservation procedure for in vitro- grown shoot-tips of chrysanthemum. We are now going to study which processes or which factors cause a decrease in the survival rates of cryopreserved shoot-tips.

Conclusion The preservation of the valuable germplasm of cultivated crops and their wild relatives is imperative for the maintenance of a broad genetic base in order to meet future demands. Maintaining vegetatively propagated crops has become very important now. However, we have to consider other ways in order to complement the field genebank. During the last decade, there has been increased interest in mass propagation, and biotechnology of plants and cryopreservation of embryogenic callus, meristems, multiple shoots, adventitious buds and somatic embryos has become important. The use of in vitro conservation techniques, including cryopreservation and slow growth conservation, represents an attractive option for the long- and medium-term conservation of genetic resources of vegetatively propagated crops. Some people doubt that long-term preservation is possible at super-low temperature. We have clearly demonstrated and confirmed that mulberry winter buds can be cryopreserved in a deep-freezer at –135°C for 8 years. In addition, in vitro shoot-tips Cryopreservation techniques 199 of mulberry cryopreserved in LN for 3 years could be regenerated into plantlets after plating on culture medium. No morphological abnormalities were observed in plants developed from the cryopreserved shoot-tips. Cryopreservation of vegetative winter buds is suitable for germplasm preservation of woody plants. The vitrification methods also appear to be suitable for routine cryopreservation of in vitro-grown shoot-tips of plants when an in vitro system is developed. Further technical improvements, systematic observation and large-scale experiments may contribute to the wider applicability of cryogenic techniques to various species. We recommend that all the genetic resources of vegetatively propagated crops be stored by cryopreservation to complement the field genebanks. The establishment of a national cryogenebank in Japan is expected for the germplasm of vegetatively propagated crops, of plants with recalcitrant seeds and of valuable culture materials.

References Matsumoto, T., A. Sakai and K. Yamada. 1994. Cryopreservation of in vitro-grown apical meristems of wasabi (Wasabia japonica ) by vitrification and subsequent high plant regeneration. Plant Cell Reports 13:442–446. Niino, T, K. Shirata and S. Oka. 1995. Viability of mulberry winter buds cryopreserved for 5 years at –135°C. Journal of Sericicultural Science of Japan 64:370–374. Niino, T., A. Sakai, H. Yakuwa and K. Nojiri. 1992b. Cryopreservation of in vitro-grown shoot tips of apple and pear by vitrification. Plant Cell, Tissue and Organ Culture 28: 261–266. Niino, T., A. Sakai, S. Enomoto, J. Magoshi and S. Kato. 1992a. Cryopreservation of in vitro-grown shoot tips of mulberry by vitrification. Cryo–Letters 13:303–312. Niino, T., H. Yakuwa, S. Oka and A. Ishihara. 1990. Survival and shoot formation in vitro in raspberry and blueberry winter buds cryopreserved in liquid nitrogen. Pp. 78–79 in Proceedings of the 2nd Plant Tissue Culture Colloquium of the Japanese Plant Tissue Cul ture Association. 6–7 Aug. 1990, Hananomaki, Japan. Committee of the Japan Association for Plant Tissue Culture. Niino, T., K. Tashiro, M. Suzuki, O. Susumu, J. Magoshi and T. Akihama. 1997a. Cryopreservation of in vitro grown shoot tips of cherry and sweet cherry by one-step vitrification. Scientia Horticulturae 70:155–163. Niino, T., T. Murayama and M. Suzuki. 1997b. Cryopreservation of in vitro-grown shoot tips of yacon and Jerusalem artichoke by vitrification. Pp. 258–259 in Proceedings of 8th SABRAO Congress, 24–28 Sept. 1997, Seoul, Korea. Oka, S. and T. Niino. 1997. Preservation of multiple bud bodies of mulberry (Morus alba L.) under low and ultra-low temperatures. Journal of Sericicultural Science of Japan 66:467–472. Sakai, A., S. Kobayashi and I. Oiyama . 1990. Cryopreservation of nucellar cells of nable orange (Citrus sinensis Osb. var. brasiliensis Tanaka) by vitrification. Plant Cell Reports 9:30–33. Suzuki, M., T. Niino, T. Akihama and S. Oka. 1997. Improved procedures for the cryopreservation of shoot tips of pear winter buds by extra-organ freezing step. Journal of Japan Society of Horticultural Science 66:29–34. Tyler, N. and C. Stushnoff. 1988a. The effect of prefreezing and controlled dehydration on cryopreservation of dormant vegetative apple buds. Canadian Journal of Plant Science 68:1163–1167. Tyler, N. and C. Stushnoff. 1988b. Dehydration of dormant apple buds at different stages of cold acclimation to induce cryopreservation of clonal materials. Canadian Journal of Plant Science 68:1169–1176. 200 Cryopreservation of Tropical Plant Germplasm

Genotype considerations in temperate fruit crop cryopreservation Barbara M. Reed USDA-ARS National Clonal Germplasm Repository, Corvallis, OR 97333-2521 USA

Introduction When we look for success in plant germplasm cryopreservation, genotype must certainly be taken into account. One of the main problems when freezing apical meristems is variability between as well as within genotypes. Variability between replications may be due to physiological condition (Dereuddre et al. 1988; Chang and Reed 1995). The growth response of plants in culture varies over time in the growth cycle. Healthy, vigorous plants in an exponential stage of growth respond differently than those in either earlier or later stages. Timing of experiments so the plants are always in the same stage may decrease variability between experiments. Such factors as growth habit and general morphology of a plant apical meristem may affect its cold tolerance and the ease of dissection. Cold-hardiness is genetically conferred and is probably due to several factors. Dehydration tolerance may affect cold tolerance, and membrane composition has been shown to influence recovery from freezing.

Genetic variability We have noted genetic variability in many experiments in our laboratory. All of the examples below involve slow freezing techniques. The first involves response to freezing rate by four pear genotypes (Reed 1990). Non-hardened plants of Pyrus communis L. cv. Beurre Hardy had moderate survival at 0.3°C and P. cossonii Rehder survived at low rates with the slowest freezing rate while P. koehnei C. Schneider and P. species (faurei C. Schneider x hondoensis Kikuchi & Nakai) had very low or no survival at any rate (Table 1). Following cold-hardening for 1 week, all survival was above 50% and three were 75–95%. In this case similar environmental and freezing rate treatments were used to overcome vast differences in genotypic response. Another example is for recovery media and Rubus (Table 2). For half of the genotypes there was no significant difference between the two media tested; however, for the remaining genotypes recovery on MS medium (Murashige and Skoog 1962) was double that of Anderson's (Anderson 1980). Recovery medium is often important whether meristems or cell cultures are involved (Reed 1993). Development of methods can of course only be done on a few genotypes at a time. However it is very important to know how a diverse germplasm collection will respond if storage is a goal. For germplasm storage we need to retain a relatively high survival rate so as not to preselect as we store. If we look at 40% survival as a guideline we find 60% of pears with regrowth suitable for storage. Cryopreservation techniques 201

Table 1. Survival and shoot production from apical meristems of Pyrus spp. after cooling at four controlled freezing rates and immersion in liquid nitrogen, following a 1-week pretreatment of cold acclimatization or growth at 25°C Cooling rate Meristem survival (% ± SEM of shoot production) (°C/min) P. communis P. hybrid P. koehnei P. cossonii Pregrowth at 25°C 0.1 23 ± 12 (100) 8 ± 5 (100) 0 26 ± 0 (100) 0.3 51 ± 21 (88) 10 ± 3 (75) 0 3 ± 3 (100) 0.5 3 ± 2 (100) 10 ± 7 (100) 3 ± 2 (100) 13 ± 5 (100) 0.8 0 0 0 5 ± 3 (100) Cold-acclimatized 0.1 95 ± 1 (81) 55 ± 15 (100) 75 ± 0 (100) 95 ± 0 (100) 0.3 82 ± 2 (91) 56 ± 15 (64) 31 ± 7 (100) 33 ± 2 (100) 0.5 27 ± 7 (96) 7 ± 3 (100) 5 ± 2 (100) 12 ± 3 (100) 0.8 10 ± 1 (100) 7 ± 1 (35) 5 ± 0 (50) 5 ± 0 (100)

Table 2. Survival following cryopreservation of meristems of Rubus species and cultivars grown for 4 weeks on two types of recovery media Percent survival † Genotype Anderson Murashige and Skoog Hillemeyer 48.2 ± 3.3 a 56.2 ± 9.8 a Kotata 26.7 ± 7.3 b 62.1 ± 3.1 a Mandarin 10.0 ± 5.0 b 33.3 ± 15.6 a ORUS 1362 16.7 ± 10 a 33.3 ± 25.4 a Silvan 24.7 ± 14 b 65.4 ± 4.8 a R. hirsutus Thunb. 3.3 ± 1.7 a 10.7 ± 3.2 a † n=60 Means in rows followed by the same letter are not significantly different (P< 0.05) (Reed 1993).

We see a very different distribution of survival among 53 Fragaria (strawberry) genotypes using a technique developed with five genotypes. For this genus we see a greater distribution at the low end of the range. Nearly 60% had low survival (<50%) while only 40% produced high recovery rates. The difference between these two genera illustrates some of the difficulties we are facing in germplasm storage. To this point, cold-hardy plants are easier to store than non-hardy plants. The group of Fragaria genotypes surveyed here includes cultivars developed for Canada where cold-hardiness is important and for California where cold-hardiness is not part of the genetic make-up.

Variability in techniques We have looked at the genotypic variability inherent in the system, now let us look at the techniques available. Slow freezing has been used for over 20 years and many different variations exist. Vitrification techniques are now widely used. Alginate encapsulation is also a useful technique which is sometimes combined with vitrification or slow freezing. Slow freezing requires pregrowth of meristems on medium with DMSO or with high osmotic potential followed by slow dehydration in mild cryoprotectants. 202 Cryopreservation of Tropical Plant Germplasm

Freezing is usually less than 1 degree per minute to –35°C or –40°C followed by a plunge in LN. Crystallization occurs in the intercellular spaces and the cytoplasm probably vitrifies. Rapid thawing is necessary for survival. Growth of surviving meristems is noticeable in 3–4 weeks. Advantages are that there is good survival in many genotypes and it is often easy to slightly modify the technique to increase survival rates. One of the main drawbacks is the need for a controlled-rate freezer. This equipment is expensive but necessary for consistency between trials. Vitrification often utilizes pregrowth dehydration or conditioning. Dehydration is accomplished with vitrification solutions which are highly concentrated and often toxic to the plant material. Ethylene glycol, glycerol and DMSO combined with high concentrations of sucrose are commonly used. These solutions do not crystallize when quenched in LN. Fast thawing is required for high survival. Recovery of frozen meristems can be seen in 1 to 2 weeks. There are many different vitrification techniques being developed at this time. The advantage of vitrification is that little equipment is needed for the cryopreservation process, but one disadvantage of this technique is the toxicity of the cryoprotectants and the need to move materials through the system quickly. This toxicity may dictate that only a few vials be processed at one time. Alginate encapsulation encloses the meristem in an alginate bead which provides protected dehydration. Plantlets are cold-hardened in advance, the meristems are excised, encapsulated and dehydrated overnight in a concentrated sucrose solution. Additional dehydration is obtained by air-drying in the laminar flow hood. After drying, beads are transferred to a cryovial and quenched in LN. Slow thawing is at room temperature for 15 minutes. New growth is apparent at 1-2 weeks after thawing. Encapsulation allows the meristem to be protected as it is dehydrated. The meristems are mixed with the alginate solution and dropped from the pipette into a high-calcium solution to form the beads. One problem is the amount of handling required. Each bead must be moved individually four or five times so it is very labour-intensive. Often the meristem must be removed from the bead for continued development to take place. This is a very useful system for less hardy materials.

Comparing methods and genotypes As these new systems have developed, we were curious about how they compared. Since each technique was developed on a different genus, we looked at the effect of the three techniques on several genotypes of one genus. The first step was to study each technique and determine the procedures to be used. The genotypes used were red and black currants and gooseberries. All were cold-hardened for 1 week before excision of meristems. The vitrification method was that of Yamada (Yamada et al. 1991), the alginate beads of Dereuddre (Dereuddre et al. 1990) and slow freezing of Reed (Reed 1988). Slow freezing protocols can be modified by altering the freezing rate or the precooling temperature. In this case we compared two freezing rates. In most cases 0.3°C/min was the most successful; however, differences were not significant for Ribes species. In all cases survival was over 50%. For the vitrification technique the Cryopreservation techniques 203 pretreatment and the amount of exposure to the cryoprotectants were important. Pretreatment in DMSO was generally more successful than sorbitol. A 20-min exposure to the cryoprotectant PVS2 was found to be optimal. The length of dehydration in the laminar flow hood was compared for the alginate bead method. A 3-h dehydration produced higher survival than did 2 h. Ribes diacantha Pall. results were not significantly different for the two time periods. As a final comparison of the three techniques, the best trial from each technique was compared. Survival of individual genotypes varied with the technique used.

Table 3. Regrowth of Ribes meristems following cryopreservation by three techniques: Slow freezing at 0.3°C/min, vitrification in PVS2, or encapsulation-dehydration in alginate beads dried for 4 h in the laminar flow hood % Regrowth of meristems Genotype Type Origin Slow Vitrify Alginate R. sativum cv. Cherry Red Italy 80 6 24 Currant R. diacantha Gooseberry USSR 88 57 48 R. species Gooseberry Idaho 65 49 90 R. odoratum Black currant Kansas 50 31 61

Each cryopreservation technique used has distinct advantages and disadvantages. Slow freezing is effective for many taxa but requires expensive equipment. This would limit its use in some laboratories. Vitrification is quick; however, the solutions tend to be toxic to the plants and careful timing is necessary for successful recovery. Alginate beads are successful for many genera but require more handling and are thus more time-consuming and tedious than the other methods.

References Anderson, W.C. 1980. Tissue Culture Propagation of Red Raspberries. Pp. 27–34 in Proceedings of the conference on nursery production of fruit plants through tissue culture- Application and feasibility. U.S. Department of Agriculture, Science and Education Administration, Agricultural Research Results ARR–NE–11, December 1980. Chang, Y. and B.M. Reed. 1995. Improved shoot formation of blackberry and raspberry meristems following cryopreservation. Cryobiology 32:581. Dereuddre, J., J. Fabre and C. Bassaglia. 1988. Resistance to freezing in liquid nitrogen of carnation (Dianthus caryophyllus L. Var eolo) apical and axillary shoot tips excised from different aged in vitro plantlets. Plant Cell Reports 7:170–173. Dereuddre, J., C. Scottez, Y. Arnaud and M. Duron. 1990. Resistance of alginate-coated axillary shoot tips of pear tree (Pyrus communis L. cv. Beurré Hardy) in vitro plantlets to dehydration and subsequent freezing in liquid nitrogen. Comptes Rendus de l’Académie des Sciences Paris 310:317–323. Luo, J. and B.M. Reed. 1997. Abscisic acid-responsive protein, bovine serum albumin, and proline pretreatments improve recovery of in vitro currant shoot-tip meristems and callus cryopreserved by vitrification. Cryobiology 34:240–250. 204 Cryopreservation of Tropical Plant Germplasm

Murashige, T. and F. Skoog. 1962. A revised medium for rapid growth and bio assays with tobacco tissue cultures. Physiologia Plantarum 15:473–497. Reed, B.M. 1988. Cold acclimation as a method to improve survival of cryopreserved Rubus meristems. Cryo–Letters 9:166–171. Reed, B.M. 1990. Survival of in vitro-grown apical meristems of Pyrus following cryopreservation. HortScience 25:111–113. Reed, B.M. 1993. Responses to ABA and cold acclimation are genotype dependent for cryopreserved blackberry and raspberry meristems. Cryobiology 30:179–184. Reed, B.M. and X. Yu. 1995. Cryopreservation of in vitro-grown gooseberry and currant meristems. Cryo–Letters 16:131–136. Yamada, T., A. Sakai, T. Matsumura and S. Higuchi. 1991. Cryopreservation of apical meristems of white clover (Trifolium repens L.) by vitrification. Plant Science 78:81–87. Cryopreservation techniques 205

Cryopreservation of in vitro-grown meristems of potato (Solanum tuberosum L.) by encapsulation-vitrification Dai Hirai¹ and Akira Sakai² ¹ Hokkaido Prefectural Plant Genetic Resources Center, Hokkaido, 073-0013, Japan ² 1-5-23, Asabu-cho, Kita-ku, Sapporo, Hokkaido, 001-0045, Japan

Introduction Maintenance of potato germplasm in the field is a major consumer of time, manpower and space aside from diseases and environmental stresses. Conservation of in vitro-cultured potato also entails high maintenance costs, risks of somaclonal variation and genetic instability, especially when growth retardants are used (Harding 1991). Thus, cryopreservation appears to be a logical choice for long-term storage of potato germplasm, with minimum space and maintenance requirements without genetic instability (Bajaj 1978, 1985; Harding and Benson 1994). However, the conventional slow freezing method, which is very complicated and time-consuming, produced low rates of shoot formation and growth recovery took place through callusing (Towill 1983, 1984; Benson et al. 1989). For long-term conservation, the availability and development of safe and cost-effective techniques and subsequent high plant regeneration are the basic requirements. Recently, some simplified and reliable cryogenic procedures such as vitrification, encapsulation-drying and encapsulation- vitrification have been developed and the number of species or cultivars that have been cryopreserved has sharply increased (Sakai 1997). We found that encapsulated potato meristems that were osmoprotected and then dehydrated with PVS2 solution at 0°C prior to a plunge into LN produced high levels of shoot formation without intermediary callus formation. Thus, we report an effective cryogenic procedure, the encapsulation-vitrification method, for long-term conservation of potato germplasm using meristems.

Materials and methods

Plant materials Potato (Solanum tuberosum L.) cv. Danshakuimo was mainly used. Apical buds of plantlets were subcultured every 10 days (each plantlet had 3 to 4 nodes and apical bud) on MS medium (Murashige and Skoog 1962) with 0.5 g/L casamino acids, 30 g/L sucrose, 2.5 g/L gellan gum at 23°C under 16-h light/8-h dark photoperiod under a light intensity of 96 µmol m-2 s-1 . This medium was used as a basal medium. Nodal segments were transferred on basal medium in plastic dishes (90 x 20 mm) and cultured to induce axillary buds. These segments were cold-hardened at 4°C for 3 weeks under a 12-h light/12-h dark photoperiod with a light intensity of 20 µmol m-2 s-1 . Non-hardened nodal segments were cultured for 2 to 10 days under the previous conditions. Then, axillary meristems with five leaf primordia (about 1 mm size) were excised for experiments. The excised meristems were precultured on basal medium supplemented with 0.3M sucrose, 206 Cryopreservation of Tropical Plant Germplasm

1 mg/L GA 3 (gibberellic acid), 0.01 mg/L BAP (6–benzylaminopurine) and 0.001 mg/L NAA (1–naphthaleneacetic acid) at 23°C for 16 h. Eleven other varieties were also used in this study.

Encapsulation, osmoprotection and dilution Precultured meristems with or without cold-hardening were encapsulated in 2% Na–alginate gel beads (Hirai et al. 1998) and then osmoprotected with MS medium (the same plant hormones as the preculture medium) supplemented with 0.4–1.6M sucrose ± 2M glycerol (LS solution) for 90 min at 25°C (50 rpm on a rotary shaker) before dehydration with the PVS2 solution. Encapsulated dehydrated meristems were placed in a 1.8-ml cryotube with 1 ml PVS2 solution, then plunged into LN (liquid nitrogen). The cryotubes were rapidly rewarmed in a water-bath at 38°C and the PVS2 solution was drained from the cryotubes and replaced twice with 1 ml of 1.2M sucrose solution and held for 10 min.

Encapsulation-dehydration procedure Meristems with three leaf primodia (about 0.5 mm size) were used for this experiment. Encapsulated meristems were treated in MS medium with 0.75M sucrose for 48 h at 90 rpm on a rotary shaker at 25°C. They were subjected to air- drying for 3.5 h in Petri dishes containing 50 g of dried silica gel. The water content of beads after air-drying was about 23% (FW basis). Dried, encapsulated meristems were placed in a 1.8-ml cryotube and directly plunged into LN and held for at least 1 h. Cryotubes were rewarmed in a water-bath at 38°C for 3 min.

Viability and plant regrowth The cryopreserved meristems were plated on basal medium in plastic dishes with the same plant hormones as the preculture medium and cultured for 1 d (encapsulation-vitrification) or 7 d (encapsulation-dehydration) under the conditions described before. Then they were transpla nted on basal medium supplemented with 0.0005 mg/L GA3. The rate of shoot formation was expressed as a percentage of the total number of meristems forming normal shoots 2 weeks after plating. In every experiment approximately 10 meristems were treated for each of three replicates.

RAPD analysis Genomic DNA was isolated using an ISOPLANT kit (Nippon Gene, Tokyo, Japan) from cryopreserved and non-treated (control) plantlets. DNA amplifications using 17 primers of 10 bases each (Operon technologies, CA, USA) were performed according to Williams et al. (1991). Differential bands were detected by staining with SYBR Green I nucleic acid gel-stain. Cryopreservation techniques 207

Results In preliminary experiments, little or no differences were observed in cryopreservability among the nodal segments (1st to 3rd rank from the top of the plantlets) and the days of culture of nodal segments (2 to 10 d). The optimal duration of exposure to PVS2 solution was 3 h for both non- hardened and cold-hardened meristems. Cold-hardened and non-hardened meristems treated with PVS2 solution for up to 4 h without cooling (treated control) retained high levels of shoot formation. However, after 4 h, shoot formation decreased in line with increasing exposure time. The effects of cold-hardening and osmoprotectant on shoot formation of encapsulated vitrified meristems cooled to –196°C were investigated. As shown in Figure 1, non-hardened meristems osmoprotected with a mixture of 2M glycerol plus 0.6M sucrose for 90 min before dehydration with PVS2 solution produced 70.0% of shoot formation, while the percentage of shoot formation of cold-hardened meristems was 62.8%. When osmoprotected with sucrose alone, the meristems with or without cold-hardening produced much lower rates of shoot formation than those treated with 2M glycerol in combination with any concentration of sucrose. Successfully encapsulated vitrified meristems cooled to –196°C resumed growth within 3 days and developed shoots and leaves within 10 days after plating without intermediary callus formation. All these shoots formed roots on basal MS medium and successfully produced microtubers. No morphological abnormalities were observed during the regrowth period and no differences in RAPD analysis were detected between cryopreserved and control plants for 17 primers used in this study. The encapsulation-vitrification protocol established in the present study was successfully applied to 12 other varieties tested (Fig. 2). The rate of shoot formation and fresh weight of plants was compared for two different cryogenic procedures (Fig. 2). The encapsulated vitrified meristems gave much higher shoot formation and much faster growth than the encapsulated dried meristems in all varieties.

Discussion In the vitrification procedure, meristems should be sufficiently dehydrated osmotically by exposure to a highly concentrated vitrification solution (PVS2). However, the exposure of meristems to the PVS2 solution without osmoprotection causes harmful osmotic stress. Thus, the key of success for cryopreservation by vitrification is to precondition meristems to induce osmotolerance to PVS2. In the present study, this limitation was almost overcome by preculturing meristems with 0.3M sucrose for 16 h, followed by osmoprotection with a mixture of 2M glycerol plus 0.6M sucrose for 90 min. However, without glycerol, no concentration of sucrose increased the osmotolerance of potato meristems to the PVS2 solution. Recently, the LS solution (a mixture of 2M glycerol plus 0.4M sucrose) was reported to be very effective in increasing osmotolerance to PVS2 solution of taro (Takagi et al. 1997), banana, orchid and pineapple (Thinh 1997), and strawberry (Hirai et al. 1998). For 208 Cryopreservation of Tropical Plant Germplasm potato meristems, 0.6M sucrose in combination with 2M glycerol was needed to significantly enhance osmotolerance. The cells of meristems which were immersed in a mixture of 0.6M sucrose plus 2M glycerol were osmotically dehydrated and plasmolyzed to a considerable extent for 90 min. These cells were then successively dehydrated with PVS2 solution. Under these conditions, the apical meristems remained osmotically concentrated and the increase in the cytosolic concentration required for vitrification was attained by osmotic dehydration. In the vitrification procedure, plasmolysis may play an important role in dehydration by mitigating its injurious effects (Steponkus et al. 1992; Jitsuyama et al. 1997; Matsumoto et al. 1998). It is particularly important that cryopreserved meristems directly produce plants identical to the non-treated controls (Haskins et al. 1980; Kartha et al. 1980; Towill 1984). In the present study, successfully vitrified and rewarmed meristems vigorously developed shoots directly within a week after plating. No morphological abnormalities and callus formation were observed during recovery growth. The same results were reported in many plants. Benson et al. (1996) also reported that vitrification and encapsulation dehydration exceeded the conventional freezing method in terms of shoot formation and recovery growth in Ribes. Thus, the vitrification method with or without encapsulation certainly offers a considerable advantage over slow freezing method.

Fig. 1. Effect of osmoprotectant(s) and cold-hardening on the rate of shoot formation of encapsulated vitrified meristems cooled to –196°C. Material: cultivar Danshakuimo. Cold-hardened (right side) and non-hardened (left side) meristems were encapsulated in alginate beads, osmoprotected at 25°C for 90 min and treated with PVS2 solution at 0°C for 3 h prior to a plunge into LN. Approximately 10 meristems were used for each of three replicates. Bars represent the standard errors. Cryopreservation techniques 209

Fig. 2. Rate of the shoot formation (o, n) and recovery growth (m, l) of encapsulated vitrified or encapsulated dried meristems of 13 varieties cooled to –196°C. Encapsulation-vitrification (o, m) : Encapsulated meristems in alginate gel beads were osmoprotected with a mixture of 0.6M sucrose plus 2M glycerol in MS medium at 25°C for 90 min and dehydrated with PVS2 at 0°C for 3 h prior to a plunge into LN. Encapsulation-drying (n, l): Encapsulated meristems in alginate gel beads were suspended at 90 rpm in MS liquid media with 0.75M sucrose for 48 h at 25°C before air- drying in silica gel for 3.5 h prior to a plunge into LN. Approximately 10 meristems were treated for each of three replicates. Bars represent standard errors. 210 Cryopreservation of Tropical Plant Germplasm

Harding (1996) reported that PCR and RAPD technology are useful for the detection of genetic changes and the assessment of the genetic stability of plants recovered from in vitro cultures. Within the primers used in this study, no differences were observed in RAPD analysis between cryopreserved and non- preserved (control) plantlets. However, further study is necessary to confirm their genetic stability by other analyses. The encapsulation-dehydration method has been successfully applied to a wide range of materials. However, the problems are a lower rate of shoot formation and later recovery growth compared with the meristems cryopreserved by vitrification (Matsumoto et al. 1995; Hirai et al. 1998). In this study, the encapsulation-dehydration method also showed the same tendency. Thus, the treatment to induce dehydration tolerance before air-drying appears to be insufficient to produce a higher level of recovery growth. The encapsulation-vitrification method is easy to handle and can be used with a large number of meristems at the same time. Besides, regrowth is achieved much earlier than with the encapsulation-dehydration method. Thus, the encapsulation-vitrification method appears to be a potentially valuable cryogenic protocol for large-scale cryopreservation of potato germplasm.

Acknowledgements The authors wish to thank Ms Noriko Otsuki for her technical assistance and Mr Takeshi Itoh, Kitami A.E.S., Mr Akihiro Arihara, Hokuren A.R.I. and Dr Takeshi Matsumura, Hokkaido Green-bio institute for supplying potato varieties. We also thank Mr Tadahiko Kiguchi, Chuo A.E.S., for teaching the principles and technique of PCR and RAPD analysis. I am especially grateful to Dr Hiroko Takagi-Watanabe, JIRCAS, for giving the opportunity to present this article.

References Bajaj, Y.P.S. 1978. Tuberization in potato plants regenerated from freeze-preserved meristems. Crop Improvement 5:137–141. Bajaj, Y.P.S. 1985. Cryopreservation of germplasm of potato (Solanum tuberosum L.) and cassava (Manihot esculenta Crantz): viability of excised meristems cryopreserved up to four years. Indian Journal of Experimental Biology 23:285–287. Benson, E.E., K. Harding and H. Smith. 1989. Variation in recovery of cryopreserved shoot-tips of Solanum tuberosum exposed to different pre- and post-freeze light regimes. Cryo–Letters 10:323–344. Benson, EE., B.M. Reed, R.M. Brennam, K.A. Clacher and D.A. Ross. 1996. Use of thermal analysis in the evaluation of cryopreservation protocols for Ribes nigrum L. germplasm. Cryo–Letters 17:347–362. Haskins, R.H. and K.K. Kartha. 1980. Freeze preservation of pea meristems: cell survival. Canadian Journal of Botany 58:833–840. Harding, K. 1991. Molecular stability of the ribosomal RNA genes in Solanum tuberosum plants recovered from slow growth and cryopreservation. Euphytica 55:141–146. Harding, K. 1996. Approaches to assess the genetic stability of plants recovered from in vitro culture. Pp. 135–168 in In-vitro conservation of plant genetic resources (M.N. Normah, M.K. Narimah and M.M. Clyde eds.). University Kebangsaan, Malaysia. Cryopreservation techniques 211

Harding, K. and E.E. Benson. 1994. A study of growth, flowering, and tuberization in plants derived from cryopreserved potato shoot-tips: Implications for in vitro germplasm collections. Cryo–Letters 15: 59–66. Hirai, D., K. Shirai, S. Shirai and A. Sakai. 1998. Cryopreservation of in vitro-grown meristems of strawberry (Fragaria x ananassa Duch.) by encapsulation vitrification. Euphytica 101:109–115. Jitsuyama, Y., T. Suzuki, T. Harada and S. Fujikawa. 1997. Ultrastructural study on mechanism of increased freezing tolerance due to extracellular glucose in cabbage leaf cells. Cryo–Letters 18:33–44. Kartha, K.K., L. Leung and K. Pahl. 1980. Cryopreservation of strawberry meristems and mass propagation of plantlets. Journal of the American Society of Horticultural Science 105:481–484. Matsumoto, T., A. Sakai and K. Yamada. 1995. Cryopreservation in vitro-grown apical meristems of lily by vitrification. Plant Cell, Tissue and Organ Culture 41: 237–241. Matsumoto, T., A. Sakai and Y. Nako. 1998. A novel preculturing for enhancing the survival of in vitro-grown meristems of wasabi (Wasabia japonica) cooled to –196°C by vitrification. Cryo–Letters 19:27–36. Murashige, T. and F. Skoog. 1962. A revised medium for rapid growth and bioassay with tobacco tissue culture. Physiologia Plantarum 15:473–497. Sakai, A. 1997. Potentially valuable cryogenic procedures for cryopreservation of cultured plant meristems. Pp. 53–66 in Conservation of Plant Genetic Resources In Vitro. Volume 1: General Aspects. M.K. Razdan and E.C. Cocking (eds.). Science Publishers Inc., Enfield, USA. Steponkus, P.L., R. Langis and S. Fujikawa. 1992. Cryopreservation of plant tissues by vitrification. Pp. 1–61 in Advances in Low-Temperature Biology, Vol. 1. P.L. Steponkus (ed.). JAI Press Ltd., Hampton Mill, UK. Takagi, H., N.T. Thinh, O.M. Isulam, T. Senboku and A. Sakai. 1997. Cryopreservation of in vitro-grown shoot tips of taro (Colocasia esculenta (L.) Schott) by vitrification. 1. Investigation of basic conditions of the vitrification procedure. Plant Cell Reports 16:594–599. Thinh, N.T. 1997. Cryopreservation of germplasm of vegetatively propagated tropical monocots by vitrification. Doctoral Papers of Kobe University, Department of Agronomy, Japan. Towill, L.E. 1983. Improved survival after cryogenic exposure of shoot tips derived from in vitro plantlet culture of potato. Cryobiology 20:567–573. Towill, L.E. 1984. Survival of ultra-low temperatures of shoot-tips from Solanum tuberosum groups andigena, phureja, stenotomum and other tuber-bearing Solanum species. Cryo–Letters 5: 319–326. Williams, J.G.K., A.R. Kubelik, K.J. Livak, J.A. Rafalski and S.V. Tingey. 1991. DNA polymorphisims amplified by arbitrary primers are useful as genetic markers. Nucleic Acids Research 18:6531–6535. 212 Cryopreservation of Tropical Plant Germplasm

Cryopreservation of in vitro-cultured meristems of wasabi Toshikazu Matsumoto Shimane Agricultural Experiment Station, Shimane, 693-0035, Japan

Introduction Wasabi, Japanese horseradish, is an important crop in Japan. The roots contain a pungent ingredient (sinigrin), which is an essential spice used for typical Japanese foods. Since the seeds are sensitive to desiccation, the cultivars and strains of wasabi are maintained as living plants in the field of some repositories in Japan. Over the last several years, newly developed cryogenic procedures, such as vitrification (Sakai et al. 1990; Yamada et al. 1991) and encapsulation-dehydration (Fabre and Dereuddre 1990), have had dramatic effects on facilitating the progress of cryopreservation. These new procedures remove a major part of freezable water of specimens at non-freezing temperatures and permit them to be cryopreserved by a direct plunge into liquid nitrogen (LN). To develop an ideal cryogenic protocol for wasabi germplasm, a comparative approach on different cryogenic procedures was performed using apical meristems from in vitro-grown wasabi plants.

Materials In vitro-grown plantlets (Wasabi japonica Matsumura) were used in the present study. Apical meristems approximately 1 mm in length were dissected from 30 to 40-d-old plantlets (approx. 30 mm long). Meristems were precultured in Petri dishes on modified Murashige and Skoog medium (1962) (half-strength of KNO 3 and NH4NO 3, termed ½ MS medium) containing 0.3M sucrose and 0.2% (w/v) gellan gum at 20°C under a photoperiod of 16-h light/8-h dark.

Vitrification procedure To induce full dehydration tolerance of excised meristems to PVS2 solution, meristems were precultured on solidified ½ MS medium supplemented with 0.3M sucrose for 16 h, and treated with a mixture of 2M glycerol plus 0.4M sucrose (LS solution) for 20 min at 25°C. Meristems were then dehydrated with the PVS2 solution at 25°C for 10 min prior to a plunge in LN. The vitrified meristems produced a high level of shoot formation (approx. 95%, Matsumoto et al. 1994). Thinh (1997) also successfully applied this protocol to some tropical plants with a slight modification. In the vitrification procedure, the LS solution was first reported to be very effective in enhancing dehydration tolerance (Nishizawa et al. 1993). With wasabi apical meristems, a treatment with LS solution for 20 min at 25°C had a considerable effect in enhancing osmotolerance to PVS2 solution. Wasabi meristems precultured with 0.3M sucrose were treated with LS solution for 20 min. The cells of the meristems that were immersed in LS solution were osmotically dehydrated and plasmolyzed to a considerable extent for 20 min. Cryopreservation techniques 213

The cells were then successively dehydrated with PVS2 solution for 10 min. Under these conditions, the apical meristems remained osmotically concentrated, and the increase in the cytosolic concentration required for vitrification was attained by osmotic dehydration. In the vitrification procedure, plasmolysis may play an important role in dehydrating by mitigating its injurious effects during dehydration (Tao et al. 1983; Jitsuyama et al. 1997). During the preculture of wasabi meristems with 0.3M sucrose for 16 h, sugar and proline concentration considerably increased (Matsumoto et al. 1998a). Reinhoud (1996) clearly demonstrated that the development of osmotolerance of tobacco cultured cells to PVS2 solution during preculture with 0.33M mannitol solution for 1 d appeared to be the combined results of cellular response to mild osmostress and the accumulation of mannitol. To eliminate the osmoprotection procedure with LS solution, excised meristems were precultured on solidified medium containing 0.3M sucrose plus 0.5M glycerol for 16 h. These meristems were directly dehydrated with PVS2 solution for 10 min at 25°C prior to a plunge into LN. This simplified procedure produced shoot formation of approximately 85%.

Encapsulation-vitrification procedure Meristems precultured with 0.3M sucrose for 16 h were encapsulated in alginate- beads (3 mm in diameter) containing 2M glycerol plus 0.4M sucrose. Encapsulated meristems were dehydrated with PVS2 solution in a glass beaker at 100 rpm on a rotary shaker at 0°C for 100 min prior to a plunge into LN. Shoot formation after cryopreservation reached approximately 95% (Matsumoto et al. 1995) compared with encapsulated dried meristems (approx. 65%). This encapsulation-vitrification method was applied for meristems of lily (Matsumoto et al. 1996), statice (Matsumoto et al. 1998b), strawberry (Hirai et al. 1998) and shoot primordia of horseradish (Phunchindawan 1997).

Encapsulation-dehydration. procedure Meristems that were precultured with 0.3M sucrose were trapped into alginate gel beads (5 mm in diameter). Encapsulated meristems were treated with ½ MS medium containing 0.8M sucrose for 16 h to induce dehydration tolerance (Fabre and Dereuddre 1990). Encapsulated meristems dried on silica gel at 25°C for 7 h were plunged into LN. The highest shoot formation was about 60% (water content: about 33% FW) (Matsumoto et al. 1995). To further enhance dehydration tolerance, encapsulated meristems were treated with a mixture of 0.8M sucrose plus 1M glycerol for 16 h before air- drying. This revised procedure produced a considerably higher recovery growth (approx. 80%) compared with meristems treated with 0.8M sucrose alone. This revised procedure was successfully applied to meristems of lily (Matsumoto and Sakai 1995), statice (Matsumoto et al. 1998b) and shoot primordia of horseradish (Phunchindawan 1997). However, the optimal glycerol concentration supplemented with 0.8M sucrose medium for meristems of statice was found to be 0.5M (Matsumoto et al. 1998b). Gonzalez–Benito et al. (1997) reported that little or no 214 Cryopreservation of Tropical Plant Germplasm improvement was observed in the nodes of Centawrium riguelli by the addition of glycerol. Thus, the additional effect of glycerol appears to be species-specific.

Recovery growth of meristems cryopreserved by different cryogenic procedures The recovery growth of meristems cooled to –196°C was compared for the four different cryogenic procedures. As shown in Figure 1, the rate of shoot formation was highest for vitrification (97.5%) and encapsulation-vitrification (EV; 96.7%), followed by the revised encapsulation-dehydration (ED; 79.1%) and lowest for ED (67.1%). Vitrified meristems with or without encapsulation produced shoots much earlier than encapsulated dried meristems (Table 1). Of the latter, the revised ED was much earlier than ED. The time used for dehydration of meristems was the shortest in the vitrification procedure.

100

80

60

40 Shoot formation (%)

2 0

0 V EV ED revised ED Cryogenic protocol

Fig. 1. Shoot formation of wasabi meristems cooled to –196°C by four different cryogenic protocols. V=vitrification, EV=encapsulation-vitrification, ED=encapsulation-dehydration. Precultured meristems were (a) treated with a mixture of 2M glycerol plus 0.4M sucrose at 25°C for 20 min, and then dehydrated with PVS2 solution, (b) encapsulated with alginate gel beads, and then treated with 0.8M sucrose, or (b+) 0.8M sucrose plus 1M glycerol at 25°C for 16 h, prior to a plunge into LN. Shoot formation (%): percent of meristems that produced normal shoots 21 days after reculture. Vertical bars represent standard errors. Cryopreservation techniques 215

Table 1. Shoot length and time used for dehydration of wasabi meristems cooled to -196°C by four different protocols Time used for Cryogenic protocols† Shoot length (mm ± SE) dehydration (min) Vitrification (a) 10.6 ± 4.0 10 at 25°C Encapsulation-vitrification (a) 12.2 ± 3.6 100 at 0°C Encapsulation-dehydration (b) 6.3 ± 3.6 420 at 0°C Revised encapsulation- 9.0 ± 2.6 420 at 0°C dehydration (b+) † Precultured meristems were (a) treated with a mixture of 2M glycerol plus 0.4M sucrose at 25°C for 20 min, and then dehydrated with PVS2 solution, (b) encapsulated with alginate gel beads, and then treated with 0.8M sucrose, or (b+) 0.8M sucrose plus 1M glycerol at 25°C for 16 h, prior to a plunge into LN. Shoot length: measured after 21 days of reculture.

To understand why there is a difference in recovery growth between vitrified and encapsulated-dehydrated meristems, both vitrified and encapsulated dehydrated meristems were examined histologically 6 days after thawing. Thin longitudinal sections, cut through the dome of the meristems were analyzed. Microscopic examination revealed that for vitrified meristems, nearly all of the cells remained via ble, while massive structural damage was observed in the encapsulated-dehydrated meristems, except for the dome area. The ED technique does not use any cryoprotectant such as glycerol, DMSO or ethylene glycol but does use sucrose. However, the induction of dehydration tolerance by 0.8M sucrose alone may be insufficient for encapsulated-dehydrated meristems to produce high levels of recovery growth in many plants. The vitrification protocol with or without encapsulation offers various advantages over the encapsulation-dehydration technique with wasabi meristems. It can be concluded that the most promising cryogenic procedure for wasabi meristems appears to be vitrification in terms of both its high recovery growth and the simplicity of the procedure.

References Fabre, J. and J. Dereuddre. 1990. Encapsulation-dehydration: A new approach to cryopreservation of Solanum shoot tips. Cryo–Letters 11: 413–426. Gonzalez–Benito, M.E., C. Perez and A.B. Viviani. 1997. Cryopreservation of nodal explants of an endangered plant species (Centanurium rigualii Esteve) using the encapsulation-dehydration method. Biodiversity and Conservation 6: 583–590. Hirai, D., K. Shirai, S. Shirai and A. Sakai. 1998. Cryopreservation of in vitro-grown meristems of strawberry (Fragaria x ananassa Duch.) by encapsulation vitrification. Euphytica 101:109–115. Jitsuyama, Y., T. Suzuki, T. Harada and S. Fujikawa. 1997. Ultrastructural study of mechanism of increased freezing tolerance due to extracellular glucose in cabbage leaf cells. Cryo–Letters 18: 33–44. Matsumoto, T., A. Sakai and K. Yamada. 1994. Cryopreservation of in vitro-grown apical meristems of wasabi (Wasabia japonica ) by vitrification and subsequent high plant regeneration. Plant Cell Reports 13:442–446. 216 Cryopreservation of Tropical Plant Germplasm

Matsumoto, T. and A. Sakai. 1995. An approach to enhance dehydration tolerance of alginate-coated dried meristems cooled to –196°C. Cryo–Letters 16: 299–306. Matsumoto, T., A. Sakai, C. Takahashi and K. Yamada. 1995. Cryopreservation of in vitro- grown apical meristems of wasabi (Wasabia japonica) by encapsulation-vitrification method. Cryo–Letters 16: 189–196. Matsumoto, T., A. Sakai, C. Takahashi and K. Yamada. 1996. Cryopreservation of in vitro- grown apical meristems of lily (Lilium L.) by encapsulation-vitrification method. Plant Tissue Culture Letters 13: 29–34. Matsumoto, T., A. Sakai and Y. Nako. 1998a. A novel preculturing for enhancing the survival of in vitro-grown meristems of wasabi (Wasabia japonica) cooled to –196°C by vitrification. Cryo–Letters 19: 27–36. Matsumoto, T., C. Takahashi, A. Sakai and Y. Nako. 1998b. Cryopreservation of in vitro- grown apical meristems of hybrid statice by three different procedures. Scientia Horticulturae 76: 105–114. Murashige, T. and F. Skoog. 1962. A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiologia Plantarum 15: 473–497. Nishizawa, S., A. Sakai, Y. Amano and T. Matsuzawa. 1993. Cryopreservation of asparagus (Asparagus officinalis L.) embryogenic suspension cells and subsequent plant regeneration by vitrification. Plant Science 91: 67-73. Phunchindawan, M. 1997. Cryopreservation of useful plant resources: Application of encapsulation-dehydration method to preservation of hairy root cultures and microalgae. Doctoral Thesis, Osaka University, Faculty of Pharmaceutical Sciences, Osaka, Japan. Reinhoud, P.J. 1996. Cryopreservation of tobacco suspension cells by vitrification. Doctoral Thesis, Leiden University, Institute of Molecular Plant Sciences, Leiden, the Netherlands. Sakai, A., S. Kobayashi and I. Oiyama. 1990. Cryopreservation of nucellar cells of navel orange (Citrus sinensis Osb. var. brasiliensis Tanaka) by vitrification. Plant Cell Reports 9: 30–33. Tao, D., P.H. Li and J.V. Carter. 1983. Role of cell wall in freezing tolerance of cultured potato cells and their protoplasts. Physiologia Plantarum, 58: 527–532. Thinh, N.T. 1997. Cryopreservation of germplasm of vegetatively propagated tropical monocots by vitrification. Doctoral Papers of Kobe University, Department of Agronomy, Japan. Yamada, T., A. Sakai, T. Matsumura and S. Higuchi. 1991. Cryopreservation of apical meristems of white clover (Trifolium repens L.) by vitrification. Plant Science 78: 81–87. Cryopreservation techniques 217

Cryopreservation of citrus apices using the encapsulation- dehydration technique Maria Teresa Gonzalez–Arnao¹, Florent Engelmann², Caridad Urra Villavicencio¹, Marlene Morenza³ and Alba Rios³ ¹ Centro Nacional de Investigaciones Científicas, La Habana, Cuba ² IPGRI, 00145 Rome, Italy ³ Instituto de Investigaciones de Cítricos, Miramar, La Habana, Cuba

Introduction Seeds of many citrus species display recalcitrant or intermediate storage behaviour, and therefore cannot be stored dry at low temperature. In addition, the seeds produced are heterozygous and particular gene combinations cannot be conserved through seed storage. Some cultivars are seedless and are thus propagated vegetatively. Citrus genetic resources are thus currently conserved as whole plants in field genebanks where they remain exposed to pests, diseases and other natural hazards such as drought, weather damage, human error and vandalism (Withers and Engels 1990). The only current alternative for long-term conservation of problem crops is cryopreservation. Cryopreservation of citrus germplasm has been performed using seeds (Mumford and Grout 1979), ovules (Bajaj 1984), embryonic axes (Radhamani and Chandel 1992), somatic embryos (Marin and Duran–Vila 1988), embryogenic calluses and cell suspensions ((Sakai et al. 1990, 1991; Niino and Sakai 1992; Engelmann et al. 1994). However, these protocols were applied to juvenile material only, which requires several years before flowering and fruit production. Apical meristems represent the material of choice for citrus germplasm conservation, since plants regenerated from apices of adult cultivars will not present juvenility characteristics and will be true to type, in contrast to plants produced from any other type of material (Navarro et al. 1985). The encapsulation-dehydration technique has led to successful results with apices of numerous temperate and tropical plant species (Engelmann 1997). In this paper we report the successful application of cryopreservation to citrus apices using the encapsulation-dehydration technique.

Materials and methods

Plant material The plant material consisted of apices sampled on in vitro plantlets of citrus rootstocks: Poncirus trifoliata (L.) Raf., Troyer and Carrizo citranges obtained from seeds germinated in vitro. Plantlets were cultivated as described by González– Arnao et al. (1998) and subcultured every 45 d. 218 Cryopreservation of Tropical Plant Germplasm

Cryopreservation experiments For cryopreservation experiments, apices (size: 0.5–1 mm) were excised 20 d after the last subculture and left overnight on standard medium for recovery. Apices were encapsulated in alginate (3%) beads and precultured in liquid medium with various sucrose concentrations (0.3–1M) for different durations (1–10 d). A progressive pregrowth by daily transfer of apices to medium with higher sucrose concentration from 0.3M up to 1M also was tested. Encapsulated apices were then dehydrated at room temperature down to 20– 25% moisture content (MC, fresh weight basis) under the sterile air current of the laminar flow cabinet and transferred to sterile 2-ml polypropylene cryotubes. Samples submitted to preculture with progressive increase in sucrose concentration were desiccated under the same conditions down to a series of different MCs, ranging from 36 to 15%. Freezing was performed either rapidly by direct immersion of the cryotubes in liquid nitrogen or slowly, by cooling at 0.5°C/min from +20 to –40°C before immersion in liquid nitrogen, using a programmable freezer (Bio-Cool, FTS Systems, USA). Samples were kept for at least 1 h at –196°C. For thawing, the cryotubes were placed under the air current of the laminar flow cabinet for 2–3 min. Beads were then transferred in Petri dishes on standard semi-solid medium. Apices were cultured for the first week in the dark, then transferred under standard lighting conditions. Survival was evaluated after 1 month by counting the number of apices which had developed into shoots. Fifteen to 20 apices were employed per experimental condition, and experiments were replicated 2 to 3 times. The cryopreservation protocol was set up using apices of Poncirus trifoliata and the most efficient procedure was then applied to other rootstock species.

Results The survival rate of encapsulated apices after pregrowth varied depending on the sucrose content in the preculture medium. Preculture for 1 d with 0.75 and 1M sucrose was detrimental to survival. By contrast, apices could withstand extended preculture durations (up to 10 d) in media with lower sucrose concentrations (0.3 and 0.5M). Preculture durations longer than 2 d resulted in survival rates around 80% after desiccation. After slow freezing, survival was achieved for all pregrowth durations tested, ranging between 10 and 50%, whereas after rapid freezing, survival was noted for 3 and 4 d of pregrowth only. The highest survival rate with slow and rapid freezing was achieved after 3 and 4 d of preculture, with 50 and 40% survival, respectively. Preculture involving a progressive increase in sucrose concentration improved the tolerance to high sucrose levels in comparison with direct preculture in medium with the same final sucrose concentration. A 1M final sucrose concentration in the preculture medium was detrimental to survival. Survival of cryopreserved apices was higher after slow freezing than after rapid freezing. Cryopreservation techniques 219

Table 1. Effect of bead moisture content on the survival (%) of apices after dehydration, rapid or slow freezing. Apices were submitted to a progressive increase in sucrose concentration during preculture (0.3M/24 h + 0.5M/24 h + 0.75M/24 h). Bead moisture content (%) Treatment 36 28 23 17 15 Dehydration 95±2.6 90±6.2 88±4.6 50±5.6 20±4.6 Rapid freezing 0 15±5.3 20±2.7 0 0 Slow freezing 0 40±6.2 40±4.6 20±4.0 0 Reprinted from Gonzalez–Arnao et al. 1998, with permission.

Survival of desiccated apices decreased in line with decreasing bead moisture content (Table 1). After cryopreservation, the highest survival was obtained after slow freezing, with beads dehydrated down to 23 or 28% MC. For recovery, control and cryopreserved apices were cultured on standard semi-solid medium. It was not necessary to extract the apices from the beads since they broke the alginate capsule without difficulty and developed into new plants without even transitory callus formation. Apices which did not survive became totally black or remained white. The optimal conditions established for freezing apices of Poncirus trifoliata achieved survival with apices of two additional citrus rootstocks (Table 2).

Table 2. Effect of cryopreservation protocol on the survival (%) of apices from three different citrus species after dehydration, rapid or slow freezing. Apices were submitted to a progressive increase in sucrose concentration during preculture (0.3M/24 h + 0.5M/24 h + 0.75M/24 h), then desiccated to 20–25% MC before rapid freezing. Survival (%) Species Pregrowth Dehydration Slow freezing Poncirus trifoliata 100 90±5 44±4 Troyer citrange 100 83±4 36±3 Carrizo citrange 100 95±5 55±5

Discussion Up to now, all previous attempts to cryopreserve citrus apices had been unsuccessful (Pérez 1995). The cryopreservation protocol established for apices of Poncirus trifoliata rootstock comprised a preculture for 3 d in liquid medium with 0.5M sucrose or in medium with progressively increasing sucrose concentration (up to 0.75M), desiccation to 20–25% MC followed by slow freezing. This procedure allowed survival with apices of two additional rootstocks. Citrus apices displayed high sensitivity to sucrose since exposure to a concentration of 0.75M was tolerated only after a progressive increase in sucrose concentration. Slow freezing produced better results than rapid freezing, suggesting that not all freezable water had been extracted from beads/apices during desiccation to 20–25%, and that further freeze-induced dehydration during slow prefreezing was necessary to achieve optimal survival. 220 Cryopreservation of Tropical Plant Germplasm

Growth recovery of cryopreserved citrus apices occurred directly without callus formation. This allows us to assume that most cells of the apical region were only slightly or not at all damaged during the cryopreservation process, as observed by histocytological examination with sugarcane apices cryopreserved using the encapsulation-dehydration technique (González–Arnao et al. 1993). This protocol, established for apices of juvenile plants, proved unsuccessful when tested with apices sampled on adult plants. However, preliminary positive results have been obtained recently with adult material using encapsulation- vitrification. Indeed, apices of three species (Nules clementine, Fortune mandarin and Pineapple sweet orange), when pretreated with a loading solution containing 0.7M sucrose and 2M glycerol, encapsulated and treated with the PVS3 vitrification solution, remained green after cryopreservation but did not regrow. Additional experiments are under way to optimize different steps of the protocol.

Acknowledgements This work was performed in the framework of the IPGRI-funded project No. 96/098 entitled “Development of cryopreservation techniques for the long-term conservation of Citrus germplasm in Cuba”.

References Bajaj, Y.P.S. 1984. Induction and growth in frozen embryos of coconut and ovules of citrus. Current Science 53: 1215–1216. Engelmann, F. 1997. In vitro conservation methods. Pp. 119–162 in B.V. Ford–Lloyd, J.H. Newbury and J.A. Callow (eds.), Biotechnology and Plant Genetic Resources: Conservation and Use. CABI, Wellingford. Engelmann, F., D. Dambier and P. Ollitrault. 1994. Cryopreservation of embryogenic cell suspensions and calluses of citrus using a simplified freezing process. Cryo–Letters 15: 53–58. González–Arnao, M.T., F. Engelmann, C. Huet and C. Urra. 1993. Cryopreservation of encapsulated apices of sugarcane: Effect of freezing procedure and histology. Cryo– Letters 14: 303–308. González–Arnao, M.T., F. Engelmann, C. Urra, M. Morenza and A. Ríos 1998. Cryopreservation of citrus apices using the encapsulation-dehydration technique. Cryo–Letters 19: 177–182. Marín, M.L. and N. Duran–Vila. 1988. Survival of somatic embryos and recovery of plants of sweet orange (Citrus sinensis (L) Osb.) after immersion in liquid nitrogen. Plant Cell Tissue Organ Culture 14: 51–57. Mumford, P.M. and B.W.W. Grout. 1979. Desiccation and low temperature (–196°C) tolerance of Citrus limon seed. Seed Science and Technology 7: 407–410. Navarro, L., JM. Ortiz and J. Juárez. 1985. Aberrant citrus plants obtained by somatic embryogenesis of nucelli cultured in vitro. HortScience 20: 214–215. Niino, T. and A. Sakai. 1992. Cryopreservation of alginate-coated in vitro grown shoot tips of apple, pear and mulberry. Plant Science 87: 199–206. Pérez, R.M. 1995. Crioconservación de recursos genéticos de cítricos. Tesis Doctoral Universidad Politécnica de Valencia, Spain. Cryopreservation techniques 221

Radhamani, J. and K.P.S. Chandel. 1992. Cryopreservation of embryonic axes of trifoliate orange (Poncirus trifoliata (L.) Raf.). Plant Cell Reports 11: 372–374. Sakai, A., S. Kobayashi and I. Oiyama. 1990. Cryopreservation of nucellar cells of navel orange (Citrus sinensis var. Brasilensis Tanaka) by vitrification. Plant Cell Reports 9: 30– 33. Sakai, A, S. Kobayashi and I. Oiyama. 1991. Survival by vitrification of nucellar cells of navel orange (Citrus sinensis var. Brasilensis Tanaka) cooled to –196ºC. Plant Physiology 137: 465–470. Withers, LA. and J.M.M. Engels. 1990. The test tube genebank – a safe alternative to field conservation. IBPGR Newsletter for Asia, the Pacific and Oceania 3: 1–2. 222 Cryopreservation of Tropical Plant Germplasm

Development of cassava cryopreservation Roosevelt H. Escobar, D. Debouck and William M. Roca Centro Internacional de Agricultura Tropical (CIAT), Cali, Colombia

Introduction Vegetative propagation, bulkiness of planting material and risk of genetic erosion make cassava an ideal candidate for the application of innovative germplasm conservation techniques. Cryopreservation is considered as the most economical and safest method for the long-term conservation of cassava genetic resources. Our efforts at CIAT, in collaboration with IBPGR/IPGRI, started with the development of a basic cassava cryopreservation protocol which was established in 1990, and consisted of the application of chemical dehydration of shoot-tips using a moderate concentration of sorbitol, DMSO and sucrose. Several factors were considered critical for the process. Cassava shoot-tips are affected by osmotic agents in different ways according to concentration and type of compound. High concentration of sorbitol changes the morphogenic response of shoot-tips, callus induction being the most typical. When sucrose was used in combination with 1M sorbitol, shoot-tips did not grow, and DMSO reduced callus formation. The best combination of osmotic agents tested with cassava shoot-tips consisted of 1M sorbitol, 0.1M DMSO and 0.11 M sucrose (medium C4) during 3 days. Other factors appeared to be critical, including the level of tissue dehydration and the size of shoot-tips. We found that dehydration for 1 h on filter paper could improve shoot recovery after freezing; dehydration at room temperature (26–28°C) favoured elimination of water from the explant surface, allowing a gradual loss of water from within the tissue as well. A relationship between the extent of cell damage due to freezing and the size of explants was established. Small shoot-tips (1–2 mm) gave better results than larger ones (3–4 mm). Based on these parameters we established a basic protocol for cassava (Escobar et al. 1997) (Fig. 1). By preculturing shoot-tips on C4 medium for 3 days, and using a two-step cryoprotective treatment, it was possible to recover viable cassava plants from frozen shoot-tips with cv. MCol 22 (50–60% as shoot formation); thereafter the method was reproduced with several cultivars representing a wide geographic distribution. Some cultivars were sensitive to osmotic concentration applied during the preculture medium. It seems that for some cassava cultivars, the edaphoclimatic origin affected response after freezing (Escobar et al. 1997). Sakai and Sugawara (1973) found a narrow correlation between the hardening behaviour and geographic origin of the plant, and Reed (1990) did not observe this relationship with Pyrus meristems. By adjusting the osmotic concentration in the preculture medium (0.25–0.5M sorbitol, 1x10 -3 – 1x10 -4 M DMSO and 0.11– 0.25M sucrose for 5 days) it was possible to increase shoot recovery in low responding cassava cultivars like MVen 232 and MMex 71 (Fig. 2). In cassava Cryopreservation techniques 223 there exists a close relationship between osmotic concentration, exposure time and response after freezing. Preculture media containing high osmotic concentration for long periods reduces shoot recovery, even prior to freezing (Escobar et al. 1997).

1-2 mm shoot tips Preculture on C4 For 3 days Dehydration 1 h (26-28oC)

Cryoprotection for 2 h / ice

In vitro cultures

Shoot recovery Callus

Slow freezing program

Recovery steps L.N. 37oC/45 s Thawing Plant Cell Reports, 1997, 16:474-478

Fig. 1. Protocol established for cassava cryopreservation (from Escobar et al. 1997).

80 70 60 50 C4 40 M6 30 M7

% shoot recovery 20 10 0 MCOL 22 MMEX 71 MVEN 232

C4: 1M Sorb, 0.1M DMSO, 0.1 M Suc M6: 0.5M Sorb, 0.001M DMSO, 0.1M Suc M7: 0.5M Sorb 0.0001M DMSO, 0.25M Suc

Fig. 2. Preculture medium and shoot recovery of recalcitrant cassava cultivars. 224 Cryopreservation of Tropical Plant Germplasm

In order to increase the recovery rate of viable plants, and to minimize genotypic differences, we found that modifications of pre- and post-freezing conditions affected the recovery of shoots. Adjustment of growth conditions of donor cultures can influence shoot recovery after freezing. Lower temperature (21–23°C) and higher illumination (75 µE m-2 s-1 ) of cassava donor cultures increased shoot recovery after freezing consistently (50–60% rate). Benson et al. (1989) found the same response with shoot-tips of Solanum tuberosum, with different pre- and post-freeze light regimes. We found that younger tissues (3–4 months of in vitro culture) were more reactive than older ones, and pregrowing shoots on CIAT–4E medium (Roca 1984) for 3 days before freezing resulted in a significant increase in survival. Medium 4E induces a higher amount of meristematic cells, able to support desiccation and with good response after freezing. Henshaw et al. (1985) showed that a pregrowth period is critical in determining the survival of cryopreserved shoot-tips of S. tuberosum. In the post-freezing phase we found that low agar concentration in the recovery medium affects survival after freezing; it seems that media with low agar concentration (0.35%) have a positive effect on the growth of plants recovered from frozen shoot-tips. Semi-solid medium avoids the formation of gradients, but contributes to hyperhydricity. Low agar concentration in the preculture phase increases damage due to freezing. The response after freezing was affected by the type of cytokinin used and its concentration. Kinetin at 0.5 mg/L was more efficient than 2iP, BAP, TDZ and Adenine. When BAP concentration was increased from 0.04 to 0.5 mg/L, a drastic effect was evident.

Reducing NAA (0.01 mg/L) and increasing GA3 (0.5 mg/L) in the recovery medium diminished callus growth and stimulated elongation of shoots. Ultra-rapid freezing of shoot-tips resulted in similar or higher recovery rates than slow freezing (Table 1). It would also save time and reduce the cost of introducing the entire CIAT cassava collection into cryopreservation (Escobar and Roca 1997). The encapsulation-dehydration technique was established as an alternative to slow/rapid freezing cryopreservation (Escobar et al. 1998; Palacio 1998). The technique has been shown to be expeditious and consistent; shoot growth from frozen shoot-tips was rapid and direct with less callus formation. Shoot-tip size, pretreatment with sucrose, and dehydration time to achieve optimal water content of beads were found critical for the success of the encapsulation-dehydration technique. It seems that a gradual increase in sucrose concentration could improve the recovery of osmo-sensible cassava cultivars. Furthermore, increasing cytokinins and reducing auxin level in the recovery media favoured shoot response after freezing and the consistency of response (Fig. 3). We moved plants from 16 cassava cultivars to the field after recovery from frozen storage and compared them with the respective non-cryopreserved in vitro propagated plants. We are monitoring morphological traits and will test possible genetic changes assisted by molecular markers. Cryopreservation techniques 225

Table 1. Effect of freezing technique on cryopreservation of cassava shoots Genotype Freezing† % Viability % Shoot recovery MCol 304 Programmed 63.6 18.1 Rapid 100 54.5 MCol 1389 Programmed 90.9 9.1 Rapid 90 10 MCol 1468 Programmed 53.8 0 Rapid 84.6 0 MPar 71 Programmed 84.6 76.9 Rapid 92.8 71.4 MCol 22 Programmed 76.5 49.75 Rapid 80.25 55.5 † Programmed protocol, Escobar et al. 1997; rapid freezing (direct immersion), Escobar and Roca 1997.

90 80 70 60 50 40 30 20 % shoot recovery 10 * recalcitrant 0 MCOL 22 MVEN MBRA MCOL MMEX ^ low response to 232* 507 1468^ 71* in vitro Fig. 3. Response of cassava genotypes to encapsulation-dehydration.

Cryopreservation will allow great reduction of the high costs of maintaining the cassava germplasm collections which are largely due to the labour required in the field as well as in the active in vitro bank. Cryopreservation should also reduce the laboratory space needed for a collection as large as the one maintained at CIAT (over 6000 clonal accesions). In addition, cryopreservation of pollen, seeds (Marin et al. 1990), somatic embryos and other cells and tissues will be fundamental for achieving the conservation goals, which are basic to the cassava improvement programmes at CIAT and elsewhere.

Conclusion 1. We developed three cryopreservation methodologies for cassava. Viable plants were recovered from all of them. 2. Not all methodologies can be applied to all cultivars. There are varietal differences. Adjusting osmotic concentration is a key factor. 3. Encapsulation-dehydration seems to be the fastest and cheapest method to introduce the cassava collection into cryopreservation. 226 Cryopreservation of Tropical Plant Germplasm

References Benson, E.E., K. Harding and H. Smith. 1989. Variation in recovery of cryopreserved shoot tips of Solanum tuberosum exposed to different pre- and post-freeze light regimes. Cryo–Letters 10:323–344 Escobar, R.H., G. Mafla and W.M. Roca. 1997. A methodology for recovering cassava plants from shoot tips maintained in liquid nitrogen. Plant Cell Reports 16:474–478. Escobar, R.H. and W.M. Roca. 1997. Cryopreservation of cassava shoot tips through rapid freezing. African Journal of Root and Tuber Crops 2: 214–215. Escobar, R.H., J.D. Palacio, M.P. Rangel and W.M. Roca. 1998. Crioconservación de ápices de yuca mediante encapsulación-deshidratación. In Proceedings of III Latin-american meeting on plant biotechnology REDBIO’98, Habana,Cuba 1–5 June 1998. Henshaw, G.G., J.F. O’Hara and J.A. Stamp. 1985. Cryopreservation of potato meristems. Pp. 159–170 in Cryopreservation of plant cells and organs. K.K. Kartha, ed. CRC Press, Boca Raton, Florida. Marín, M.L, G. Mafla, W.M. Roca and L.A. Withers. 1990. Cryopreservation of cassava zygotic embryos and whole seeds in liquid nitrogen. Cryo–Letters 11:257–264. Palacio, J.D. 1998. Crioconservación de ápices de yuca (Manihot esculenta Crantz) utilizando la técnica de encapsulación- deshidratación. Tesis CIAT, Cali, Colombia. Reed, B.M. 1990. Survival of in vitro grown apical meristems of Pyrus following cryopreservation. HortScience 25:111–113. Roca, W.M. 1984. Cassava. Pp. 269–301 in Handbook of plant cell culture: Crop species. Vol 2. (Sharp W.R., D.A. Evans, R.V. Ammirato and Y. Yamada, eds.). MacMilliam Publ., New York. Sakai, A. and Y. Sugawara. 1973. Survival of poplar callus at super-low temperatures after cold acclimatation. Plant and Cell Physiology 14:1201–1204. Cryopreservation techniques 227

Cryopreservation of in vitro-grown apical meristems of some vegetatively propagated tropical monocots by vitrification Nguyen Tien Thinh, Hiroko Takagi and Akira Sakai JIRCAS, Okinawa Sub-Tropical Research Station, Ishigaki, Okinawa 907, Japan

Introduction Currently, most of the reports of successful cryopreservation of plant meristems/meristems deal with temperate species. Tropical plant germplasm, of which the vegetatively propagated monocots are an important part, has received little attention or has even been considered as recalcitrant to cryostorage (Takagi et al. 1997). We have established an efficient vitrification procedure to cryopreserve the germplasm of taro [Colocasia esculenta (L.) Schott Araceae], a common vegetatively propagated tropical monocot (referred to as VPTM). The application of this procedure to meristems of four other VPTM (banana, Cymbidium , Cymbopogon, pineapple) was also successful. In this report, technical factors in the procedure observed to affect the success of the vitrification process are discussed. Using these results, possible practical steps in the development of a vitrification procedure for meristems of other similar VPTM are suggested.

Materials and methods

Materials From in vitro-grown plants, three kinds of apical meristems, i.e. meristems with apical domes (i) uncovered (O type), (ii) partly covered (PC type), and (iii) fully covered (FC type) by the outer leaf primordia (hereafter LP), were dissected and used as explants. The roughly relative numbers of LP in these meristems were one for the O type, two for the PC type and three for the FC type.

Method of vitrification Details of different experimental steps in the vitrification procedure are shown in Figure 1.

Results and discussion During the development of the vitrification technique for all the tested VPTM, preconditioning, meristem structure, preculture, loading treatment and dehydration with PVS2 were observed to affect post-thaw survival of cryopreserved meristems. These factors, based on their importance to the final results, are discussed below. 228 Cryopreservation of Tropical Plant Germplasm

Preconditioning Preculture Loading Dehydration 0.7ml O

PC

FC Meristem selection

Recover Blotting Unloading Thawing LN storage y

Fig. 1. General view of the applied materials and method. Preconditioning: 1 month, MS medium with 6–12% sucrose. Meristem selection: see 'Materials" in text. Preculture: 1–2 nights, MS medium with 0.3–0.7M sucrose. Loading: 20 min, in solution of 2M glycerol + 0.4M sucrose. Dehydration: 0–60 min at 25°C or 0–80 min at 0°C, in PVS2. LN storage: use only the 0.7-ml cryotube, rapid cooling. Thawing: rapid, shaking 80 sec, in water at 40°C. Unloading: 15 min, in liquid MS medium with 1.2M sucrose. Blotting: 1–2 nights, on filter paper on MS medium with 0.3M sucrose. Recovery: different media depending on species, but always with 3% sucrose.

Meristem structure – the first important factor for successful vitrification Among the three types of meristems, FC expressed the highest tolerance to dehydration by PVS2 (see below for details), followed by the PC, whereas the O ones were the most sensitive. However, regardless of various trials of sucrose preculture, loading treatment and PVS2 dehydration, the post-thaw survival rates were always highest with the PC, followed by the O, and lowest with the FC ones. Under optimal conditions, for the order of banana–Cymbidium– Cymbopogon–pineapple–taro, the average post-thaw survival rates (%) of the PC, the O and the FC meristems were 66.7–94.5–67.9–53.3–77.6, 43.5–56.6–34.5–24.7– 33.5, 6.2–35.7–6.8–5.8–4.6, respectively. The five tested VPTM share a common shoot structure, i.e they all have apical meristems very well covered by the outer leaves, which are tubular in shape and have interfolded, thick leaf bases. There exist also closed, empty spaces between the two neighbouring LPs of an apex. Thus, in relation with the increasing survival achieved from LP to O and FC Cryopreservation techniques 229 meristems, the above described leaf morphology could make the apical domes of these different meristems differently loaded and dehydrated by the loading and PVS2 solutions. In this study, FC meristems, with the apical dome hidden in the outer LP, may not have been appropriately dehydrated before being cooled in LN. The O meristems, with the whole fragile apical dome in direct contact with PVS2, may have sometimes been overdehydrated and/or damaged during explant handling. The PC meristems, with only part of the dome in contact with the external agents, appeared to be the most suitable cryogenic explants. Therefore, for further experiments, PC meristems were selected as explants.

Loading treatment – the second important factor to ensure high rates of post-thaw survival The second decisive factor to obtain high post-thaw survival of meristems was the loading treatment. With all the VPTM tested, loading enhanced greatly both the tolerance to PVS2 dehydration and the post-thaw survival of meristems. For the similar order of plants as described above, the post-thaw survival rates (%) of unloaded and loaded meristems were 0.0–46.7–25.5–11.5–33.3 vs. 66.7–94.5–67.9– 53.3–77.6, respectively. Observations on similar effects of loading treatment were reported for cells and meristems of other species, such as rye protoplasts (Langis and Steponkus 1991) and meristems of wasabi and lily (Matsumoto et al. 1994, 1995). In this study, it appears that meristems of dehydration-sensitive species (see PVS2 dehydration below for details), such as banana and pineaple, required much more loading than those of PVS2-tolerant ones (Cymbidium , Cymbopogon, taro) to be able to overcome the high osmosis of the PVS2. In the latter group of plants, loading treatment also resulted in highest post-thaw survival. Therefore, loading is an essential step in the cryopreservation of meristems of the VPTM by the vitrification method.

PVS2 dehydration – the third important factor to obtain post-thaw survival Dehydration of meristems with a highly concentrated and viscous solution is the main step in the vitrification process. Successful dehydration would result in cells with minimal injury, with the ability to be vitrified upon cooling in LN. In some preliminary trials using three different vitrification solutions to dehydrate the meristems of taro, the best results in post-thaw survival were always obtained with the PVS2 solution developed by Sakai et al. (1990). Thus, PVS2, which consists of 30% glycerol + 15% ethylene glycol + 15% DMSO + 0.4M sucrose, was used throughout this study to dehydrate the meristems. The results showed that the five tested VPTM could be divided into two groups according to their tolerance to PVS2 dehydration. Cymbidium, Cymbopogon and taro formed a group with high tolerance. The meristems of these plants could be treated with PVS2 at 25°C for up to 40–60 min with little effect on plant recovery. Pineapple and particularly banana formed another group which was much more sensitive. At 25°C, recovery was dramatically reduced and/or death of meristems of these plants occurred if meristems were dehydrated for only 5–20 min. Thus, 230 Cryopreservation of Tropical Plant Germplasm sensitivity to PVS2 made it difficult to obtain enough dehydration of meristems of this second group prior to LN cooling. As mentioned above, it was found that loading greatly enhanced the tolerance of meristems to PVS2. This tolerance was increased further if dehydration was carried out at reduced temperature, i.e. 0°C. Using banana meristems as an example, the survival rates after having either been directly (non-loaded) dehydrated for 10 min at 25°C, loaded then dehydrated for 15 min at 25°C or loaded then dehydrated for 40 min at 0°C were 0, 100 and 100%, respectively. Upon cooling in LN, meristems of the first plant group showed high post-thaw survival rates (70–90%, depending on the species) after they had been treated with PVS2 for 10 min at 25°C or for 20–40 min at 0°C, whereas for the second group, good results (50–70%, depending on species) were only derived from the dehydration for 20–30 min at 0°C. Thus, although longer treatment durations were needed, dehydration at reduced temperature appeared to be safer to the meristems and ensured high recovery rates. Similar positive effects of dehydration at low temperature were shown for meristems of wasabi and lily (Matsumoto et al. 1994, 1995).

Sucrose preculture – an additional factor enhancing post-thaw survival Among different sucrose preculture regimes, preculture of meristems for one night on MS medium supplemented with 0.3M sucrose was found to enhance post-thaw survival of meristems of most of the VPTM tested. However, in the case of banana, different regimes of sucrose preculture always led to the rapid and toxic browning of explants during their post-thaw blotting and subculture for recovery. For the same described order of species, the post-thaw survival rates (%) of non- and precultured meristems were 65.7–80.8–43.3–21.2–54.6 vs. 22.1–94.5–67.9–53.3–77.6, respectively. The positive effect of sucrose preculture of meristems has already been reported for various cold-hardened species, such as wasabi, lily (Matsumoto et al. 1994, 1995) and mulberry (Niino et al. 1992a). In this study, with the exception of the banana case, sucrose preculture was also effective for meristems of the tested VPTM.

Sucrose preconditioning – a promising way to harden the VPTM Cold-hardening (4–5°C) was successfully applied to meristem-donor plants (referred to as MDPs) of several temperate species prior to the cryopreservation of meristems (Bajaj 1995). In our study, growth of MDPs of taro, cold-hardened at 10–15°C, was severely affected and the meristems possessed significantly lower rates of post-thaw survival. We found that preconditioning taro MDPs for one month on MS medium supplemented with increasing concentrations (6, 9 and 12%) of sucrose could be a promising alternative to cold-hardening for this tropical species. Compared with the control plants grown on standard concentration (3%) of sucrose, the preconditioned MDPs appeared to be shorter and compact. They possessed larger cormels, shorter petioles, thicker leaf bases, smaller leaf blades and more rigid tissues. These characteristics made the dissection of the tiny, fragile meristems easier, resulting in less damage. Cryopreservation techniques 231

Moreover, meristems dissected from preconditioned MDPs (hereafter preconditioned meristems) looked morphologically more uniform and much less watery than the non-preconditioned counterparts. Analysis of shoot parts of the MDPs showed that preconditioning also significantly reduced the water content, but enhanced the accumulation of stress-responsive solutes (soluble sugars, free proline). After LN storage, the post-thaw survival rates of preconditioned tips were significantly higher than that of the controls (i.e. 97 vs. 77%). Preconditioning was also observed to give rise to similar effects on the MDPs of banana and Cymbopogon. Though results were not statistically significant, the preconditioned meristems of these two VPTM also survived the LN cooling with slightly higher rates than the non-preconditioned ones. Thus, preconditioning appears to be a promising step to be included in the vitrification process of the VPTM tested.

Conclusions Based on the above results, we conclude that it is possible to cryopreserve the meristems of the tested VPTM by vitrification. The practical working steps in the development of vitrification technique for meristems of the tested VPTM are summarized in Figure 2. Experiments should be carried out to check whether this procedure can be extrapolated to other VPTM, such as bamboo, curcuma, sugarcane, canna, ginger, etc.

Fig. 2. Suggested working steps in the development of vitrification procedure for apical meristems of the vegetatively propagated tropical monocots. 232 Cryopreservation of Tropical Plant Germplasm

Acknowledgements Thanks are due to Dr S.D. Hamill (Department of Primary Industry, Queensland Horticulture Institute, Australia) for correcting the English of the manuscript and to Dr B. Panis (KUL, Leuven, Belgium) for useful comments and discussions. Nguyen Tien Thinh is grateful to JIRCAS for granting him a Visiting Research Fellowship to carry out most of the present research.

References Bajaj, Y.P.S. (ed.) 1995. Biotechnology in Agriculture and Forestry Vol. 32. Cryopreservation of Plant Germplasm I. Springer, Berlin. Langis, R. and P.L. Steponkus. 1991. Vitrification of isolated Rye protoplasts: Protection against dehydration injury by ethylene glycol. Cryo–Letters 12: 107–112. Matsumoto, T., A. Sakai and K. Yamada. 1994. Cryopreservation of in vitro-grown apical meristems of wasabi (Wasabia japonica ) by vitrification and subsequent high plant regeneration. Plant Cell Reports 13: 442–446. Matsumoto, T., A. Sakai and Y. Yamada. 1995. Cryopreservation of in vitro-grown apical meristems of lily by vitrification. Plant Cell Tissue and Organ Culture 41: 237–241. Sakai, A., S. Kobayashi and I. Oiyama. 1990. Cryopreservation of nucellar cells of navel orange (Citrus sinensis Osb. var. brasiliensis Tanaka) by vitrification. Plant Cell Reports 9: 30–33. Takagi, H., N.T. Thinh, A. Sakai and T. Senboku. 1997. Cryopreservation of in vitro-grown shoot tips of taro (Colocasia esculenta (L.) Schott) by vitrification. 1. Investigation of basic conditions of the vitrfication procedure. Plant Cell Reports 16: 594–599. Cryopreservation techniques 233

Cryopreservation of yam apices: a comparative study with three different techniques B.B. Mandal NBPGR, Pusa Campus, New Delhi-12, India

Introduction Cryopreservation is becoming a very important tool for long-term conservation of germplasm of clonally propagated species and species bearing recalcitrant seeds. Yams (Dioscorea spp.), being clonally propagated, are conventionally conserved in field genebanks as living collections. Field maintenance is expensive and presents high risks of loss of materials due to biotic and abiotic stresses. In vitro conservation under slow growth demands periodic subculturing and regular attention. Therefore, the development of an efficient and reliable protocol for cryopreservation of yam is essential for long-term conservation of its germplasm. As shoot apices are the most suitable explants for in vitro conservation of clonally propagated species, cryopreservation of yams using shoot apices has received wide attention in recent years. Shoot apices used to be cryopreserved using classical methods including pretreatment with cryoprotectant in liquid nitrogen and controlled slow freezing. However, classical methods need expensive equipment for programmable freezing. Survival rates were also generally low, recovery growth was slow, often including transitory callusing phase and results were not always reproducible (Gonzalez Arnao et al. 1993). Recently, new techniques such as vitrification, simplified freezing, encapsulation-dehydration and encapsulation-vitrification were presented. Successful use of these techniques on several plant species was also reported (Matsumoto et al. 1995). In yams (Dioscorea spp.) successful cryopreservation of apices using encapsulation-dehydration technique was reported from our laboratory earlier (Mandal et al. 1996a). More recently, the use of the same technique for cryopreservation of yam was also reported from another laboratory (Malaurie et al. 1998). However, in both studies the frequency of survival of frozen apices and subsequent shoot regeneration achieved were not uniformly adequate with all the species. More importantly, many of the shoots that recovered from frozen apices regenerated through an intermediary callus phase. So, to develop an efficient protocol for yam apices, two other cryogenic techniques, vitrification and encapsulation-vitrification, were tested. In the present report, results of a comparative study of the effects of vitrification, encapsulation-dehydration and encapsulation-vitrification on survival of frozen yam apices and subsequent shoot regeneration are presented. An attempt has also been made to compare the effects of the former two techniques on the morphological pattern of growth of the thawed apices during regeneration. 234 Cryopreservation of Tropical Plant Germplasm

Materials and methods The plant materials consisted of in vitro plantlets of two edible yams (Dioscorea alata, D. wallichii) and one medicinal yam (D. floribunda) which are being maintained at the tissue culture repository of NBPGR. The in vitro plantlets of these species were established by culturing nodal explants on Murashige and Skoog (MS) medium (Murashige and Skoog 1962) supplemented with 30 g/L sucrose, 8 g/L bacto agar, naphtalene acetic acid (NAA) and benzylaminopurine (BAP). These cultures were maintained through periodic subculture on the same media. Shoot apices measuring 0.6–2.5 mm, depending on the procedure and/or species, were dissected from 6 to 8-week-old plantlets and were used as explants for cryopreservation.

Cryopreservation procedures

Encapsulation-dehydration technique The procedure used in this technique was described in detail in our earlier report (Mandal et al. 1996a). The encapsulated shoot apices were precultured for 3 d in liquid medium with 0.75M sucrose followed by 6 h of desiccation in sterile airflow, rapid freezing and slow thawing.

Vitrification procedure Isolated precultured apices were pretreated with a mixture of 2M glycerol plus 0.4M sucrose for 20 min at 25°C. These apices were then dehydrated using cryoprotectant mix such as PVS2 and PVS3 (Sakai et al. 1990) at 0°C for 90 min prior to immersion in liquid nitrogen. After apices were kept for at least 1 h in liquid nitrogen, they were rewarmed at 37°C for 10–15 min and treated with 1.2M sucrose solution for 20 min before plating on recovery medium.

Encapsulation-vitrification procedure The precultured shoot apices were encapsulated in alginate beads containing 2M glycerol and 0.4M sucrose. These beads were then dehydrated in PVS2 for 90 min at 0°C before plunging in liquid nitrogen. After apices were kept for at least 1 h in liquid nitrogen, beads were thawed at 37°C and then treated with 1.2M sucrose before plating on recovery medium.

Growth recovery For growth recovery, agar-solidified MS media supplemented with zeatin/6– benzyl amino purine, naphthalene acetic acid and gibberellic acid in various combinations and concentrations were used depending on the species as detailed in our earlier report (Mandal et al. 1996a). Initially, the thawed apices were kept in the dark for 2 weeks followed by exposure to light during recovery growth. Cryopreservation techniques 235

Results

Encapsulation-dehydration Experimental results of our earlier studies allowed us to determine the optimal preculture and desiccation conditions and duration such as 3 or 7 d culture in medium with 0.75M sucrose, followed by 6 h of desiccation under sterile airflow (Mandal et al. 1996a). In that study, survival frequencies of cryopreserved apices ranged between 71 and 58% for the three Dioscorea species (D. wallichii, D. alata and D. floribunda) and the regeneration frequencies from 37 to 0%. However, in the present study the survival percentage of cryopreserved apices did not increase but regeneration of shoots from thawed apices of D. floribunda could be achieved by culturing them on a medium with modified concentration of growth regulators. In all experiments the majority (57%) of the plants that were obtained from frozen apices of D. wallichii and D. alata regenerated through an intermediary callus formation. Microscopic study of morphological pattern and histology performed during the recovery growth phase of thawed apices revealed that the surviving apices contained a considerable number of damaged cells. The extent of damage perhaps played a significant role in regeneration of a shoot either directly or through callus formation (Mandal et al. 1996b).

Vitrification In the vitrification technique, resistance to deep-freezing was induced by dehydration with two different cryoprotectant solutions of which the best results were obtained with PVS2. Survival of thawed apices was recorded in all the three species. However, high frequency survival of thawed apices and subsequent regeneration of shoots were recorded in D. floribunda (Table 1). The most interesting observation recorded was that direct regeneration of shoots from thawed apices was noted with D. floribunda (Table 2). Shoots emerged even when the original intact explants were clearly visible.

Encapsulation-vitrification The results of experiments with encapsulation-vitrification on the survival of cryopreserved shoot apices and subsequent shoot regeneration revealed that survival could be obtained with all the three species and subsequent shoot regeneration with two species, although the frequencies were relatively lower (Table 1). 236 Cryopreservation of Tropical Plant Germplasm

Table 1. Survival and shoot regeneration rate (%) of apices of three yam (Dioscorea spp.) species cooled to –196°C by three different cryopreservation techniques (encapsulation-dehydration; vitrification; encapsulation-vitrification) Cryopreservation technique Encap.-dehy. Vitrification Encap.-vitrif. Shoot Shoot Shoot Species Survival regen. Survival regen. Survival regen. D. alata 64 21 33 0 20 0 D. wallichii 71 37 71 28 33 14 D. floribunda 58 – † 87 30 50 16 † Frequency was not recorded.

Table 2. Rate (%) of direct and callus-mediated regeneration of shoots from apices of two yam (Dioscorea spp.) species cooled to –196°C using two different cryogenic techniques (encapsulation-dehydration; vitrification; encapsulation-vitrification) Cryopreservation technique Encapsulation-dehydration Vitrification Species Direct regen. Regen. via callus Direct regen. Regen. via callus D. wallichii 43 57 50 50 D. floribunda – – 75 25

Discussion During the present study it was demonstrated that use of the encapsulation- dehydration technique could produce moderate to high rates of survival of cryopreserved apices and subsequent plant regeneration in all the three yam species tested (D. alata, D. wallichii and D. floribunda). However, use of the vitrification technique resulted in equally good results with D. wallichii, significantly higher survival and regeneration frequency with D. loribunda but very low frequency with D. alata. These results indicate that yam apices can withstand deep-freezing in liquid nitrogen. However, there was a clear genotypic variation in response to one cryogenic technique or the other. Therefore, further studies are needed with more genotypes to develop an efficient protocol applicable to a wide range of genotypes/species of yams. With D. alata, neither vitrification nor encapsulation-dehydration could produce very high frequency of plant regeneration from thawed apices. Therefore, further studies are still required to establish if D. alata has inherent problems in withstanding deep- freezing or if survival and regrowth can be improved by modifying the cryogenic procedure and/or recovery growth media. Use of the encapsulation-vitrification technique resulted in relatively low frequency of survival of frozen apices and subsequent plant regeneration in all the three species tested. This could perhaps be improved by further experimentation or modification of the procedure. One of the most interesting observations recorded during this study was the high frequency of direct regeneration of shoots which was obtained from frozen apices of D. floribunda when the vitrification technique was employed. Even with D. wallichii the frequency of direct regeneration was apparently higher with the Cryopreservation techniques 237 vitrification technique. Though the pattern of recovery growth, i.e. direct or callus-mediated regeneration, depends largely on the concentration and composition of growth hormones of the culture medium as shown with potato (Harding and Benson 1994), it may also depend on the extent of damage that occurs in the thawed apices (Mandal et al. 1996b). Therefore, further studies are required to establish if there is any relation between the use of a particular cryogenic procedure, the extent of damage that occurs during cryopreservation and the pattern of recovery growth of the thawed apices. In conclusion, it can be said that yam apices can be cryopreserved using various cryogenic techniques. However, critical studies are still required to develop an efficient cryogenic protocol applicable to a wide array of genotypes that also produce a high frequency of survival and direct regeneration of shoots. In this respect, the vitrification technique may be promising, because the frequency of direct regeneration with D. floribunda was significantly higher when this technique was used.

Acknowledgements I would like to gratefully acknowledge Dr P.L. Gautam, Director, NBPGR for his encouragement during this study. Financial support of DFID/IPGRI (Holdback project R6110(H)) and of DBT, Govt. of India (NFPTCR Project) is duly acknowledged.

References Gonzalez Arnao, M.T., F. Engelmann, C. Huet and C. Urra. 1993. Cryopreservation of encapsulated apices of sugarcane: Effect of freezing procedure and histology. Cryo– Letters 14: 303–308. Harding, K. and E.E. Benson. 1994. A study of growth, flowering, and tuberization in plants derieved from cryopreserved potato shoot-tips: Implications for in vitro germplasm collections. Cryo–Letters 15: 59–66. Malaurie, B., M.F. Trouslot, F. Engelmann and N. Chabrillange. 1998. Effect of pretreatment conditions on the cryopreservation of in-vitro culture of yam (Dioscorea alata ‘Brazo Fuerte’ and D. bulbifera ‘Noumea Imboro’) shoot apices by encapsulation- dehydration. Cryo–Letters 19:15–26. Mandal, B.B., K.P.S. Chandel and S. Diwvedi. 1996a. Cryopreservation of yam (Dioscorea spp.) shoot apices by encapsulation-dehydration. Cryo–Letters 17: 165–174. Mandal, B.B., S.K. Malik and K.P.S. Chandel. 1996b. Cryopreservation of encapsulated apices of yams (Dioscorea spp.) ultra structural studies on recovery growth. SLTB Annual Meeting, Dundee, Scotland. Selected Abstracts, Cryo Letters 18: 73. Matsumoto, T., A. Sakai, C. Takahashi and K. Yamada. 1995. Cryopreservation of in vitro grown apical meristems of wasabi (Wasabia japonica) by encapsulation-vitrification method. Cryo–Letters 16: 189–196. Murashige, T. and F. Skoog. 1962. A revised medium for rapid growth and bioassays with tobacoo tissue cultures. Physiologia Plantarum 15:473–497. Sakai, A., S. Kobayashi and Y. Oiyama. 1990. Cryopreservation of nucelar cells of naval orange (Citrus sinensis var. brasiliensis Tanaka ) by vitrification. Plant Cell Reports 9: 30– 33. 238 Cryopreservation of Tropical Plant Germplasm

Cryopreservation of proliferating meristem cultures of banana Bart Panis¹, Hilde Schoofs¹, Nguyen Tien Thinh² and Rony Swennen¹ ¹ Laboratory of Tropical Crop Improvement, Catholic University of Leuven, 3001 Heverlee, Belgium ² Department of Biotechnology and Nuclear Techniques, Nuclear Research Institute Dalat, Dalat City, Viet Nam

Introduction In theory, any regenerable tissue is suitable for storage of germplasm through cryopreservation. Cryopreservation of seed (Chin 1996) or zygotic embryos (Abdelnour et al. 1992) is only applicable for wild diploid bananas. Also, cryopreservation of regenerable banana cell suspensions is possible (Panis et al. 1990). However, their initiation is very time-consuming. Therefore, shoot meristematic tissues are the only acceptable alternative for long-term storage of banana germplasm. Two types of highly meristematic and regenerable in vitro tissues can be obtained in banana: (i) tiny meristems isolated from in vitro plants, and (ii) proliferating meristem cultures containing 'cauliflower-like' meristem groups. Shoot meristem tips of 0.5–1 mm diameter excised from rooted in vitro plants were successfully cryopreserved using the vitrification method. Post-thaw survival rates ranged between 41.1 and 91.6% depending on the cultivar (Thinh et al. 1999). Preliminary experiments carried out at K.U. Leuven proved that slow freezing in the presence of DMSO was totally ineffective for the cryopreservation of proliferating banana meristems. Ice crystallization in the extracellular solution during slow freezing was always lethal (Panis 1995). Encapsulation-dehydration (Dereuddre et al. 1990), which proved to be highly successful for meristems of many plant species, only resulted in a maximal post-thaw recovery rate of 8.1% in banana (Panis 1995). In this report we describe two successful cryopreservation methods applied to highly proliferating 'cauliflower-like' meristem cultures, i.e. simple freezing which involves a sucrose preculture (Panis et al. 1996) and a cryopreservation method that combines this preculture with the vitrification method.

Material and methods

Simple freezing method

Starting material Cauliflower-like meristem clusters can be obtained on Murashige and Skoog (1962) semi-solid medium supplemented with 10 or 100 µM benzyladenine (BA), depending on the cultivar, 1 µM indole acetic acid (IAA) and 3% sucrose. Every 1 to 2 months, the material was subcultured but only small clumps of 'cauliflower- like' meristems were selected and transferred to fresh medium. Cryopreservation techniques 239

Preculture As soon as ‘cauliflower-like’ clumps were obtained, white meristematic clumps of about 4 mm diameter, each containing at least four apical domes, were excised 6 weeks after the last subculture. They were transferred to a preculture medium containing Murashige and Skoog salt and vitamins, 10 µM BA, 1 µM IAA, 0.4M sucrose and 2 g/L Gelrite. At the end of the pregrowth phase, 2 weeks after inoculation, meristem survival and growth were determined visually.

Cryopreservation Small clumps of 5–15 mg, containing 3 to 6 meristematic domes, were excised from the precultured buds. Brown tissues were removed and only the most healthy, as indicated by a white-yellowish colour, were retained. These clumps were transferred to 2-ml sterile cryovials (Greiner, Germany) and directly plunged into liquid nitrogen. Each cryotube contained 7 to 10 clumps. Samples were stored for at least 1 h in liquid nitrogen.

Post-thaw recovery After storage, rapid thawing took place by stirring the frozen cryotubes for 1.5 min in a water-bath set at 40°C. Control (precultured but non-frozen) and frozen meristems were transferred to semi-solid or liquid regeneration medium. After 1 week of culture in the dark, dishes and flasks were transferred to continuous light. Four weeks after freezing, regrowth was determined under a binocular microscope. Two types of surviving tissues were distinguished, i.e. shoots and callus. Recovering clumps were further regenerated in test tubes on regeneration medium. As soon rooted plants reached the top of the test tube, they were planted in the soil.

Vitrification of proliferating meristems

Starting material Proliferating ‘cauliflower-like’ meristem cultures, with or without a 2-week sucrose preculture, were prepared according to the procedures described above. From these cultures, meristem clumps of 1.5 to 3 mm diameter (containing 3 to 5 meristematic domes) were excised.

Loading Loading solution was added to the meristem clumps and kept for 20 min at room temperature. The filter-sterilized loading solution contained 2M glycerol and 0.4M sucrose dissolved in MS medium (pH 5.8).

Treatment with vitrification solution (dehydration) and freezing The meristems were then subjected to the PVS2 solution for 60 min at 0°C. The PVS2 solution consists of 30% glycerol, 15% ethylene glycol (EG), 15% DMSO and 0.4M sucrose (Sakai et al. 1990). After the PVS2 treatment, cryotubes were plunged into liquid nitrogen. 240 Cryopreservation of Tropical Plant Germplasm

Thawing and deloading The tubes were rapidly thawed by stirring the cryotubes in a warm water-bath (40°C) for 1 min and 20 s. Directly after thawing, the toxic PVS2 solution needs to be removed and replaced by the unloading solution. The filter-sterilized unloading solution consists of 1.2M sucrose dissolved in MS medium (pH 5.8). The meristems were unloaded for 15 min at room temperature.

Regeneration Control and frozen meristems were placed on two sterile filter papers on top of a semi-solid hormone-free MS medium containing 0.3M sucrose. After 2 days, the meristems were transferred onto regeneration medium (also in Petri dishes) without filter papers. The first week of culture always took place in the dark.

Results

Simple freezing Previous experiments revealed that preculture on proliferation medium with 0.4M sucrose for 2 weeks was optimal to obtain the highest rate of post-thaw survival (Panis et al. 1996). However, we observed that thawed meristem clumps that were transferred to semi-solid medium often became black due to oxidation of polyphenols. This observation was often correlated with the outgrowth of non- regenerable watery callus. In order to dilute the polyphenols released, we therefore transferred the thawed meristem clumps also to a liquid regeneration medium. The results are indicated in Table 1. For 8 out of 12 cultivars tested, we observed that regeneration in liquid regeneration medium results in higher viability rates compared with regeneration on semi-solid medium. What is more important, the frequency of non-regenerable callus was considerably reduced. The best results were obtained for the three ABB cultivars – Bluggoe, Monthan and Cachaco – which are also known to possess a high resistance to drought and pathogen stress in the field. Three Hand Planty, Nakitengwa (only successful in liquid medium), Kisubi and Guyod responded very poorly. The two accessions of the cultivar Prata and the two AAA, non-highland bananas showed an intermediate behaviour. Only for the cultivar Mbwazirume was no survival obtained regardless of the regeneration conditions. Up to now 23 accessions have been frozen according to the simple freezing protocol (results not shown). Viability rates vary between 0 and 75% and are highly influenced by the genomic constitution of the accession (see also results in Table 1). Proliferating banana meristems can be classified in six classes, based on morphological characteristics and oxidation of polyphenols (Schoofs 1997). The class that proliferates best and exhibits the lowest degree of polyphenol oxidation (containing most of the ABB cultivars) also results in the highest post-thaw viability rates. Further improvement of this protocol can thus be expected from an optimization of the proliferation degree by, for example, the application of different plant growth regulator balances in the culture media. Cryopreservation techniques 241

Table 1. Post-thaw viability rates (%) of precultured proliferating banana meristems on solid or in liquid regeneration medium. Values in parentheses represent the frequency of viable clumps giving rise to non-regenerable callus. Each value is the result of the regeneration of 4 to 31 cryotubes each containing 8 to 10 meristem clumps. Cultivar Group Solid (%) Liquid (%) Bluggoe ABB 65.8 ± 27.6 (31) 67.2 ± 13.2 (1) Monthan ABB 25.2 ± 19.6 (61) 34.0 ± 28.7 (9) Cachaco ABB 30.9 ± 29.3 (34) 59.1 ± 17.0 (3) Three Hand Planty AAB Plantain 20.8 ± 26.0 (60) 9.0 ± 9.3 (27) Prata AAB 29.6 ± 26.1 (72) 34.2 ± 25 (42) Prata JD AAB 22.0 ± 18.8 (83) 25.5 ± 17.3 (31) Grande Naine JD AAA 27.8 ± 24.1 (55) 20.7 ± 15.5 (16) Williams JD AAA 21.9 ± 12.6 (69) 35.0 ± 23.1 (20) Nakitengwa AAA highland 0 ± 0 (0) 11.7 ± 14.4 (39) Mbwazirume AAA highland 0 ± 0 (0) 0 ± 0 (0) Kisubi AB 25.0 ± 37.6 (63) 14.0 ± 11.7 (68) Guyod AA 6.3 ± 8.8 (100) 16.7 ± 23.6 (0)

Vitrification of proliferating meristems Preliminary experiments using the vitrification method applied on proliferating meristem cultures were not successful since these tissues proved to be very susceptible to dehydration with PVS2. After a 20-min exposure, without freezing, 75% of the meristems failed to recover (Panis 1995). Toxicity of PVS2 to banana meristems excised from rooted plants could be overcome by using an additional loading phase prior to dehydration and the application of the vitrification solution at reduced temperature (0°C) (Thinh et al. 1999). Tolerance to highly concentrated vitrifying (dehydrating) solutions is an essential step in the vitrification protocol since only after a sufficient dehydration will the concentrated cell content be able to vitrify upon rapid freezing without the formation of lethal ice crystals. The effect of preculture of proliferating meristems on 0.4 M sucrose for 2 weeks on their resistance to the PVS2 solution is shown in Figure 1A. The sucrose treatment had a clear positive effect on the survival after dehydration. The viability rates for all cultivars exceeded 80% except for Mbwazirume where 68% of the meristematic clumps survived. Also, the frequency of non-regenerable callus was considerably reduced. We also observed that for the two AAA highland bananas, a sucrose treatment was almost essential for obtaining a sufficient amount of regenerable regrowth. Figure 1B indicates that a sucrose preculture was not only improving tolerance of meristem clumps to PVS2 but was also increasing their freezing tolerance. The frequency of callus regrowth, however, was high. When we compare these results with those obtained using the simple freezing method (results not shown), an increase of post-thaw viability rates for all cultivars was observed. This increase ranged from 2% (for Prata JD) to 35% (for Three Hand 242 Cryopreservation of Tropical Plant Germplasm

A

100 90 callus 80 shoots 70

60 50

40 viability (%) 30 20

10 0 p4 THP p4 MON p4 NAKI p4 MBW p4 WI JD sucr THP p4 PR JD p4 GN JD sucr MON sucr NAKI sucr MBW sucr WI JD sucr PR JD sucr GN JD

B

100 90 callus 80 shoots 70

60 50

40 viability (%) 30 20

10 0 p4 THP p4 MON p4 NAKI p4 MBW p4 WI JD sucr THP p4 PR JD p4 GN JD sucr MON sucr NAKI sucr MBW sucr WI JD sucr PR JD sucr GN JD

Fig. 1. Survival rates, indicated by callus and shoot regeneration, of proliferating meristems of different banana cultivars, without (A) or with (B) freezing. The meristem cultures were subjected to dehydration with PVS2 after a 2-week culture on p4 medium (p4) and medium with 0.4M sucrose (sucr). MONT, Monthan (ABB group); THP, Three Hand Planty (AAB plantain); PR JD, Prata JD (AAB group); GN JD, Grand Naine JD (AAA group); WI JD, Williams JD (AAA group); NAKI, Nakitengwa (AAA highland banana) and MBW, Mbwazirume (AAA highland banana).

Planty). Also the frequency of regenerable regrowth was higher for all cultivars. An increase between 3 and 20% was observed. Mbwazirume, a cultivar that Cryopreservation techniques 243 previously proved to be totally recalcitrant to cryopreservation, was able to regrow after vitrification, although still at a very low frequency. We believe that a further improvement of this vitrification protocol with emphasis on the recovery conditions will lead to a cryopreservation protocol suitable for most banana cultivars.

Conclusion We demonstrated that a sucrose preculture of proliferating meristems is not only an essential step in the simple freezing protocol, but also increases post-thaw recovery rates after vitrification. The mode of action of sucrose with respect to cryoprotection is not very well understood. Preliminary experiments indicated that a sucrose preculture results in a lower moisture content and higher sugar and protein contents of proliferating meristems (Panis 1995; Panis et al. 1998). Recently, we found that preculture also affects the membrane lipid composition (Panis et al. 1998). The membrane fatty acids became shorter, thereby increasing the fluidity of the membrane. It is likely that a better knowledge of the mechanisms of sucrose preculture will lead to a more efficient optimization of different cryopreservation protocols.

Acknowledgements The author thanks INIBAP (International Network for the Improvement of Banana and Plantain), the Belgian Administration for Development Cooperation (BADC/ABOS) and the ‘Onderzoeksfonds’ of the Catholic University of Leuven for their financial support. SGRP (System-wide Genetic Resources Programme) is gratefully acknowledged for providing travel funds to participate in the workshop.

References Abdelnour–Esquivel, A., A. Mora and V. Villalobos. 1992. Cryopreservation of zygotic embryos of Musa acuminata (AA) and Musa balbisiana (BB). Cryo–Letters 13: 159–164. Chin, H.F. 1996. Germination and storage of banana seeds. Pp. 218–227 in Proceedings of the workshop on 'New Frontiers in Resistance Breeding for Nematode, Fusarium and Sigatoka', 2–5 October 1995, Kuala Lumpur, Malaysia. E.A. Frison, J.P. Horry and D. De Waele (eds.). INIBAP, Montpellier, France. Dereuddre, J., C. Scottez, Y. Arnaud and M. Duron. 1990. Resistance of alginate-coated axillary shoot tips of pear tree (Pyrus communis L. cv. Beurré Hardy) in vitro plantlets to dehydratation and subsequent freezing in liquid nitrogen: effects of previous cold hardening. Comptes Rendus de l’Académie des Sciences Paris, 310, Série III: 317–323. Murashige, T. and F. Skoog. 1962. A revised medium for rapid growth and bioessays with tobacco tissue cultures. Physiologia Plantarum 15: 473–497. Panis, B. 1995. Cryopreservation of banana (Musa spp.) germplasm. Dissertationes de Agricultura 272, Catholic University of Leuven, Belgium. Panis, B., N. Totté, K. Van Nimmen, L.A. Withers and R. Swennen. 1996. Cryopreservation of banana (Musa spp.) meristem cultures after preculture on sucrose. Plant Science 121: 95–106. Panis, B., K. Vandenbranden, H. Schoofs and R. Swennen. 1998. Conservation of banana germplasm through cryopreservation. Acta Horticulturae 461: 515–521. 244 Cryopreservation of Tropical Plant Germplasm

Panis, B., L.A. Withers and E. De Langhe. 1990. Cryopreservation of Musa suspension cultures and subsequent regeneration of plants. Cryo–Letters 11: 337–350. Sakai, A., S. Kobayashi and I. Oiyama. 1990. Cryopreservation of nucellar cells of navel orange (Citrus sinensis Osb. var. brasiliensis Tanaka) by vitrification. Plant Cell Reports 9: 30–33. Schoofs, H. 1997. The origin of embryogenic cells in Musa. Dissertationes de Agricultura 330, Catholic University of Leuven, Belgium. Thinh, N.T., H. Takagi and S. Yashima. 1999. Cryopreservation of in vitro-grown shoot tips of banana (Musa spp.) by vitrification method. Cryo–Letters 20: (in press). 246 Cryopreservation of Tropical Plant Germplasm

Application of cryopreservation protocols at a clonal genebank Barbara M. Reed, Jeanine DeNoma and Yongjian Chang USDA-ARS National Clonal Germplasm Repository, Corvallis OR 97333-2521, USA and Department of Horticulture, Oregon State University, Corvallis OR 97331, USA

Introduction For the past 20 years scientists have talked about the promise of using cryopreservation as a long-term storage technique (Withers 1980, 1991; Engelmann 1991). Now cryopreservation of plant germplasm is actively used throughout the world. Methods for storage of plants as seeds, pollen, dormant buds and apical meristems are in use in many facilities (Razdan and Cocking 1997). Although long-term storage is already in place for many seed-propagated crops, organized short-, medium- and long-term preservation of clonally propagated plants has mostly developed in the last 20 years. Development of efficient methods for germplasm preservation is a high priority for many nations. Recent improvements in cryopreservation techniques now make it possible to protect germplasm collections through base (long-term back-up) storage in liquid nitrogen. Active collections of clonal germplasm are growing plants available for evaluation and distribution while base collections are in long-term storage and used only if the active collections are lost. With seeds a base collection can usually be stored frozen at –80 or – 196°C, but with clonally propagated crops living plant material must be maintained. Back- up collections of clonally propagated crops are field collections at a second site, or in vitro collections. Cryopreservation is the newest addition to the germplasm storage system. Storage of clonal materials in liquid nitrogen as a base collection is the goal of many genebanks. Storage of many species and cultivars as dormant buds, seeds, pollen and shoot-tips is being investigated at this time. In furthering the thought that cryopreservation should be used rather than discussed, we initiated a long-term base collection of pear meristems and pollen in liquid nitrogen.

Materials and methods

Pollen storage Pollen was collected from unopened "popcorn stage" blossoms and the anthers separated from the flowers using sieve screens; the undehisced anthers dried and dehisced overnight on the lab bench (Craddock 1987). Initial moisture content (MC) of air-dried pollen was 25–35% with 85–95% germination. Pollen/anther mixtures could be cryopreserved after 24 h air-drying or stored in a desiccator at 5°C until cryopreserved. Vials were directly submerged in liquid nitrogen for storage. Thawing was at room temperature. Ongoing cryopreservation projects 247

Meristem storage Pear genotypes were randomly chosen from 168 accessions available in the in vitro collection of the National Clonal Germplasm Repository – Corvallis (NCGR) and screened for response to cryopreservation techniques. Culture conditions and plant condition were evaluated to produce plants suitable for cryopreservation. Healthy plants were required for the best survival. Initially all cultures were screened, but after initial tests, only healthy, actively growing plants with no obvious growth inhibition were used. Contraindications included hyperhydration and shoot-tip necrosis.

Plant culture In vitro plantlets were multiplied on Cheng's basal medium (Cheng 1979) with per liter: 1 mg N–benzyladenine, 3 g agar (Bitek, Difco, Detroit, MI) and 1.75 g Gelrite (Schwizerhall, South Plainfield, NJ) adjusted to pH 5.2. Cultures were grown in Magenta GA7 culture vessels (Magenta Corp. Chicago, Ill.) at 25°C -2 -1 under a 16-h photoperiod with 25 µmol m s using cool white florescent bulbs. Growth regulators were obtained from Sigma Chemical Co., St. Louis, MO. Plantlets were given 1-3 weeks of cold acclimation (CA) in an incubator -2 -1 with 22°C, 8-h days (3 µmol m s) and –1°C, 16-h nights before cryopreservation (Reed 1988).

Slow freezing Meristems were pretreated for 2 days in the CA incubator on medium with 5% DMSO and additional Gelrite (0.3 g/L), then transferred to 0.25 ml liquid medium in 1.2-ml plastic cryotubes (Reed 1990). One millilitre of the cryoprotectant PGD [10% each polyethylene glycol (MW 8000), glucose and DMSO in liquid medium] was added over a half-hour period (Finkle and Ulrich 1979). Samples were equilibrated at 4°C for 30 min after which the cryoprotectant was drawn down to 1 ml, the samples frozen at 0.3 or 0.5°C/min to –40°C and then plunged into LN. Samples were thawed for 1 min in 45°C water then 1 min in 22°C water, rinsed in liquid medium and plated on recovery medium.

Vitrification A modification of the technique for white clover was used (Yamada et al. 1991). Meristems were pretreated for 2 days in the CA incubator on medium with 5% DMSO and additional Gelrite (0.3 g/L), then transferred to 1.2-ml plastic cryotubes with PVS2 cryoprotectant (30% glycerol, 15% ethylene glycol and 15% DMSO in liquid medium with 0.4M sucrose) on ice and stirred. After 20 min, vials were submerged in LN. Samples were rewarmed as in slow freezing then rinsed in liquid MS medium with 1.2M sucrose.

Screening Each technique was tested in single experiments with 20 meristems of each accession. Controls exposed five meristems to cryoprotectants but not to liquid nitrogen. 248 Cryopreservation of Tropical Plant Germplasm

Storage Genotypes with greater than 40% regrowth were candidates for storage. The 150 meristems cryopreserved (25/vial) for storage were transferred to a travel dewar for shipping. Twenty-five meristems were removed from the dewar before shipping, thawed and regrown as controls. The remaining 125 meristems were sent to the National Seed Storage Laboratory (NSSL) in Fort Collins Colorado.

Shipping The shipping dewar was prepared in advance by filling and refilling it with LN until some LN remained in the well. Cryovials on canes were transferred to the shipping dewar and sent by express mail with the required hazardous materials shipping documents. The shipping dewar retains LN temperatures for 15 days, so it could also be used for international shipments.

Handling at NSSL Canes were removed from the travel dewar and placed in long-term storage in LN. One tube was thawed after one or more weeks of storage as a control for travel and storage and regrown. The remaining 100 meristems remained in storage as part of the base collection.

Results and discussion Development of methods can only be done on a few genotypes at a time; however, it is very important to know how a diverse germplasm collection will respond if base storage is a goal (Reed et al. 1997). Regrowth among 93 genotypes screened varied widely with 12 showing no regrowth, 41 with 1– 39% regrowth, and 40 genotypes with 40–100% regrowth. We chose a moderate regrowth rate (³40%) as the minimum for germplasm storage so as not to preselect as we store and also so that a smaller number of meristems could be stored. About half of the tested genotypes were suitable for storage using the 40% minimum as a guideline. Only 17 of 73 genotypes had less than 25% survival rates following slow freezing, and 33% had greater than 50% survival. A comparison of 28 genotypes with vitrification and slow freezing found greater than 50% recovery for 17 genotypes with slow freezing and 12 with vitrification. Six genotypes did poorly (<50% recovery) with both techniques. Each method available has distinct advantages and disadvantages. Slow freezing is effective for many taxa and material handling is easy, but it requires expensive equipment. This would limit its use in many laboratories. Vitrification is quick and requires no special equipment, but the solutions tend to be toxic to the plants and careful timing is necessary for successful recovery, thus limiting the number of vials that can be handled at one time. Encapsulation and dehydration of alginate beads is successful for many genera but requires more individual handling of meristems which makes it more labour-intensive than the other methods. We chose the slow freezing technique for several reasons. First, it worked well for over half of the genotypes tested. Second, it was easy to use with large Ongoing cryopreservation projects 249 numbers of samples. Third, the cryoprotectants were less toxic to the plants. Fourth, the timing of addition and removal of cryoprotectant were not highly critical, so survival was less likely to be affected by interruptions during handling. More than 100 Pyrus meristem accessions (73 cultivars and 31 samples of species accessions) were shipped to NSSL for base storage from 1993 to 1998 and additional accessions are sent as they become available. Pollen storage at NCGR consists of one to three vials each of 37 accessions (13 cultivars and 24 Pyrus species).

Conclusions Long-term base storage of Pyrus clonal germplasm as meristems and pollen is now a reality. Germplasm conservation in liquid nitrogen is now possible owing to the many techniques available. The hundreds of genotypes and species successfully cryopreserved in individual experiments should now be stored as base collections. Field, greenhouse and in vitro genebanks are important as working collections, but require safe back-ups in case of insects, diseases or natural disasters. Cryopreservation is now a viable technique to provide a reliable back-up for these working collections in a form that requires little maintenance. Implementation of base storage must now be factored into the budgets of genebanks to provide for the safety of these invaluable collections.

References Cheng, T.Y. 1979. Micropropagation of clonal fruit tree rootstocks. Compact Fruit Tree 12:127–137. Craddock, W.J.H. 1987. Cryopreservation of Pollen. Master of Science Thesis, Oregon State University, Corvallis. Engelmann, F. 1991. In vitro conservation of horticultural species. Acta Horticulturae 298:327–332. Finkle, B.J. and J.M. Ulrich. 1979. Effects of cryoprotectants in combination on the survival of frozen sugarcane cells. Plant Physiology 63:598–604. Razdan, M.K. and E.C. Cocking (eds.). 1997. Conservation of Plant Genetic Resources In Vitro, Vol. 1. Science Publishers, Inc, Enfield, New Hampshire. Reed, B.M. 1988. Cold acclimation as a method to improve survival of cryopreserved Rubus meristems. Cryo–Letters 9:166–171. Reed, B.M. 1990. Survival of in vitro-grown apical meristems of Pyrus following cryopreservation. HortScience 25:111–113. Reed, B.M., J. DeNoma, J. Luo, Y. Chang and L. Towill. 1997. Cryopreserved storage of a world pear collection. In Vitro Cellular Development Biology 33:51A. Withers, L.A. 1980. Low temperature storage of plant tissue cultures. Pp. 101–150 in Advances in Biochemical Engineering, Vol. 18. (A. Fiechler ed.). Springer Verlag, Berlin. Withers, L.A. 1991. In-vitro conservation. Biological Journal of the Linnean Society 43:31–42. Yamada, T., A. Sakai, T. Matsumura and S. Higuchi. 1991. Cryopreservation of apical meristems of white clover (Trifolium repens L.) by vitrification. Plant Science 78:81–87. 250 Cryopreservation of Tropical Plant Germplasm

Advances in potato cryopreservation at CIP Ali M. Golmirzaie and Ana Panta International Potato Center (CIP), Lima, Peru

Introduction CIP, during its more than 25 years of working on potato conservation, is continuously looking for more efficient conservation methods. Currently, the in vitro CIP potato collection contains over 7000 accessions and is rapidly increasing with new improved material. For this reason, cryopreservation, which is considered the safest and less labour-consuming method for the long- term conservation, may be the best alternative to store potato germplasm for an indefinite period without genetic erosion. In 1995, in a United Nations Development Program (UNDP) collaborative project with Cornell University, CIP started testing cryopreservation by vitrification, a method developed by P. Steponkus (Cornell University, USA). With this method, shoot-tips (the meristem dome and several leaf primordia) are dehydrated by exposure to concentrated solutions of sugars and cryoprotectants, then frozen rapidly in liquid nitrogen (–196°C). The use of shoot-tips has an advantage over other tissues. They can be regenerated into plants that are identical to the mother plants. This article describes attempts to assess the feasibility of applying this technology for the long-term conservation of large potato collections.

Material and methods

Mother plants Genotypes of six potato species with different ploidy levels were taken from the Potato Base Collection held at CIP. These were Solanum tuberosum subsp. andigena (tetraploid), S. chaucha (triploid), S. phureja (diploid), S. stenotomum (diploid), S. goniocalyx (diploid), natural hybrids of S. goniocalyx × S. stenotomum (diploid) and S. stenotomum × S. goniocalyx (diploid). This material is being conserved in long-term in vitro storage by slow growth at low temperature on a medium containing sorbitol as an osmotic growth retardant.

In vitro growth Plants were micropropagated by single-node stem segments in tubes containing an MSA medium (Golmirzaie and Panta 1997b). The medium was poured into magenta jars in two layers. First, semi-solid MSA medium (15 ml) was poured, replacing the gelling agent phytagel with agar. Then nine plant single nodes were planted. After 4–5 days, liquid MSA medium (10 ml) was added. Magenta jars were covered with a transparent polypropylene film with a filter (SIGMA, C6920). Ongoing cryopreservation projects 251

Cryopreservation by vitrification The vitrification method, based on Steponkus’ method (Steponkus et al. 1992; Golmirzaie and Panta 1997a), involves removing axillary shoot-tips (1.5 mm long) from plantlets grown in vitro for 30–45 days. The shoot-tips consist of 4– 5 leaf primordia and the apical dome. They are first precultured on modified Murashige-Skoog medium supplemented with 0.04 mg/L kinetin, 0.5 mg/L indol acetic acid, 0.2 mg/L gibberellic acid and 0.09M sucrose, for 24 h under proper incubation conditions for micropropagation, and then on the same medium containing 0.06M sucrose for 5 h at room temperature. After preculture, the shoot-tips are dehydrated by placing them in a vitrification solution containing ethylene glycol: sorbitol: bovine serum albumin (50:15:6 wt%) for 50 min at room temperature. The shoot-tips are then transferred to 0.25-ml propylene straws with 150 µl of vitrification solution, and the straws are rapidly quenched in liquid nitrogen. Following storage in liquid nitrogen, the shoot-tips are thawed, expelled from the straws into a hypertonic (1.5 osmolal) sorbitol solution at room temperature, and incubated for 30 min. The shoot-tips are then plated on M1 semi-solid potato meristem medium containing Murashige-Skoog salts supplemented with 0.04 mg/L kinetin, 0.1 mg/L gibberellic acid and 25 g/L sucrose (M1), and maintained under normal incubation conditions for micropropagation. After 4–6 weeks, survival is evaluated by counting plantlets that grow from the shoot-tips. This protocol was applied following the next steps.

Stage 1: Testing the vitrification protocol with 80 potato genotypes Eighty different genotypes were processed by following the above-mentioned protocol and stored in liquid nitrogen. For each genotype, 120 shoot-tips were stored, 10 shoot-tips were loaded into each straw. For dehydration control, 20 shoot-tips (two repetitions of 10 samples) were treated with the vitrification solution and then directly expelled in the sorbitol solution and plated on semi- solid medium after 30 min. Survival of frozen material was evaluated twice. For each evaluation, two straws containing 10 shoot-tips each were removed from liquid nitrogen and thawed. First thawing was after 1 day of storage; second thawing was after 3 months. Survival was evaluated 6 weeks after thawing. For those accessions that did not respond even when the assay was repeated once or twice, the assay was performed again by using dehydration periods of 45, 55 and 60 min.

Stage 2: Improvement of cryopreservation method

Use of apical shoot-tips from vigorous in vitro plants Seven genotypes chosen randomly were micropropagated by several subcultures of apical tips in magenta jars. The growth time between subcultures was 3 weeks. After seven subcultures, 140 apical shoot-tips from vigorous plants of each genotype were isolated. They were processed by the vitrification method, using 50 min of dehydration. 252 Cryopreservation of Tropical Plant Germplasm

Post-thaw culture medium improvement From 4-week-old plantlets of seven genotypes, 140 apical shoot-tips (1.5 mm long) were isolated and processed by the vitrification method using 50 min of dehydration. After 1 day in liquid nitrogen storage, 100 shoot-tips were thawed, 50 of them were cultivated on M1 post-thaw culture medium. Another 50 were cultivated on medium M2 (M1 plus a supplement of vitamins 100 mg/L inositol, 0.5 mg/L nicotic acid, 0.5 mg/L pyridoxine–HCl, 0.1 mg/L thiamine–HCl, and 2 mg/L glycine). For the vitrification control, 20 shoot-tips were considered per treatment and were evaluated without going through the freezing step. The survival was evaluated 6 weeks after thawing.

Cost and space reduction An assay with 10 randomly selected genotypes was carried out. The straw size and capacity tested were: 10 cm, 6.5 cm and 3.7 cm long with 250 ml, 100 ml and 70 ml, respectively. One hundred apical shoot-tips per treatment were tested. They were processed by the vitrification protocol with 50 min of dehydration. Ten shoot-tips were loaded per straw. M2 medium was used for post-thaw culture. Survival was evaluated 6 weeks after thawing.

Stage 3: Cryopreserving potatoes on a large scale Utilizing the modified Steponkus' vitrification protocol, eight potato genotypes were cryopreserved weekly at CIP. For this purpose, 290 apical shoot-tips were isolated, 20 were treated as vitrification controls and 270 were frozen. From that amount, 20 were thawed 1 day after freezing. Post-thaw culture was made on M2 medium and shoot-tips were transferred to fresh medium 1 week after thawing. Survival evaluation was made 6 weeks after thawing. Finally, 250 shoot-tips of each genotype that had a survival rate over 20% were stored in a 130-L liquid nitrogen tank.

Results and discussion During the initial propagation of mother plants, some genotypes showed symptoms of weakness and loss of apical dominance. This lack of vigour could be due to the sorbitol content in the medium used for long-term storage, which could affect the plants’ hormone balance. After culturing (2–3 times) the genotypes in magenta jars covered with a transparent polypropylene film and a filter that increases air exchange, plants grew stronger, stems were thicker, leaves were bigger, and the lack of apical dominance decreased. With regards to the testing of 80 genotypes, during the first evaluation only 69% of them were successfully recovered. Of those recovered, the average survival was 46% in the first evaluation (1 day after freezing), and 40% after 3 months. This difference, however, was not statistically significant. Theoretically, the survival rate should not change even if the plant material was stored for many years. To confirm this hypothesis, a third evaluation after 1 year of freezing is being carried out. Testing the recalcitrant accessions with other dehydration times (45, 55 and 60 min) showed an increase of the percentage of surviving genotypes (75%). After this testing, it was evident that plant vigour is a bottleneck in the cryopreservation process and that the survival rate obtained Ongoing cryopreservation projects 253 by using axillary shoot-tips varies in some genotypes. With apical shoot-tips from vigorous plants, seven genotypes tested showed higher survival; survival of two recalcitrant genotypes was over 50%, and the average survival rate increased from 31 to 67%. In the assay for improving the recovering culture medium we found that M2 increased the survival by 10%. After these experiments, the feasibility of applying this protocol for the long- term conservation of potato was confirmed. However, some refinements for reducing cost and storage space were needed. According to the original protocol, three shoot-tips are stored in plastic straws of 250-ml capacity and 10 cm long. Straws (not identified) are stored in open goblets. With this procedure, a lot of storage space is needed for storing large collections, and there is a high risk of mixing samples. In a test with straws of smaller size, no differences were found in survival rate. Therefore, CIP adopted the use of 3.7- cm long straws, which can be stored in 2-ml cryovials with cap. Using cryovials allows efficient identification and optimizes the cryotank capacity. To date, CIP's system permits storing 960 accessions (250 shoot-tips organized in five cryovials containing 50 shoot-tips each) in a liquid nitrogen tank with capacity of 130 L. By reducing the storage space and optimizing the use of vitrification solution, the freezing cost of one accession has been reduced by more than 50% (from US$80 to $35).

Table 1. Number and survival rate of potato accessions maintained in cryopreservation at CIP Surviving Average No. of genotypes in genotypes survival Genotype cryopreservation (%) rate (%)† S. tuberosum subsp. andigena 59 75 40 S. phureja 38 74 55 S. stenotomum 57 71 46 S. goniocalyx 19 78 37 S. chaucha 4 77 30 Natural hybrids S. goniocalyx × S. stenotomum 17 80 53 S. stenotomum × S. goniocalyx 8 73 60 Total 197 75 46 † Survival rate evaluated after 3 months of storage in liquid nitrogen. Source: Golmirzaie and Panta 1997a.

During this research, different survival levels were observed within the tested species; this could be related to the vigour of mother plants or genotype dependence (Table 1). To date, CIP has cryopreserved 197 potato accessions (Table 1) and tried to recover 100% of the genotypes that were cryopreserved. To reach this goal, we are improving the vitrification method and testing other methods, such as the encapsulation-dehydration and droplet methods that have been tested by other researchers for a wide range of crops (Benson et al. 1996; Schäfer–Menuhr et al. 1996). We expect to be able to safely store the Potato Base Collection (approx. 4000 accessions) using cryopreservation. This 254 Cryopreservation of Tropical Plant Germplasm collection contains materials that are not requested often by users. Since this germplasm will eventually form a foundation for potato breeding work for future generations, we are making genetic stability studies of samples of cryopreserved materials.

Acknowledgements CIP and the authors want to thank UNDP for funding the first stage of this project as well as Cornell University and Dr Peter Steponkus for their kind collaboration.

References Benson, E.E., M. Wilkinson, A. Todd, U. Ekuere and J. Lyon. 1996. Developmental competence and ploidy stability in plants regenerated from cryopreserved potato shoot- tips. Cryo–Letters 17:119–128. Golmirzaie, A.M. and A. Panta. 1997a. Advances in potato cryopreservation by vitrification. Pp. 71–76 in CIP Program Report. International Potato Center, Lima, Peru. Golmirzaie, A.M. and A. Panta. 1997b. Tissue culture methods and approaches for conservation of root and tuber crops. Pp. 123–152 in Conservation plant genetic resources in vitro. Vol. 1: General aspects. M.K. Razdan and E.C. Cocking (eds.). Science Publishers, Inc., USA. Schäfer–Menuhr, A., E. Muller and G. Mix–Wagner. 1996. Cryopreservation: an alternative for the long-term storage of old potato varieties. Potato Research 39:507–513. Steponkus, P.L., R. Langis and S. Fujikawa. 1992. Cryopreservation of plant tissues by vitrification. Advances in Low-Temperature Biology 1:1–16. Ongoing cryopreservation projects 255

The in vitro germplasm collection at the Musa INIBAP Transit Centre and the importance of cryopreservation Ines Van den houwe, Bart Panis and Rony Swennen Laboratory of Tropical Crop Improvement, Catholic University of Leuven, 3001 Heverlee, Belgium

Origin, importance and uses The geographical origin of bananas extends from the Indian subcontinent to Papua New Guinea. Distinct centres of secondary genetic diversification evolved in the Great Lakes region of East Africa where the distinct group of AAA-highland bananas flourishes and in the more humid forests of Central and West Africa where the AAB ‘plantain’ subgroup underwent locally an intensive diversification. Approximately 500 years ago, the banana plant was introduced in Central and Latin America where the crop became of major economical importance. The fruits, which are produced parthenocarpically, are consumed raw, cooked, brewed into an alcoholic beverage or processed into flour, meal and chips. The main nutritional component of banana fruits is carbohydrate – over 90% of the dry matter – but banana fruits are also rich in minerals (potassium) and vitamins A, B and C. The leaves of the plants are used as plates and food wrappers for steaming, and the terminal male bud of the inflorescence is cooked as a vegetable. The fibres of the false stem are used to produce ropes, paper and clothing. According to FAO, the total world production in 1997 was about 89 million tonnes of which 36% was plantains (FAO 1997). Smallholders for local consumption provide an estimated 90% of the world banana production. The export trade accounts for only 10% of the world production and deals almost entirely with dessert bananas (AAA Cavendish type).

Ex situ conservation of banana germplasm Around the world over 70 ex situ collections, mainly field and in vitro collections of Musa, have been identified by the International Plant Genetic Resources Institute (IBPGR 1992). The global aim of genebanks is to ensure the availability of quality material and to conserve genetic resources for future use. Field genebanks are the traditional method for preserving banana germplasm because cultivated species do not produce seeds; they are vegetatively propagated through planting young sword suckers. Field collections play an essential role in characterization and evaluation of genetic resources. A major constraint of collections of living plants in the field is that the germplasm is most exposed to a number of hazards such as diseases, pests and adverse weather conditions. Moreover, field genebanks are costly to maintain properly as plants need to be transplanted periodically and treated with fertilizers and pesticides. In addition, bananas and plantains are perennial giant herbs, which occupy a considerable surface demanding vast amounts of land. The possibility of utilising seeds for the maintenance of wild 256 Cryopreservation of Tropical Plant Germplasm relatives offers a cheap alternative to field genebanks. In vitro methods for the conservation of genetic resources are valuable complements for field and seed genebanks. Active in vitro genebanks, where the material is maintained as shoot-tip or meristem cultures, are particularly interesting as a source of disease-free plant material, readily available for international exchange. The method also allows conservation in minimal space and, under the appropriate conditions, storage duration of Musa germplasm can be extended to approximately 1 year, requiring less labour input (see below). Nonetheless, maintaining an in vitro collection remains labour-intensive and involves the risk of losing valuable germplasm through accidental contamination of cultures and human error. Another major impediment of the use of tissue culture for germplasm preservation is the occurrence of somaclonal variations. Genetic stability studies have shown that the level and type of variation is linked to the intrinsic characteristics of the plant (genotype) and may also be affected by external factors such as the culture system, conditions and its duration (Cote et al. 1993). In order to guarantee genetic integrity over a longer period of time, plant tissue storage in liquid nitrogen at – 196°C is the only viable option.

Germplasm conservation at the INIBAP Transit Centre In 1985, INIBAP (International Network for the Improvement of Banana and Plantain) established at the Laboratory of Tropical Crop Improvement at K.U. Leuven in Belgium, its Transit Centre (ITC), serving as an active in vitro genebank. This global collection of banana and plantain germplasm, comprising approximately 1100 accessions, is held under the auspices of FAO. Around 75% of the materials stored are landraces, 15% wild relatives and the remaining 10% are advanced cultivars. Accessions in the ITC genebank have been acquired through donations from existing germplasm collections, breeders and through collecting missions in the centre of origin of Musa (INIBAP/IBPGR 1990). In recent years, acquisition emphasis has been placed on elite germplasm developed by breeding programmes and genetically diverse and unique materials obtained through plant explorations conducted in the centres of diversification in collaboration with IPGRI (INIBAP 1998).

Medium-term conservation Shoot-tips are isolated from young suckers and placed on proliferation- inducing culture medium, i.e. Murashige and Skoog medium, supplemented with 30 g/L sucrose, 10 mM 6–benzyladenine (BA) and 1 mM indole–3–acetic acid (IAA), and solidified with 2 g/L Gelrite. Proliferating cultures are maintained under slow growth conditions at an ambient temperature of -2 -1 16±1°C and continuous light with an intensity of 25 µE m s . Under these conditions, the transfer period of proliferating shoot-tip cultures at the ITC is lengthened to nearly 1 year (334 days) on average, whereas at a higher ambient temperature (22±3°C) subculturing is carried out every 220 days on average. However, large differences in transfer interval, ranging between 3 and 22 months, were measured among the different genomic (sub) groups and even within the same subgroup when stored at 16°C (Van den houwe et al. Ongoing cryopreservation projects 257

1995). Several accessions in the collection have been maintained in vitro for more than 10 years now. To guarantee the genetic integrity of the stored material, it should be systematically compared with field-propagated materials from the same accessions using a combination of morphological and molecular markers. However, presently, molecular techniques for the identification of somaclonal variants are still at the research stage and monitoring of the genetic stability in the collection is limited to visual examination of the stored cultures and regenerated greenhouse plants. As a security measure, this unique collection is currently in the process of being duplicated. Over half of the ITC accessions are backed-up in the in vitro laboratories of TBRI (Taiwan Banana Research Institute), Taiwan and CATIE (Centro Agronómico Tropical de Investigación y Enseñanza), Costa Rica.

Long-term storage Currently, three methods for the cryopreservation of shoot meristematic tissues are under investigation. The first method, which relies on preculture of highly proliferating 'cauliflower-like' meristem cultures on 0.4M sucrose medium followed by rapid freezing, shows a very heterogeneous post-thaw response (Panis et al. 1996, this vol., p. 238). Viability rates are very genotype-dependent and range between 0% (for AAA highland bananas) and 75% (for ABB cooking bananas). This cryopreservation method is extremely simple and user- friendly, once 'cauliflower-like' meristem clumps are obtained. In the second method tiny apical meristems of about 1 mm diameter excised from rooted in vitro plants are subjected to the vitrification procedure (Thinh et al. 1999; this vol., p. 227). Viability rates are less cultivar-dependent and range between 41 and 91%. Its main disadvantage is that the excision of apical meristems at a size suitable for cryopreservation is very labour-intensive and can only be executed efficiently by skilled personnel. The third method is a combination between the two previous ones and involves preculture of cauliflower-like meristem clumps on high sucrose medium followed by vitrification (Panis et al., this vol., p. 238). Post-thaw viability ranges between 14 and 74% and is less cultivar-dependent than the first cryopreservation method. All three cryopreservation methods are still subject to further improvement. Post-thaw regeneration frequencies, availability of starting material and user- friendliness of the protocol will decide which cryopreservation method will be chosen for routine application at the ITC. However, a possible solution might be that the collection stored in liquid nitrogen will be a combination of the methods described above. Accessions belonging to the ABB group could be stored through simple freezing, AAA highland bananas and AAB plantains through vitrification of apical meristems, and AAA and AAB bananas using the combined method. 258 Cryopreservation of Tropical Plant Germplasm

Dissemination and use of disease-free material from ITC collection In vitro culture is known to reduce the risk of spreading fungal diseases and harmful insects but virus and endogenous bacteria are able to pass symptomless through tissue culture. In particular, viruses pose a special risk as they may hinder the intercontinental transfer of banana germplasm. Therefore, samples of newly introduced materials are checked for viruses at one of the three INIBAP virus-indexing centres (VICs): CIRAD (Centre de coopération internationale en recherche agronomique pour le développement), France, QDPI (Queensland Department of Primary Industries), Australia or PPRI (Plant Protection Research Institute), South Africa. The VICs operate in accordance with the protocols for virus-indexing provided by FAO/IPGRI Technical Guidelines for the Safe Movement of Germplasm (Diekmann and Putter 1996). Samples of the stored accessions are grown in a screenhouse and subjected to serological tests for cucumber mosaic virus (CMV) and banana bunchy top virus (BBTV) and to ISEM for banana streak virus (BSV) and banana bract mosaic virus (BBrMV), 3 and 6 months after planting. In 1996, an INIBAP research project funded by the Belgian BADC (Belgian Administration for Development Cooperation), was started at the University of Gembloux, Belgium, to develop methods for virus eradication in Musa germplasm. Also, newly introduced materials are tested in-house for endogenous bacteria by streaking the basis of the shoot-tip onto a bacteriological medium. In general, infected cultures are immediately discarded, but sometimes re- initiation is not possible. In these cases, antibiotics and meristem culture are used to produce clean cultures (Van den houwe et al. 1998). Clones in the active germplasm collection are regularly accessed and multiplied for distribution to national and international agricultural research centres and other interested parties in developed and developing countries. The ITC provides, subject to availability and a Material Transfer Agreement, small quantities of in vitro cultures, free of charge for bona fide use, together with available associated data. This FAO/INIBAP Material Transfer Agreement binds recipients of FAO-designated germplasm not to claim ownership or intellectual property rights over the germplasm received. During 1985–97, more than 5000 accessions were distributed from the collection to users in more than 50 countries (Table 1). Plant materials are supplied mainly for research and breeding activities, for characterization and field evaluation. Within the scope of INIBAP’s International Musa Testing Program (IMTP), plantlets of virus-tested advanced cultivars produced by banana breeding programmes as well as of some natural varieties and wild species, are distributed by the ITC for evaluation under different ecological and pathogenic conditions. The first phase of this programme started in 1989 and resulted in the recommendation of some exceptional and good-performing hybrids, with high resistance to the fungal disease black Sigatoka. Since then, these valuable hybrids have been disseminated from the ITC to more than 50 countries worldwide for evaluation at the national level (INIBAP 1996), distribution to farmers and large-scale production. Ongoing cryopreservation projects 259

Table 1. Number of accessions exported from the INIBAP Transit Centre genebank Year America W-C Africa E Africa Asia/Pacific Europe Total 1985 0 20 0 12 0 32 1986 0 27 0 0 0 27 1987 13 154 0 3 10 180 1988 127 105 105 5 14 356 1989 88 109 69 48 34 348 1990 111 54 42 68 122 397 1991 68 79 21 50 103 321 1992 61 0 48 83 104 296 1993 99 24 66 75 179 443 1994 155 158 19 154 119 605 1995 183 41 93 436 183 936 1996 261 55 27 71 247 661 1997 78 37 63 88 292 558 Total 1244 863 553 1093 1407 5160 % 24 17 11 21 27 100

In recent years, efforts have been made to document the plant material in the ITC collection and to improve access to and dissemination of available information. The overall goal is to develop a fully operational Musa germplasm database system that will be linked with databases of regional and national collections. This system will include management of passport data, characterization and evaluation data, and will encompass genetic and molecular information. Passport and characterization data for all ITC accessions will also be included in the System-wide Information Network for Genetic Resources (SINGER) database of the CGIAR (Consultative Group on International Agricultural Research) which is accessible via Internet (http://www.cgiar.org/singer).

Acknowledgements The authors thank INIBAP (International Network for the Improvement of Banana and Plantain), the Belgian Administration for Development Cooperation (BADC/ABOS) and the ‘Onderzoeksfonds’ of the Catholic University of Leuven for their financial support. SGRP (System-wide Genetic Resources Programme) is also gratefully acknowledged for providing travel funds for one co-author (BP) to participate in the workshop.

References Cote, F.X., J.A. Sandoval, Ph. Marie and E. Auboiron. 1993. Variation chez les bananiers et les plantains multipliés in vitro, analyse des données de la littérature. Fruits 48: 15–23. Diekmann, M. and C.A.J. Putter. 1996. FAO/IPGRI Technical Guidelines for the Safe Movement of Germplasm. No. 15. Musa (2nd edition). Food and Agriculture Organisation of the United Nations, Rome/International Plant Genetic Resources Institute, Rome. FAO. 1997. Production Year Book 1997. Food and Agriculture Organisation of The United Nations, Rome. IBPGR. 1992. Directory of germplasm collections. 6.I Tropical and Subtropical Fruits and Tree Nuts. Annona, avocado, banana and plantain, breadfruit, cashew, Citrus, date, fig, guava, mango, passionfruit, papaya, pineapple and others. International Board for Plant Genetic 260 Cryopreservation of Tropical Plant Germplasm

Resources. Bettencourt, E., Hazekamp, Th. and Perry, M.C., Rome. INIBAP. 1996. INIBAP Annual Report 1995. International Network for the Improvement of Banana and Plantain, Montpellier, France. INIBAP. 1998. Networking Banana and Plantain. INIBAP Annual Report 1997. International Network for the Improvement of Banana and Plantain, Montpellier, France. INIBAP/IBPGR. 1990. Musa conservation and documentation. Proceedings of a workshop held in Leuven, Belgium, 11–14 December 1989. INIBAP, Montpellier, France. Panis, B., N. Totté, K. Van Nimmen, L.A. Withers and R. Swennen. 1996. Cryopreservation of banana (Musa spp.) meristem cultures after preculture on sucrose. Plant Science 121: 95–106. Thinh, N.T., H. Takagi and S. Yashima. 1999. Cryopreservation of in vitro-grown shoot tips of banana (Musa spp) by vitrification method. Cryo–Letters, in press. Van den houwe, I., K. De Smet, H. Tezenas du Montcel and R. Swennen. 1995. Variability in storage potential of banana shoot cultures under medium term storage conditions. Plant Cell, Tissue and Organ Culture 42: 269–274. Van den houwe, I., J. Guns and R. Swennen. 1998. Bacterial contamination in Musa shoot tip cultures. Acta Horticulturae (in press). Ongoing cryopreservation projects 261

Cryopreservation: roles in clonal propagation and germplasm conservation of conifers David R. Cyr Tissue Culture BC Research Inc., Vancouver, BC, Canada V6S 2L2

Introduction Conifer somatic embryogenesis (SE), with the potential for capturing genetic diversity and providing for selection of elite genotypes (Park et al. 1998), is emerging as a key component of advanced forestry programmes (Aitken– Christie et al. 1994; Gupta et al. 1994; Handley et al. 1995). Targets for selection, developed through conventional breeding or genetic engineering, include growth, form, wood quality and resistance to insects or disease. Cryopreservation of embryogenic cultures is a critical platform for the development of a successful conifer SE strategy. It facilitates long-term storage of genetic resources, alleviates the effects of tissue culture induced somaclonal variation, enables efficient recovery of propagules and supports sustained high rates of multiplication (Sutton et al. 1998). Additionally, it provides for dynamic and changing deployment populations, as determined by data accumulating from long-term field trials (Cyr 1998). The ability to maintain donor tissue juvenility through cryopreservation represents an immeasurable advantage over propagation programmes based on rooted cuttings (Grossnickle et al. 1996), and the genotype response supersedes that of systems based on organogenesis (Smith et al. 1994; Menzies and Aimers–Halliday 1997). As in other plant species, cryopreservation of more complex conifer tissues is problematic. For example, in axillary shoot multiplication, germplasm conservation is dependent on short-term cold storage of metabolically active shoots (Menzies and Aimers–Halliday 1997). Within the clonal forestry context, large-scale cryopreservation programmes are underway in Canada (Park et al. 1993; Adams et al. 1994; Cyr 1998), France (Bercetche and Paques 1995), New Zealand (Aitken–Christie et al. 1994), Scandinavia (von Arnold et al. 1995) and the United States (Gupta et al. 1994; Handley et al. 1995). At present, at least 16 organizations (biotechnology firms, government organizations and forest companies) are known to have active programmes. Published information indicates the application of cryopreservation to eight species of spruce (Picea), eight species of pine (Pinus), four species of larch (Larix), two species of fir (Abies), one species of Douglas-fir (Pseudotsuga), and numerous hybrids of Larix and Picea (Cyr 1988). By 1998, an estimated 8000 to 10 000 genotypes had been stored worldwide. Approximately 50–60% of the germplasm stored is represented by spruce species, with pines and Douglas-fir at 30% and 10% of the total, respectively. Evidence suggests that the proportion of stored germplasm from pine species will increase significantly over the next few years. 262 Cryopreservation of Tropical Plant Germplasm

Review of cryopreservation technology

Culture variation Long-term maintenance of germplasm in vitro has been limited by variation which is inherent to tissue culture (Kartha et al. 1988). In conifer SE this has been associated with fluctuation and eventual decline in culture productivity (Fourre et al. 1997). Genetic stability of embryogenic cultures has been investigated at several levels of organization. No evidence of somaclonal variation has been found in spruce sublines at the isozyme level (Eastman et al. 1991), among spruce genotypes before and after cryopreservation using RFLP analysis (Cyr et al. 1994), in spruce somatic embryos using RAPDs (Isabel et al. 1993), and at the ploidy level in larch and spruce species after extended culture at ambient conditions (Nkongolo and Klimaszewska 1994, 1995). By contrast, a low frequency of ploidy abnormalities has been detected in radiata pine (Maddocks et al. 1995) and Norway spruce (Fourre et al. 1997). In the latter case, the variation was associated with long-term subcultures, thus underscoring the importance of cryopreservation in minimizing the associated risks. The suggested strategy in conifer SE is to restrict culture maintenance to an annual plant production cycle (Smith et al. 1994) for embryogenic cultures derived from freshly initiated embryogenic lines or from germplasm that has been regenerated from a cryopreservation clone bank.

Cryopreservation methods Efforts in conifer SE cryopreservation have focused primarily on cultures which are comprised of early stage somatic embryos, commonly referred to as embryonal suspensors masses (ESM). However, the storage of mature somatic embryos, using vitrification with and without prior desiccation (Charest et al. 1996), is a potential strategy for re-establishment of juvenile cultures via secondary embryogenesis (or organogenesis) or for creating an inventory of artificial seed (Florin et al. 1993). In a recent example, Percy (1997) demonstrated recovery of viable plantlets from spruce somatic embryos that had been dried to remove free water and immersed in liquid nitrogen without the use of cryoprotectants. These results have implications for germplasm conservation of non-commercial or rare species via cryostorage of mature zygotic embryos. Cryopreservation protocols for conifer SE cultures are derived, for the most part, from the work by Kartha et al. (1988) on white spruce. Most typically, 0.4M sorbitol and 5% DMSO (final concentrations) are used as osmotic agents and cryoprotectants; and slow cooling rates of 0.3–0.5°C/min are applied to reach terminal temperatures of –35 to –40°C prior to immersion in liquid nitrogen (Cyr 1998). The thawing process is conducted for 1.5–2 min at 37-40°C, with culture conditions facilitating the removal of osmotic and cryoprotectant agents. Regeneration of embryogenic tissue occurs from the cells which survive the preculture and freezing process, specifically the densely cytoplasmic embryogenic cells (Kartha et al. 1988). Cultures are stored in the liquid (–196°C) Ongoing cryopreservation projects 263 or vapour (–140°C) phase of nitrogen. Temperatures higher than –140°C are considered to be suboptimal (Charest and Klimaszewska 1995). Variations in the freezing protocols include a complex freezing programme designed to compensate for temperature changes during phase transition (Cyr et al. 1994) and a simplified freezing protocol (Hargreaves and Smith 1994). The simplified method involves the incubation of vials at –80°C for 1 h in vessels insulated with isopropanol and providing a cooling rate of 0.99°C/min. This approach has been adopted at BC Research for routine use on a variety of conifer species (Cyr 1998). For recalcitrant conifer genotypes the method described by Kartha et al. (1988) is applied.

Culture effects Differences in line cryotolerance have been reported in some cases (Adams et al. 1994; Hargreaves and Smith 1994); however these results were associated with a suboptimal freezing protocol and declining culture quality, respectively. Most typically, 90% or greater success has been reported, e.g. Douglas-fir (Gupta et al. 1994), interior spruce (Cyr et al. 1994), white spruce (Park et al. 1994) and maritime pine (Bercetche and Paques 1995). In general, studies with large arrays of genotypes and families suggest that culture quality, not genetics, is the major factor in cryopreservation success (Park et al. 1994, 1998). Improved embryogenic potential has been reported for cryopreserved cultures (Gupta et al. 1994). Most studies, however, indicate that cryopreservation has no long-term effect on culture performance (Klimaszewska et al. 1992; Norgaard et al. 1993; Cyr et al. 1994; von Arnold et al. 1995). A lag-phase is often observed during regeneration (Norgaard et al. 1993; Cyr et al. 1994), with recovery of pre-thaw growth rates occurring within a few days to 2 weeks (Park et al. 1994; Cyr 1998). The proportion and organization of embryogenic cells (Norgaard et al. 1993), culture growth stage (Charest and Klimaszewska 1995), and nurse cultures and vial source (Hargreaves et al. 1995) have all been cited as factors affecting post-thaw regeneration. Gupta et al. (1994) cite the occurrence of vial to vial variability within a genotype. This effect is likely due to the relative heterogeneity of conifer embryogenic cultures. Similar observations have been made at BC Research and its commercial affiliate, Silvagen Inc. At these organizations this variability is minimized by pooling 3–5 g of ESM from several Petri plates and distributing a suspension of the tissue among 10–12 vials (Cyr 1998). Somatic embryos and plants have been generated after storage up to 1 year (Kartha et al. 1988; Cyr et al. 1994). In general, plants have been found to be phenotypically normal (Find et al. 1993; Gupta et al. 1994; Hargreaves and Smith 1994) and have been successfully established in field trials (Klimaszewska et al. 1992; von Arnold et al. 1995). Most recently, genetic stability was demonstrated for 12 white spruce lines after 3 and 4 years of cryopreservation (Park et al. 1998). In vitro germination assessments, ex vitro survival and morphological characterization after 5 months of nursery growth were the parameters assessed. 264 Cryopreservation of Tropical Plant Germplasm

Applications in cryopreservation In this section the interior spruce (Picea glauca/engelmannii complex) programme conducted at BC Research Inc from 1991 to 1998 will be used to illustrate the role of cryopreservation in a clonal selection programme based on somatic embryogenesis.

Clonal selection From 1991 to 1992 a pilot SE programme was applied to 12 full-sib families ranked from low to high for growth and weevil resistance. Approximately 260 genotypes representing all families were established and subsequently cryopreserved (Cyr et al. 1994) (Table 1). Plants were regenerated from non- frozen cultures and 181 genotypes were delivered to clonal field trials in 1994 and 1995. Top-ranked parents for the subsequent clonal selection programme were selected via progeny testing from a British Columbia Ministry of Forests (BCMoF) base population of 173 based on 15-year height and 10-year weevil- resistance (Sutton et al. 1993). The programme strategy called for the establishment, cryopreservation and plant delivery of 1000 embryogenic lines from 30 to 40 top-ranked families, with the aim of selecting 30 to 50 operational elite genotypes. From 1993 to 1996 more than 1700 genotypes from 36 top-ranked families were established and cryopreserved (Cyr et al. 1995, 1997; Grossnickle et al. 1996; Sutton et al. 1998) (Table 1). This effort has resulted in the delivery of greater than 1200 genotypes from 36 families to clonal trials in 1996, 1997 and 1998. The net result of the two SE programmes was the cryopreservation of more than 2000 lines from 48 families and the delivery of 75% of these to clonal field trials. Up to 40 genotypes from previous- year trials (1994–97) were retrieved from cryostorage for inclusion as check- lines in next-year clonal trials (1996–98). This effort will allow an evaluation of the performance of plants derived from non-frozen and frozen cultures.

Table 1. Summary of cryopreserved lines and clonal trials for interior spruce Trial year Families No. of lines stored No. of lines in trials 1994–95 12 260 181 1996–97 21 941 571 1998 15 842 714 Total 48 2093 1466

Cryopreservation strategy The cryopreservation strategy management under development at BC Research and Silvagen Inc. is being applied in three areas: clonal trials, research and large-scale production. For all of these applications a database linking all activities and outputs to the cryopreservation clone bank is the process of implementation. The data linked to the cryopreservation clone bank include genetic information, historical in vitro culture and nursery performance, and field trial locations and results. The interior spruce clonal programme has been the initial focus species. Ongoing cryopreservation projects 265

Staff training, standard operating procedures (SOP) and quality control standards (QC) have been established for cryopreservation protocols and all other components of the SE process. An integrated electronic monitoring and alarm system has been established for the cryopreservation storage facilities (remote monitoring, on-site audible alarms, download to clone bank database, SOP response protocols). Off-site storage is an integral component of the risk- management strategy. For example, all interior spruce field trial genotypes are stored in a BCMoF cryopreservation storage facility. All clonal and operational plant production programmes emphasize regeneration and plant production within an annual cycle. For clonal programmes, 10 vials per genotype are prepared for cryopreservation and all germplasm is stored in the vapour phase. Two vials are used for viability assessments and the remaining eight vials are distributed among three separate storage tanks. Six vials are reserved as parent germplasm, with the remaining two reserved for short-term regeneration needs. These needs include the establishment of off-site germplasm, check-lines, research material and production populations. Two vials are used for off-site storage. Figure 1 illustrates the history of culture retrieval for interior spruce for approximately 160 genotypes stored originally in 1994. Approximately 50% of the cultures regenerated once were used for the establishment of a production clone bank. For research studies, six genotypes are considered as the standard complement, with two vials stored per regeneration. Regeneration intervals range from 3 to 6 months, dependent on the species, and research programmes for a given set of genotypes typically range from 3 to 6 years. The establishment of a separate cryopreservation clone bank of large-scale production of interior spruce was completed in 1998 (Silvagen Inc.). The number of vials stored per genotype is based on requirements for annual production, assuming the use of any given germplasm in a defined time frame, and on long-term commercial market projections. Typically, three vials per genotype per regeneration event are stored; multiplication rates are factored into the equation.

120

90 1500 60 1000

No. of lines 30 500 No. of lines S1 0 0 1 2 3 92 93 94 95 96 97 98 No. of regenerations Year Fig. 1. Regeneration of interior spruce Fig. 2. Storage of lines, 1992–98. lines stored in 1994. 266 Cryopreservation of Tropical Plant Germplasm

Additional cryopreservation efforts The international efforts in conifer somatic embryogenesis have brought cryopreservation technology forward into an operational arena, and the evidence to date suggests that this technology has matured and is not affected by genetic influences. Since the inception of SE cryopreservation at BC Research in 1992, the annual storage of new genotypes has approached 2000 genotypes per year (Fig. 2). To date, approximately 5000 genotypes representing 14 conifer species (pine, spruce and Douglas-fir) have been stored. For BC Research and other organizations, this progress is accompanied by the need for continued development of tools for monitoring the output of material regenerated from cryopreservation (i.e. genetic fidelity), as well as the implementation of appropriate risk-management strategies for these genetic resources.

References Adams, G.W., M.G. Doiron, Y.S. Park, J.M. Bonga and P.J. Charest. 1994. Commercialization potential of somatic embryogenesis in black spruce tree improvement. The Forestry Chronicle 70: 593–598. Aitken–Christie, J., K. Gough, D. Maddocks, M. Sigley, F. Burger and P.C.S. Carter. 1994. Towards commercialization of conifer embryogenesis. Pp. 181–190 in Proceedings of the 1994 Second International Symposium on the Applications of Biotechnology to Tree Culture, Protection, and Utilization. (C.H. Michler, M.R. Becwar, D. Cullen, W. Nance, R.R. Sederoff and J.M. Slavicek, eds.). October 2–6, 1994. Bloomington, MIN. Bercetche, J. and M. Paques. 1995. Somatic embryogenesis in maritime pine (Pinus pinaster). Pp. 221–242 in Somatic Embryogenesis in Woody Plants Volume 3 – Gymnosperms. (S.M. Jain, P.K. Gupta and R.J. Newton, eds.). Kluwer Academic Publishers, Dordrecht, Boston and London. Charest, P.J. and K. Klimaszewska. 1995. Cryopreservation of germplasm of Larix and Picea species. Pp. 191–203 in Biotechnology in Agriculture and Forestry, Vol. 30 Somatic Embryogenesis and Synthetic Seed I. (Y.P.S. Bajaj, ed.). Springer–Verlag, New York. Charest, P.J., J. Bonga and K. Klimaszewska. 1996. Cryopreservation of plant tissue cultures: the example of embryogenic tissue cultures from conifers. Pp. 1–27 in Plant Tissue Culture Manual. Section C: Propagation and Conservation of germplasm. 9. K. Lindsey, ed. Kluwer Academic Publisher, Dordrecht. Cyr, D.R., W.R. Lazaroff, S.M.A. Grimes, G.Q. Quan, T.D. Bethune, D.I. Dunstan and D.R. Roberts. 1994. Cryopreservation of interior spruce (Picea glauca engelmanni complex) embryogenic cultures. Plant Cell Reports 13: 574–577. Cyr, D.R., S. Fan, S. Grossnickle, C. Hawkins, J. Russell and A. Yanchuk. 1995. Development of a clonal selection program for interior spruce using somatic embryogenesis. P. 31 in Abstracts of Conifer Biotechnology Working Group 7th International Conference, June 26–30 Surfers Paradise, Queensland, Australia. Cyr, D.R., S.B. Binnie, K. Finstad, S. Grimes, K. Klimaszewska, I. Loyola, R. Percy, G. Quan and A. Valentine. 1997. From the cradle to the forest: advances in conifer propagation. Pp. 199–202 in Proceedings, 1997 Biological Symposium. 1997 October 19–23; San Francisco. TAPPI Press (Atlanta). Cyr, D. 1998. Cryopreservation of embryogenic cultures of conifers and its application to clonal forestry. In press in Somatic Embryogenesis in Woody Plants Volume 4. S.M. Jain, P.K. Gupta and R.J. Newton, eds. Kluwer Academic Publishers, Dordrecht, Boston and London. Ongoing cryopreservation projects 267

Eastman, P.A.K., F.B. Webster, J.A. Pitel and D.R. Roberts. 1991. Evaluation of somaclonal variation during somatic embryogenesis of interior spruce (Picea engelmannii complex) using culture morphology and isozyme analysis. Plant Cell Reports 10:425–430. Find, J.I., F. Floto, P. Krogstrup, J.D. Moller, J.V. Norgaard and M.M.H. Kristensen. 1993. Cryopreservation of an embryogenic suspension culture of Picea sitchensis and subsequent plant regeneration. Scandinavian Journal of Forestry Research 8: 156–162. Florin, B., H. Tessereau, C. Lecouteux, D. Courtois and V. Petiard. 1993. Long-term preservation of somatic embryos. Pp. 133–161 in Synseeds: Applications of synthetic seeds to crop improvement. (K. Redenbaugh, ed.). CRC Press Boca Raton, Ann Arbor, London, Tokyo. Fourre, J.L., P. Berger, L. Niquet and P. Andre. 1997. Somatic embryogenesis and somaclonal variation in Norway spruce: morphogenetic, cytogenetic and molecular approaches. Theoretical and Applied Genetics 94: 159–169. Grossnickle, S.C., D. Cyr and D.R. Polonenko. 1996. Somatic embryogenesis tissue culture for the propagation of conifer seedlings: a technology comes of age. Tree Planters Notes 47:48–57 Gupta, P.K., R. Timmis, K. Timmis, W. Carlson, J. Grob and E. Welty. 1994. Plantlet regeneration via somatic embryogenesis in Douglas-fir (Pseudotsuga menziesii). Pp. 35–40 in Proceedings, 1994 Biological Symposium. 1994 October 3–6; Minneapolis, MN. TAPPI Press (Atlanta). Handley, L.W., M.R. Becwar, E.E. Chesick, J.F. Coke, A.P. Godbey and M.R. Rutter. 1995. Research and development of commercial tissue culture systems in loblolly pine. Tappi Journal 78: 169–175. Hargreaves, C.L. and D.R. Smith. 1994. Techniques used for cryopreservation of Pinus radiata embryogenic tissue. Cryobiology. 31: 577. Hargreaves, C.L., A.A. Warr, L.J. Grace and D.R. Smith. 1995. Cryopreservation and plant regeneration of select genotypes and transformed embryogenic tissus of Pinus radiata. P. 40 (Abstract) in Proceedings: Conifer Biotechnology Working Group 7th International Conference, 26-30 June 1995, Surfers Paradise, Queensland, Australia. Isabel, N., L. Tremblay, M. Michaud, F.M. Tremblay and J. Bousquet. 1993. RAPDs as an aid to evaluate the genetic integrity of somatic embryogenesis-derived populations of Picea mariana (Mill.) B.S.P. Theoretical and Applied Genetics 86: 81–87. Kartha, K.K., L.C. Fowke, N.L. Leung, K.L. Caswell and I. Hakman. 1988. Induction of somatic embryos and plantlets from cryopreserved cell cultures of white spruce (Picea glauca). Journal of Plant Physiology 132: 529–539. Klimaszewska, K., C. Ward and W.M. Cheliak. 1992. Cryopreservation and plant regeneration from embryogenic cultures of larch (Larix x eurolepsis) and black spruce (Picea mariana). Journal of Experimental Botany 43: 73–79. Maddocks, D., K. Gough, M. Hopping and J. Aitken–Christie. 1995. Early screening of radiata pine embryogenic cells and occurrence of tetraploidy. P. 29 in Conifer Biotechnology Working Group 7th International Conference, June 26–30 Surfers Paradise, Queensland, Australia. Menzies, M.I. and J. Aimers–Halliday. 1997. Propagation options for clonal forestry with Pinus radiata. Pp. 256–263 in FRI Bulletin No. 203 Proc. IUFRO '97 Genetics of Radiata Pine. (R.D. Burton and J.M. Moore, eds.). Dec 1–4, 1997, Rotorua, New Zealand. Nkongolo, K.K. and K. Klimaszewska. 1994. Karyotype analysis and optimization of mitotic index in Picea mariana from seedling root tips and embryogenic cultures. Heredity. 73: 11–17. Nkongolo, K.K. and K. Klimaszewska. 1995. Cytological and molecular relationship between Larix decidua, L. leptolepis and Larix x eurolepis: identification of species-specific chromosomes and synchronization of mitotic cells. Theoretical and Applied Genetics 90: 827–834 268 Cryopreservation of Tropical Plant Germplasm

Norgaard, J.V., V. Duran, O. Johnson, P. Krogstrup, A. Baldursson and S. von Arnold. 1993. Variations in cryotolerance of embryogenic Picea abies cell lines and the association to genetic, morphological and physiological factors. Canadian Journal of Forestry Research 23: 2560–2567. Park, Y.S., S.E. Pond and J.M. Bonga. 1993. Initiation of somatic embryogenesis in white spruce (Picea glauca) : genetic control, culture treatment effects, and implications for tree breeding. Theoretical and Applied Genetics 86: 427–436. Park, Y.S., S.E. Pond and J.M. Bonga. 1994. Somatic embryogenesis in white spruce (Picea glauca): genetic control in somatic embryos exposed to storage, maturation treatments, germination, and cryopreservation. Theoretical and Applied Genetics 89: 742–750. Park, Y.S., J.D. Barrett and J.M. Bonga. 1998. Application of somatic embryogenesis in high- value clonal forestry: deployment, genetic control, and stability of cryopreserved clones of white spruce (Picea glauca). In Vitro. In press. Percy, R.E.L. 1997. Desiccation and cryopreservation of spruce embryogenic tissue and mature somatic embryos. MSc Thesis, Department of Biology, University of Victoria, Victoria, BC, Canada. Smith, D.R., C. Walter, A. Warr, C.L. Hargreaves and L.J. Grace. 1994. Somatic embryogenesis joins the plantation forestry revolution in New Zealand. Pp. 19–29 in Proceedings, 1994 Biological Symposium. 1994 October 3–6; Minneapolis, MN. TAPPI Press (Atlanta). Sutton, B., D. Polonenko, D. Cyr and S.C. Grossnickle. 1998. Commercialization of somatic embryogenesis in forestry. In press in Biotechnology International: International Developments in the Biotechnology Industry, Universal Medical Press Inc. San Francisco. Sutton, B.C.S., S.C. Grossnickle, D.R. Roberts, J.H. Russell and G.K. Kiss. 1993. Somatic embryogenesis and tree improvement in interior spruce. Forestry. 91:34–38. Von Arnold, S., U. Egersdotter, I. Ekberg, P. Gupta, H. Mo and J. Norgaard. 1995. Somatic embryogenesis of Norway spruce (Picea abies). Pp. 17–36 in Somatic Embryogenesis in Woody Plants Volume 3 – Gymnosperms. (S.M. Jain, P.K. Gupta and R.J. Newton, eds.). Kluwer Academic Publishers, Dordrecht, Boston and London. Ongoing cryopreservation projects 269

Conservation of threatened flora by cryopreservation of shoot apices Darren H. Touchell Kings Park and Botanic Garden, West Perth, 6005, Western Australia (present address: USDA-ARS National Seed Storage Laboratory, Fort Collins, CO 80521, USA)

Introduction Cryobiology has emerged as an important scientific discipline for conserving plant germplasm. Its benefits are extending beyond agriculture and economically valuable species and reaching into endangered and threatened species, as reflected by the small but growing number of institutions using this technology in their conservation research programmes. Research at Kings Park and Botanic Garden in Western Australia has seen the development and implementation of cryostorage protocols for shoot apices and seed of a growing number of endangered species. The National Seed Storage Laboratory in Fort Collins, Colorado is developing procedures for conserving endangered germplasm of recalcitrant-seeded species. Other institutions that have investigated cryostorage of seed and shoot apices for endangered species include Cincinnatti Zoological and Botanic Gardens (Pence 1991), and the Royal Botanic Gardens, Kew (Fay 1994). This paper will focus on the application of cryostorage to conserving endangered species of Western Australia. The southwest of Western Australia contains one of the richest, most diverse and most endemic floras of the world. As typical with such areas of megadiversity there is a high level of threatening processes that lead to plant endangerment and extinction. In an attempt to stem the flow of loss of biodiversity, cryostorage procedures are being developed and implemented to establish long-term ex situ conservation collections. These collections aim to contain the maximum genetic diversity of a species in order to make possible effective species translocation as a later date. Many endangered species, however, produce little or no viable seed, thus the development of cryostorage has focused on shoot apices. The development of these procedures for routine use of a large and diverse number of species needs to overcome the species- or even clonal-specific nature of cryostorage protocols for shoot apices.

Case study – Grevillea scapigera Grevillea scapigera is an extremely endangered species from the southwest of Australia with approximately 30 plants existing in the wild. In 1986, the species was classified extinct until 1989 when a population of six plants was found. Successful in vitro propagation was achieved (Bunn and Dixon 1991) as part of conservation efforts. Through population genetic studies, 10 genotypes containing 87.4% of the genetic diversity of the species (Rossetto et al. 1995) were selected to establish a conservation collection. These 10 plants were 270 Cryopreservation of Tropical Plant Germplasm selected in order to capture the maximum genetic diversity with a minimum number of plants. In order to maintain the species on a long-term basis, cryostorage of shoot apices was investigated. The cryostorage of shoot apices can be achieved using a number of different methods such as slow cooling, encapsulation-dehydration, encapsulation- vitrification and vitrification. For G. scapigera, slow cooling, encapsulation- dehydration and vitrification methods have been investigated, with the vitrification method being the most effective with up to 80% of shoot apices surviving (Touchell 1995; Touchell and Dixon 1995). Excised shoot apices were cultured on half-strength Murashige and Skoog basal salt media suplemented with 0.6M sorbitol for 48 h, followed by a 30-min treatment with a plant vitrification solution (PVS2) (Yamada et al. 1991) for 30 min at 0°C before directly immersing samples in liquid nitrogen. Thawed shoot apices were washed free of cryoprotectant and recovered on a basal medium for 7 days before being transferred to a medium containing zeatin.

Optimising the prefreezing procedures Prior to excising shoot apices for cryostorage, it is essential that plants are grown to optimize their physiological condition. A wide variety of possible treatments has been used, including cold conditioning, hormonal treatments and osmotic stress. For G. scapigera, optimal preculture conditioning involved growing plantlets on media supplemented with a low concentration of benzyl aminopurine (BAP). High level of BAP or the use of an alternative cytoknin in the culture media, such as kinetin, zeatin or 2IP, adversely effected post-thaw shoot apex survival. Kinetin in particular reduced survival significantly with only 10% of shoot apices surviving cryostorage compared with 79% obtained when plants were maintained on BAP.

Optimising post-thaw recovery Post-thaw recovery is an essential component of cryostorage procedures. Optimising post-thaw recovery procedures should promote shoot development and lead to rapid plantlet recovery and development, thus minimising callus formation and possible genetic abnormalities. In the case of G. scapigera, it was observed that the endogenous synthesis of trans zeatin riboside 4 weeks after shoot apex excision was 3-fold lower in cryostored shoot apices than in frozen control shoot apices. This correlates strongly with senescence of cryostored shoot apices as well as development of unfrozen shoot apices. Thus, optimal recovery procedures involved culturing of shoot apices on a medium containing 1 mM zeatin under reduced light. This not only prolonged shoot apex survival, but also promoted rapid shoot development and elongation. Shoot apices that were cryostored and recovered using optimal procedures grew rapidly and showed no morphological differences. Genetic studies using Random Amplified Polymorphic DNA (RAPD) also showed that there were no genetic differences between recovered cryostored shoot apices and plantlets derived from unfrozen controls. The vitrification cryostorage protocol developed for G. scapigera has now been implemented into conservation programmes at Kings Park and Botanic Ongoing cryopreservation projects 271

Garden. The protocol has been applied to the 10 G. scapigera clones, which have been identified as containing 87.4% of the known genetic variability of the species (Table 1). Variability in post-thaw survival was minimal between clones, with the exception of clone 8, which has a significantly lower survival. The small amount of variability may be attributed to clonal differences, although it is more likely that the variations observed are related to the physiological conditions of the cultures.

Table 1. Post-thaw survival of 10 clones of the endangered species Grevillea scapigera Control Survival (%) Clone 1 100 79.6 ± 4.6 Clone 2 100 54.4 ± 12.3 Clone 5 100 50 ± 4.2 Clone 8 100 8.5 ± 6.6 Clone 16 100 40 ± 0.9 Clone 23 100 78.5 ± 1.9 Clone 27 100 55 ± 2.1 Clone 33a 100 60 ± 3.6 Clone 33b 100 40 ± 6.3 Clone Q32 100 55 ± 6.6

Application to other species Despite the species-specific nature of cryopreservation protocols, the cryostorage procedure developed for Grevillea scapigera has been effectively applied to over 30 endangered and horticulturally valuable species from Western Australia. Understanding the unique qualities as well as the tissue culture constraints of each species or clone benefits in the development of optimal cryopreservation procedures applicable for a diverse range of species. For Western Australian species, optimising the prefreezing culture conditions has been of paramount importance for obtaining high post-thaw survival. For example, as with G. scapigera, high levels of cytoknin in the growth media had an adverse effect on the post-thaw survival of shoot apices of Anigozanthos viridis. Survival decreased from 29% when there was no cytokinin in the growth media to 5% when 2.5 mM BAP was added to the growth media. For other species, such as Lechenaultia formosa, it was essential to add abscisic acid to the prefreezing growth media to obtain survival after cryostorage. Through modifying prefreezing culture conditions to optimize plant growth for a diverse range of species genera and families, the implementation of vitrification has seen the establishment of conservation collections through securing germplasm in liquid nitrogen. This provides an economical means for the long-term ex situ conservation of endangered species.

Conclusion These studies have demonstrated the advantages of cryostorage as a discipline which can now be incorporated into an integrated conservation strategy, 272 Cryopreservation of Tropical Plant Germplasm providing a safe and economical means for long-term storage of an endangered species. However, cryostorage cannot be viewed as a method to replace in situ conservation. Although essential, it should only be used as a means for facilitating the conservation process by providing a secure method for storing endangered germplasm for future use or in case of catastrophic events.

References Bunn, E. and K.W. Dixon. 1991. In vitro propagation of the rare and endangered Grevillea scapigera (Proteaceae). Hortscience 27:261-262. Fay, M.F. 1994. In what situations is in vitro culture appropriate to plant conservation? Biodiversity and Conservation 3: 176–183. Pence, V.C. 1991. Cryopreservation of seeds of Ohio native plants and related species. Seed Science and Technology 19: 235–251. Rossetto, M., P.K. Weaver and K.W. Dixon. 1995. Use of RAPD analysis in devising conservation strategies for the rare and endangered Grevillea scapigera (Proteaceae). Molecular Ecology 4: 321–329. Touchell, D.H. 1995. Principles of cryobiology for conservation of threatened Australian plants. Doctoral Thesis of the University of Western Australia, Botany Division, Australia. Touchell, D.H. and K.W. Dixon. 1996. Cryopreservation for the conservation of Australian endangered plants. Pp. 169–180 in International Workshop on In Vitro Conservation of Plant Genetic Resources, Kuala Lumpur, Malaysia. Plant Biotechnology Laboratory, Faculty of Life Sciences, Universiti Kebangsaan. Yamada, T., A. Sakai, T. Matsumura and S. Higucho. 1991. Cryopreservation of apical meristems of white clover (Trifolium repens L.) by vitrification. Plant Science 78: 81–87. Ongoing cryopreservation projects 273

Cryopreservation and cassava germplasm conservation at CIAT William M. Roca, D. Debouck, Roosevelt H. Escobar, Graciela Mafla and M. Fregene CIAT, Cali, Colombia

Introduction Cassava is a staple food crop for 500 million people in tropical countries of Africa, Asia, Latin America, the Caribbean and Oceania. The crop is a chief source of calories and constitutes a unique food security resource, particularly when production of other crops has failed owing to edaphoclimatic problems like drought. In recent years, cassava has entered the commodity market as a feed, and has demonstrated potential for the starch industry. Cassava is grown on 16 million ha, with a total production of 150 million MT (FAO 1995); around 60% of the production is in Africa, 25% in Asia and 15% in Latin America.

Cassava genetic resources The genus Manihot comprises some 100 species of which M. esculenta is the only cultivated species; wild materials include herbs, shrubs and trees. Manihot species originated in tropical America, and domestication and selection of cassava preferred large storage roots and vegetative propagation. Modern cassava is monoecious, highly heterozygous and outcrossed. Vigour and productivity are maintained through vegetative reproduction. Seed production is generally low owing to poor flowering and poor seed set, but genotypes with high seed production and high yield have been selected in cassava breeding programmes like those at CIAT, IITA and several National Programs. From its primary centre of diversity in tropical America, cassava was taken by Portuguese and Spanish to Africa and Asia between the mid-1500s and mid- 1800s. Consequently secondary centres of diversity are found, particularly in Africa. Because of collecting efforts, principally between the early 1970s and early 1980s, nearly 20 000 clonal accessions of cassava (principally landraces) have been assembled in germplasm collections in nearly 40 countries (Table 1).

Table 1. National and international cassava collections assembled to 1997† Geographic area No. countries No. clonal accessions South America 6 9538 Central America 4 466 Caribbean 2 541 South Africa 5 1685 West and Central Africa 11 4710 Asia and Oceania 11 2747 Total 39 19687 † Bonierbale et al. 1997. 274 Cryopreservation of Tropical Plant Germplasm

In the CGIAR system, CIAT and IITA share the responsibility for cassava genetic resources conservation, with CIAT having the global mandate and IITA the mandate for Africa. The CGIAR international cassava collections, including over 9000 clonal accessions in total, are maintained in trust under an agreement signed with FAO in 1994.

Cassava genetic resources conservation at CIAT CIAT’s activities in cassava conservation should be seen in the context of the Center’s overall research programme on cassava genetic resources, which is designed to address key questions like the kind and amount of genetic resources to be conserved, the most appropriate methods of conservation, and the efficient utilization of cassava genetic resources. CIAT research on cassava genetic resources comprises the three areas and research activities, and the methods and techniques available at the Center to tackle the respective research areas, as indicated below.

Research type Methods/tools Research on genetic diversity GIS, molecular markers, botany, Spatial/geographic distribution ecology, participatory research Genepool structure Ex situ collections vs. in situ diversity Comparative genome mapping Research on conservation Morphology, agronomy, ecology, Rationalization of collections molecular markers, seed storage, in – Identification of redundancies vitro storage, cryopreservation – Development of core collections Conservation methodologies – Active and base genebanks Research on genetic base Molecular markers, morphology, Measure diversity within/between species agronomy, wide crossing, Trait introgression transformation

Research on cassava genetic diversity has been critical for developing scientific criteria for improving conservation strategies, ex situ and in situ, including in vitro and cryopreservation methodologies, and for enhancing utilization of cultivated and wild genetic resources in cassava improvement.

Integrated conservation of cassava genetic resources at CIAT The genetic resources available for cassava improvement at CIAT include: collected landraces, advanced breeding lines and the crop’s wild relatives. Figure 1 shows the overall scheme for a genus–wide conservation approach for cassava. The various conservation components shown in the diagram are considered complementary. They provide an integral conservation strategy for Manihot both for the long term and for immediate practical utilization of the international collections. Each of the conservation modalities shown in the scheme should be considered dependent on the dynamics of the research activities shown above, in particular on those under genetic diversity and conservation research. Ongoing cryopreservation projects 275

Low temperature Field collections $ Seed Conventional Genebanks $ Core $ (Pollen) * $ Subcores

Base collections Active collections

Cryopreservation ** Culture $ Shoot-tips in $ Slow $ (Seed pollen) * Genebanks $ Shoot plantlet $ (Leaf tissue - DNA) * $ (DNA Libraries) *

Micropropagation Seed regeneration (DNA extraction/cloning) * $ Field $ Distribution

Phytosanitary testing/cleaning

Introduction

Fig. 1. Integral, complementary, conservation methods for cassava genetic resources. (*) Indicates components of conservation scheme not yet implemented; cryopreservation is entering the implementation phase.

The conventional and in vitro genebanks are complementary, as well as the active and base collections. It is envisaged that only the core and subcore collections will be maintained in the field, at any one time, while all clonal accessions will be represented all the time in the in vitro slow growth collections. Cassava germplasm can also be conserved in the base collections as seed and pollen, particularly as a means of maintaining genepools. The role of cryopreservation in the cassava base collection scheme is considered extremely critical since all types of germplasm and material can be maintained, including all clonal accessions as shoot-tips, genepools as seed and pollen, frozen leaf tissue of any material as sources of DNA, and DNA libraries. In addition, cryopreservation, once fully developed, should also provide an expeditious and cheaper means to duplicate the base collection for safety reasons, as well as for the distribution of germplasm sets to other countries/continents. Cassava being preferentially reproduced by vegetative means, the role of micropropagation is also key to the management of the integral conservation strategy. Improvement of current micropropagation through the implementation of bioreactor technology for both shoot-tip based and somatic embryogenesis methods will be essential. Finally, current advances in cassava molecular biology will contribute to implementing DNA banks and their utilization through molecular screening and DNA cloning in cassava improvement. 276 Cryopreservation of Tropical Plant Germplasm

Cassava collections assembled and conserved at CIAT Table 2 shows the overall current status of the international cassava germplasm collections held in trust at CIAT. Over 6000 clonal accessions from 23 countries are maintained in the field and in vitro genebanks, including cultivated cassava, mostly landraces, selected improved materials, and wild Manihot genotypes representing 32 species. The greater size of the in vitro collection than the field collection is because many introductions in vitro have moved directly to the in vitro active collection (Fig. 1) and await phytosanitary testing before moving to the field for evaluation. Another reason is that a number of duplicates (identified through morphological and biochemical/molecular methods) have been eliminated from the field collection, to be maintained only in the in vitro collection.

Table 2. Current status of the cassava germplasm collections assembled and conserved at CIAT (CIAT 1998)† Number Germplasm Type In field In vitro Cultivated material ‡ Clonal accessions 4750 6017 Wild material § Genotypes 10–15 353 † From 23 primary diversity countries, mostly Latin American and Caribbean. ‡ Manihot esculenta. § 32 wild Manihot spp.

In vitro collections Between the late 1970s and early 1980s apical meristem and shoot-tip based micropropagation of cassava was implemented at CIAT and implications in genetic resources conservation established (Roca 1984; Roca et al. 1989). The techniques utilize the MS basal medium supplemented with gibberellic acid (GA), benzyl aminopurine (BAP) and naphtalene acetic acid (NAA), and proved to be universally applicable for virus elimination (associated with thermotherapy and for micropropagation (through shoot-tip and nodal microcuttings). Modifications of these methodologies, involving media composition and osmotic concentration, and physical in vitro conditions of culture, allowed the gradual implementation of an active genebank for the cassava collection. Currently, more than 6000 clonal accessions comprise the in vitro active genebank at CIAT. Each accession is represented by six 18 x 25 mm 2 test tubes, and the entire collection occupies a 50-m room. Temperature and illumination have been set at 22–23°C and 1500 and 2000 lux, respectively. Under these conditions, subculture interval ranges between 12 to 18 months, depending upon the cassava genotype. More than 1500 accessions have been pathogen-tested, including the core collection (630 accessions). The CIAT cassava in vitro genebank comprises mostly landraces collected in Latin America, a set of advanced breeding lines and 325 wild Manihot genotypes. Unlike the landraces, the latter cultures derive from zygotic embryos because the shoot-tip culture of wild Manihot is still not well developed. After 10 years of slow growth storage, involving 10–20 subculture cycles, the genotypic Ongoing cryopreservation projects 277 stability of seven varieties was assessed using molecular markers. No changes were observed in DNA fingerprintings vis-à-vis non-conserved controls (Angel et al. 1996).

The Pilot In Vitro Active Gene Bank Project Although the cassava in vitro collection has been operative at CIAT since the early 1980s, several aspects of the management of such a genebank were not established, particularly those that relate to scaling-up of the process. In order to tackle this issue, between 1988 and 1991, CIAT and IPGRI conducted a pilot project to assess the technical and logistical aspects of establishing and running an in vitro active genebank (IVAG) using cassava as a model (IPGRI/CIAT 1994). A condensed, representative sample of 100 genotypes from the cassava world collection assembled at CIAT was used for storage under slow growth conditions, and the following parameters were monitored throughout the process: phytosanitary status of cultures, culture viability, genetic stability, sample size and subculture interval. Also, the study determined needs of laboratory, equipment facilities, consumable items and staffing. A database system was designed for the management of in vitro genebanks. The project provided guidelines and testing parameters for establishing and running an IVAG (IPGRI/CIAT 1994).

Cassava cryopreservation at CIAT As show in Figure 1, cryopreservation is considered to play a critical role in the integral, complementary, conservation strategy for cassava. The technical developments in cassava cryopreservation have been described in these proceedings by Escobar et al. (this vol., p. 222). The major events in the evolution of cassava cryopreservation research at CIAT can be summarized chronologically as follows:

1985 First discussions on cryopreservation feasibility with Prof. A. Sakai and Dr K. Kartha in Hokkaido, Japan. 1988– A collaborative project between CIAT and IBPGR, with the 1990 participation of the PBI, Saskatoon, was carried out. As a result, a technique for cassava true seed cryopreservation was developed (Marin et al. 1990), and the first viable shoot-tips of cassava variety M Col22 were recovered. The classical slow freezing technique was used, with a programmable freezing apparatus donated by the Japanese government. 1991–92 Cryopreservation research continued with CIAT’s core resources. 1993–98 During this period, viable shoot-tips and plants were recovered using the slow freezing technique with a range of cassava varieties (Escobar et al. 1997), and fully grown plants were moved to the field for assessing genotypic stability. On the other hand, using a rapid freezing technique, shoot regrowth rates comparable to the slow freezing technique, were obtained. Interestingly, high genotypic response occurred 278 Cryopreservation of Tropical Plant Germplasm

with cassava landraces adapted to subtropical or high drought areas. The encapsulation-dehydration cryotechnique was also implemented in this period, with obvious advantages gained over the former two methods, in simplicity and rapidity of the technique. In addition, genotypic response was high (CIAT 1998). Informal cooperation with IPGRI took place in this period.

Future plans The assessment of the technical and logistical aspects of establishing and running a base genebank under cryopreservation will be the main CIAT activity in the period 1999–2001. Prior to this, the vitrification technique, and combinations with encapsulation, will be tested with cassava. This developmental work will be carried out as a pilot project using a subcore of the 630 cassava core accessions maintained in the in vitro active genebank. Important paramenters such as monitoring genotypic stability, culture viability, sample size per clonal accession, and number of replicates, will be key components of the project. Integration with other CGIAR-mandated vegetatively propagated crops like potato, yams and banana/plantains is considered highly appropriate, and thus a collaborative effort between CIAT, CIP, IITA and INIBAP is being planned.

The role of research in cassava genetic diversity CIAT has developed a repertoire of molecular genetic markers for cassava including RAPDs, RFLPs, AFLPs and SSRs, both nuclear and cytoplasmic, which are integrated with morphological, edaphoclimatic and ecological data. This information can be used for assessing diversity at inter- and intraspecies level, drawing phylogenetic relationships between Manihot spp. and cassava and describing structural relationships between unimproved (landraces) and improved (elite material) germplasm. And through GIS, we can describe the geographic distribution of diversity. Diversity data will serve to quantify similarities among accessions, define core and subcore collections, for short- and long-term cassava conservation. It would be possible also to relate molecular diversity and geographic origin to in vitro and cryogenic response of cassava landraces, and define sets of wild species for in situ and/or ex situ conservation. For example, recent analysis of cassava relationships with wild Manihot species using molecular markers has provided support to the existence of three subspecies: the domesticated M. esculenta subsp. esculenta, and its two wild forms M. esculenta subsp. peruviana and M. esculenta subsp. flabellifolia (Roa et al. 1996; Bonierbale et al. 1997). This information will find direct application not only in designing genus-wide conservation strategies for cassava, but also for enhancing the genetic improvement of the crop. Cassava molecular markers such as AFLPs and SSRs, and the recent development of the cassava molecular genetic map at CIAT (Fregene et al. 1997), provide powerful tools for monitoring and assessing the genotypic stability of in vitro and cryopreserved cassava germplasm, and for enhancing the utilization of wild Manihot spp. in crop improvement. Ongoing cryopreservation projects 279

References Angel, F., V.E. Barney, J. Tohme and W. Roca. 1996. Stability of cassava plants at DNA level after retrieval from 10 years in vitro storage. Euphytica 90:307–713 Bonierbale, M., C. Guevara, A.G. Dixon, N.Q. Ng, R. Asiedu and S.Y.C. Ng. 1997. Cassava. Pp. 1-20 in Biodiversity in trust: Conservation and use of plant genetic resources in CGIAR centres. D. Fucillo, L. Sears and P. Stapleton, eds. Cambridge University Press, UK. CIAT. 1998. Annual Report Project SB–02. CIAT, Cali, Colombia. Escobar, R., G. Mafla and W. Roca. 1997. A methodology for recovering cassava plants from shoot tips maintained in liquid nitrogen. Plant Cell Reports 16:474–476. FAO. 1995. Production year book for 1994. Food and Agriculture Organization of the United Nations, Rome. Fregene, M., F. Angel, R. Gomez, F. Rodriguez, P. Chavarriaga, W. Roca, J. Tohme and M. Bonierbale. 1997. A molecular genetic map of cassava (Manihot esculenta Crantz). Theoretical and Applied Genetics 95:431–441. IPGRI/CIAT. 1994. Establishment and operation of a pilot in vitro active genebank. Report of a CIAT-IBPGR collaborative project using cassava Manihot esculenta Crantz) as a model. A joint publication of IPGRI, Rome and CIAT, Cali, Colombia. Marin, M.L., G. Mafla, W. Roca and L.A. Withers. 1990. Cryopreservation of cassava zygotic embryos and whole seeds in liquid nitrogen. Cryo–Letters 11:257–264. Roa, A.C., M. Maya, M. Duque, J. Tohme, A. Allem and M. Bonierbale. 1996. AFLP analysis of relationships among cassava and other Manihot species. Theoretical and Applied Genetics 95:741–750. Roca, W. 1984. Cassava. Pp. 269–301 in Handbook of Plant Cell Culture (Sharp, W., Evans, D., P. Ammirato and Y. Yamada, eds). MacMillan, New York. Roca, W., R. Chavez, M.L. Marin, D. Arias, G. Mafla and R. Reyes. 1989. In vitro methods of germplasm conservation. Genome 31: 813–817. 280 Cryopreservation of Tropical Plant Germplasm 282 Cryopreservation of Tropical Plant Germplasm

Cryopreservation research in India: present status and future perspectives B.B. Mandal NBPGR, Pusa Campus, New Delhi-12, India

Introduction India, having a large area with humid and humid tropical climates, has many economically important plant species bearing either recalcitrant or suborthodox seeds. The country also possesses a wide array of endemic and/or other economically important species, which are predominantly vegetatively propagated. Conservation of genetic resources of the above two categories of plant species through the traditional method of field maintenance poses serious problems. Cryopreservation is perhaps the only viable method available for long-term conservation of these problem species. Cryopreservation research for conservation of plant germplasm was initiated in India in the 1970s (Bajaj 1977, 1978). However, to make a concerted research effort in various aspects of plant cryopreservation, a special programme was launched at NBPGR in 1986. During the past decade this programme has made significant progress. Several protocols have been developed for cryopreservation of recalcitrant seed and vegetatively propagated species. Base collections of plant germplasm under cryopreservation also have been established for a few species. In this report a brief description of the progress of cryopreservation research at NBPGR is presented. The status of cryopreservation research at various other research centres of the country is then summarized. Finally, the impact of these developments on the future perspectives of its application is discussed.

Cryopreservation research at NBPGR

Cryopreservation of recalcitrant / suborthodox seed species

Using zygotic embryos / embryonic axes The most simple method, i.e. desiccation of excised zygotic embryos / embryonic axes, was used to develop protocols for cryopreservation of several recalcitrant seed species. Investigations suggested that, after isolation and desiccation to a moisture content of about 14% under aseptic conditions, embryonic axes of several species could tolerate freezing in liquid nitrogen. In one of the studies with embryonic axes of three predominantly recalcitrant seed species including tea, jackfruit and cacao it was found that partially and fully mature embryonic axes of tea and jackfruit could be desiccated to about 14% moisture content and successfully cryopreserved. The degree of sensitivity of these axes to desiccation varied with physiological maturity of seeds (Chandel et al. 1995). Similarly, successful cryopreservation using embryonic axes was obtained with trifoliate orange (Radhamani and Chandel 1992) and almond (Chaudhury and Chandel 1995a). Current status of cryopreservation research 283

Using seeds Seed cryopreservation may be advantageous in certain recalcitrant/suborthodox seed species as it can avoid the inherent problems of embryo isolation and handling. Seeds of neem with or without endocarp could be desiccated and cryopreserved (Chaudhury and Chandel 1991). Suborthodox seeds of black pepper (Chaudhury and Chandel 1994) and cardamom (Chaudhury and Chandel 1995b) also could be processed and successfully cryopreserved. Even in vegetatively propagated crops such as banana, seed cryopreservation of wild species is relevant because in wild species conservation in the form of a genepool may be adequate. Cryopreservation of zygotic embryos / embryonic axes of wild banana was reported earlier (Abdelnour–Esquivel et al. 1992). However, in our laboratory, cryopreservation of wild banana (Musa balbisiana) has been simplified using seeds with high percentage of germination (Bhat et al. 1994).

Establishment of base collection of tea and other plant species At NBPGR, cryopreservation protocols have not only been developed and/or standardized but also utilized to establish base collections. In tea, 85 accessions were collected from diverse sources and conserved under cryopreservation using embryonic axes. Recently, the collections of recalcitrant/suborthodox seeds in the cryobank have been increased to over 100 accessions by the addition of several accessions of other species such as almond, neem, cardamom and black pepper.

Cryopreservation of clonally propagated species Shoot-tips of four species of yams including Dioscorea alata, D. bulbifera, D. wallichii and D. floribunda were successfully cryopreserved using the technique of encapsulation-dehydration. The survival of shoot apices ranged from 26 to 71% and regeneration of plants from thawed apices was achieved with two species (Mandal et al. 1996). Protocols for cryopreservation of shoot- tips of three of these species also have been developed using the vitrification and encapsulation-vitrification techniques. Success has been registered in cryopreservation of shoot-tips of blackberry and somatic embryos and embryogenic callus of D. bulbifera employing encapsulation-dehydration.

Cryopreservation of pollen for the establishment of a base collection Storage of pollen is a useful adjunct to conservation of plant germplasm. It requires little space and can be useful as a supplement to base collections of specific plant germplasm. Cryopreservation of pollen from various crop species of importance to plant breeders has been carried out. A total of 65 accessions of about 16 species including Brassica species and primitive landraces of maize have been conserved using pollen cryopreservation.

Cryopreservation of orthodox seed species Although cryopreservation of orthodox seed is considered to be a 284 Cryopreservation of Tropical Plant Germplasm straightforward method, real data on amenability of seeds of many species to freeze preservation are not available. Cryopreservation of seeds of many small- seeded crop species is important, as it requires very little space and the seeds can be conserved for indefinite periods. It is particularly important in the case of economically important species for which very little seed is available and for endangered species. At NBPGR, a cryobank with 1243 accessions of various orthodox seed species, particularly small-seeded/wild species of cereals, millets, pulses, vegetables, medicinal and aromatic plants has been established.

Table 1. Status of cryopreservation of recalcitrant/suborthodox seed species at NBPGR Species Explants Camellia sinensis Embryonic axes Artocarpus heterophyllus Embryonic axes Poncirus trifoliata Embryonic axes Prunus sp. Embryonic axes Azadirachta indica Seeds Musa balbisiana Seeds Piper nigrum Seeds Eletaria sp. Seeds

Table 2. Status of cryopreservation of clonally propagated species at NBPGR Species Explants used Dioscorea alata Shoot-tips D. bulbifera Shoot-tips/ somatic embryos /embryogenic callus D. wallichii Shoot-tips D. floribunda Shoot-tips

Cryopreservation activities at other research centres in India The Indian Horticultural Research Institute (IIHR), Bangalore and the Indian Institute of Technology (IIT), Kharagpur are the two premier institutes in the country where cryopreservation activities were initiated in the 1980s. At IIHR, pollen cryopreservation of various horticultural crop species such as eggplant, gladiolus, onion, papaya and tomato were carried out to establish base collections (Rajasekharan et al. 1993; Rajasekharan and Ganeshan 1995). Similarly, at IIT Kharagpur, research on cryopreservation of plant species continued and a protocol for cryopreservation of sapota (a recalcitrant seed species) using embryonic axes has been developed. Recently, cryopreservation activities have been initiated in a few other research centres. The Tropical Botanical Garden and Research Institute, Trivendrum has initiated cryopreservation research on medicinal and aromatic plant species. Similarly, the Institute for Forest Genetics and Tree Breeding, Coimbatore, the Indian Institute of Spices Research, Calicut and the University of Horticulture and Forestry, Solan, have initiated cryopreservation research on forest plant species, spices and walnut, respectively. Current status of cryopreservation research 285

Future perspectives For long-term conservation of germplasm of important recalcitrant seed and vegetatively propagated plant species, particularly the indigenous diversity, cryopreservation will receive emphasis in future. Cryopreservation protocols were not only developed for several recalcitrant seed species but also have been utilized to establish a base collection of about 100 accessions belonging to four species such as tea, jackfruit, almond, etc. Recently, NBPGR has established its expanded infrastructure (cryobank) at the new National Gene Bank, which has an estimated capacity of 0.25 million small seeds / embryonic axes / shoot-tips to be stored under cryopreservation. Also, NBPGR has been maintaining a large collection of germplasm of over about a dozen crops under in vitro slow or normal growth for over a decade. Development of cryogenic protocols for these in vitro cultures will allow their safe, long-term conservation under cryopreservation. In this direction in vitro-grown shoot-tips and somatic embryos of four yam species (Dioscorea spp.) have been successfully cryopreserved employing vitrification and encapsulation-dehydration. Besides IIHR, Bangalore has established a cryobank to conserve a base collection of horticultural crops in the form of pollen. Several research centres in the country have initiated research on their mandate crops as mentioned earlier. Several other governmental and Non-Governmental Organizations (NGOs) of the country are also coming forward for collaboration with NBPGR for cryopreservation of different crop species. For example, NBPGR has formulated collaborative cryopreservation research projects with the Coffee Biotechnology Network and Neem board and memoranda of understanding have been signed. Considering these developments, it can be envisaged that cryopreservation methods would be utilized routinely in India for long-term conservation of germplasm in the near future.

References Abdelnour–Esquivel, A., A. Mora and V. Villalobos. 1992. Cryopreservation of zygotic embryos of Musa acuminata (AA) and Musa balbisiana (BB). Cryo–Letters 13:159–164. Bajaj, Y.P.S. 1977. Initiation of shoots and callus from potato-tuber sprouts and axillary buds frozen at –196ºC. Crop Improvement 4: 48–53. Bajaj, Y.P.S. 1978. Tuberization in potato plants regenerated from freeze-preserved meristems. Crop Improvement 5: 137–141. Bhat, S.R., K.V. Bhat and K.P.S. Chandel. 1994. Studies on germination and cryopreservation of Musa balbisiana seed. Seed Science and Technology 22:637–640. Chandel, K.P.S., R. Chaudhury, J. Radhamani and S.K. Malik. 1995. Desiccation and freezing sensitivity in recalcitrant seeds of tea, cocoa and jackfruit. Annals of Botany 76:443–450. Chaudhury, R. and K.P.S. Chandel. 1991. Cryopreservation of desiccated seeds of neem (Azadirachta indica A. Juss.) for germplasm conservation. Indian Journal of Plant Genetic Resources 4:67:72. Chaudhury, R. and K.P.S. Chandel. 1994. Germination studies and cryopreservation of seeds of black pepper (Piper nigrum L.) –A recalcitrant species. Cryo–Letters 15:145–150. Chaudhury, R. and K.P.S. Chandel. 1995a. Cryopreservation of embryonic axes of almond (Prunus amygdalus Batsch.) seeds. Cryo–Letters 16:51–56. Chaudhury, R. and K.P.S. Chandel. 1995b. Studies on germination and cryopreservation of 286 Cryopreservation of Tropical Plant Germplasm

cardamom (Elettaria cardamomum Maton) seeds. Seed Science and Technology 23:235–240. Mandal, B.B., K.P.S. Chandel and S. Diwvedi. 1996. Cryopreservation of yam (Dioscorea spp.) shoot apices by encapsulation-dehydration. Cryo–Letters 17: 165–174. Radhamani, J. and K.P.S. Chandel. 1992. Cryopreservation of embryonic axes of trifoliate orange (Poncirus trifoliata (L.) RAF.). Plant Cell Reports 11:372–374. Rajasekharan, P.E. and S. Ganeshan. 1995. Conservation of haploid genetic diversity through pollen cryopreservation in Lycopersicon pimpinellifolium: evaluation of pollen viability, vigour and fertility. Journal of Palynology 31:383–388. Rajasekharan, P.E., M.P. Alexander and S. Ganeshan. 1993. Long-term pollen preservation of wild tomato, brinjal species and cultivars in liquid nitrogen. Golden Jubilee Symposium of HIS, Bangalore (May 24–28, 1993) Abstract No.22. Current status of cryopreservation research 287

Current status of cryopreservation research and future perspectives of its application in Malaysia M.N. Normah Department of Botany, Faculty of Life Sciences, Universiti Kebangsaan Malaysia, 43600 UKM, Bangi, Selangor, Malaysia

Introduction Tropical plant species that produce recalcitrant seeds include the forest trees, plantation crops and fruit species. In fact, in tropical forests, about 70% of the species produce seeds that are recalcitrant in nature (Marzalina et al. 1994). The germplasm of these species cannot be stored as seeds. At present, their conservation is dependent upon wild stands or arboreta, where they are susceptible to loss through diseases, pests, vandalism and other natural calamities. In vitro conservation techniques are the best possible alternatives for conservation of these species. Techniques such as in vitro slow growth and slow growth of seedlings are used for short- to medium-term storage, while cryopreservation offers a long-term storage method.

Cryopreservation research Research on cryopreservation in Malaysia is mainly performed on three important plant groups – fruit tree species, forest species and plantation crops. Universiti Kebangsaan Malaysia (UKM), Universiti Putra Malaysia (UPM), Forest Research Institute of Malaysia (FRIM) and Malaysian Agricultural Research and Development Institute (MARDI) are the institutions involved in this cryopreservation research. Most of the research concentrates on cryopreservation of seeds and embryonic axes/embryos. However, much attention is also given to cryopreservation of shoot-tips and buds from in vitro seedlings/explants, especially for those recalcitrant species whose embryonic axes have failed to survive cryopreservation. At the Plant Biotechnology Laboratory, UKM, cryopreservation techniques are being developed for fruit tree species such as Garcinia mangostana, Citrus spp., Lansium domesticum, Baccaurea motleyana and B. polyneura. Work on fruit tree species such as L. domesticum and Baccaurea species using embryonic axes has shown that the axes are very sensitive to desiccation and low temperatures (Normah et al. 1997). Currently, techniques are being developed for regeneration and cryopreservation of shoot-tips and buds of these species. Preliminary TTC results indicated some viability of shoot-tip tissue of Garcinia mangostana after cryopreservation. More studies need to be carried out in order to establish the most suitable method. The research is funded mainly by the National IRPA grant, while IPGRI funds the later work on Citrus which started in July 1998. Citrus species studied earlier were C. halimii, C. mitis, C. aurantifolia, C. hystrix and C. medica. Desiccation, encapsulation-dehydration and vitrification have been used for the studies. Cryopreservation of embryonic axes of C. aurantifolia, C. halimii and C. medica has been successful (80–100% 288 Cryopreservation of Tropical Plant Germplasm viability) using the desiccation technique (Normah and Serimala 1997). It was also observed that C. aurantifolia did not lose viability after cryostorage of 3 months (Haslina 1997). For C. hystrix, viability after cryopreservation was relatively low (60%), whereas for C. mitis none of the embryonic axes survived the low temperature of liquid nitrogen even when encapsulation-dehydration and vitrification were used. For the project funded by IPGRI, the objective is to determine seed desiccation and freezing tolerance of the Citrus species found in the Citrus collection at the Universiti Malaya. This is a joint project with UPM. Preliminary results have shown that Citrus madurensis and Fortunella polyandra seeds are sensitive to freezing temperature and lose viability after the moisture content is reduced to below 30%. The seeds of Triphasia buxifolia were found to be recalcitrant while those of Aegle marmelos are orthodox. Other work carried out by UPM is on plantation crops such as oil-palm, cacao, coffee and rubber. Some work on fruit trees (not necessarily recalcitrant) is also being carried out using zygotic embryos. Methods employed are desiccation, encapsulation-dehydration and vitrification. The effects of different concentrations of sucrose in the techniques are emphasized. Some of the results have shown that the techniques developed are very variety/species-dependent. The research is funded by the National IRPA Grant and IPGRI. At MARDI, two fruit tree species are being researched: durian (Durio zibethinus) and starfruit (Averrhoa carambola). Besides the three techniques mentioned earlier, MARDI is also using the encapsulation-vitrification technique and testing different cryoprotectants. Thus far, no success has been obtained with durian zygotic embryos, but with carambola more than 60% survival was obtained when seeds were desiccated to 9–10% moisture. Higher survival (80%) was obtained with encapsulation-dehydration of embryos. Funding of the research is by the National IRPA Grant. A lot of work on cryopreservation of forest species has been carried out by FRIM. Emphasis is on Dipterocarps and urban forestry species. Whole seeds, zygotic embryos and shoot-tips/meristems are used. Techniques employed are desiccation, encapsulation-dehydration and vitrification. Besides the development and regeneration of cryopreserved tissues, rapid and systematic screening techniques such as TTC are used for a preliminary indication of post- thaw survival success for forest tree species such as Shorea macrophylla, Shorea sumatrana, Hopea weightiana, Vatica cinerea and Calamus manan (Benson et al. 1995). Positive TTC tests are most promising; however, longer-term studies must be performed concerning the development and regeneration of cryopreserved material. Cryopreservation experiments carried out by FRIM involved storage from 1 month to 3 years. Results obtained on viability can be as low as 5% for a recalcitrant species like Shorea to as high as 100% for orthodox species like Albizia falcataria (Marzalina et al. 1997). Funding for the research at FRIM is mainly from the national IRPA grant. The cryopreservation protocols described are still in the developmental stage. At present there is no long-term storage of plant germplasm using cryopreservation techniques in Malaysia. Current status of cryopreservation research 289

Future perspectives and strategies Cryopreservation has found useful applications in plant germplasm conservation. However, the very large size of many recalcitrant tropical tree species poses problems for cryopreservation of seeds or embryos. There are recalcitrant seed species that contain embryos which are tolerant of desiccation and low temperatures, but many others are very sensitive to these two factors. Cryopreservation of shoot-tips has potential for these difficult species and, as mentioned earlier, much work has started using this material. Nevertheless, specific techniques need to be developed for each species. Efforts must be made to obtain cryopreservation techniques that can be used routinely across a range of genotypes or even species. Furthermore, protocols for regeneration of these tissues in culture must be established. Many tropical trees show recalcitrant behaviour in culture, thus creating another problem that needs to be overcome. Nevertheless, some positive results have been shown with regeneration of Garcinia mangostana and Baccaurea sp. shoot-tips in our laboratory at UKM. The National Policy on Biological Diversity of Malaysia was launched in April 1998 with a vision to transform Malaysia into a world centre of excellence in conservation, research and utilization of tropical biological diversity by the year 2020. With enough funding and improvement or development of techniques for cryopreservation of our tropical species, we hope to establish cryo/in vitro genebanks at each institution on a small scale or at the national level with support from the government. One of the recommendations from the International Workshop on In Vitro Conservation of Plant Genetic Resources held in 1995 in Kuala Lumpur was to update an in vitro conservation database and to publish and circulate the status of in vitro conservation periodically. With the national policy, it is hoped that this recommendation could be tackled and implemented not only for in vitro germplasm collections but also for ex situ germplasm collections as a whole. In fact, currently we are working on establishing a Network of Germplasm Collections at the national level. A committee is being set up to coordinate activities involving germplasm conservation in the country.

Acknowledgements I thank Dr Marzalina Mansor (FRIM), Dr Hor Yue Luan (UPM), Professor H.F. Chin (IPGRI) and Mrs Halimatul Saadiah (MARDI) for contributing the information in the paper.

References Benson, E.E., B. Krishnapillay and M. Marzalina. 1995. The potential of biotechnology in the in vitro conservation of Malaysian Forest Germplasm: An integrated approach. Pp. rd 76–90 in Proceedings 3 Conference on Forestry and Forest Product Research. FRIM, Kuala Lumpur. Haslina, Othman Nasri. 1997. Cryopreservation studies on Citrus aurantifolia, C. mitis and C. medica. Bachelor of Science Thesis, Universiti Kebangsaan Malaysia. Marzalina, M., M.N. Normah and B. Krishnapillay. 1994. Artificial seeds of Swietenia nd macrophylla. Pp. 132–134 in Proceedings of the 2 National Seed Symposium (B. Krishnapillay, M. Haris, M.N. Normah and K.G. Lim, eds.). FRIM, Kuala Lumpur. 290 Cryopreservation of Tropical Plant Germplasm

Marzalina, M., B. Krishnapillay and N.A. Nashatul Zaimah. 1997. In vitro conservation of th tropical rainforest germplasm via cryopreservation. Proceedings 4 Conference on Forestry and Forest Product Research, 2–4 Oct. 1997. FRIM, Kuala Lumpur. (In press). Normah, M.N., D.R. Saraswathy and G. Mainah. 1997. Desiccation sensitivity of recalcitrant seeds – a study on tropical fruit species. Seed Science Research 7:179–183. Normah, M.N., and M.N. Siti Dewi Serimala. 1997. Cryopreservation of seeds and embryonic axes of several Citrus species. Pp. 817–823 in Basic and Applied Aspects of Seed Biology (R.H. Ellis, M. Black, A.J. Murdoch and T.D. Hong eds.). Kluwer Academic Publishers, Dordrecht. Current status of cryopreservation research 291

Cryopreservation of tropical plants: current research status in Indonesia Enny Sudarmonowati Research and Development Centre for Biotechnology–Indonesian Institute of Sciences (LIPI), Cibinong 16911, Indonesia

Introduction As a country that has megadiversity, Indonesia has determined that conservation and utilization of genetic resources in a sustainable way are of primary importance. Until now, the main focus has been on in situ and ex situ conservation in field genebanks, but risks of losing genetic material exist with these conservation methods. Alternative strategies which would ensure more efficient and economic conservation and reduce germplasm loss have to be developed. Research on cryopreservation of plant species in Indonesia was not initiated until 1992 at the time when research on the effect of various osmotical agents on the growth of various plants was conducted at R&D Centre for Biotechnology–LIPI. The results of this research were then used as the basic information for conducting cryopreservation work in subsequent years. Research on cryopreservation of various plant species has been carried out intensively in this institute since then. The cryopreservation techniques used are conventional two-step freezing, rapid freezing, encapsulation-dehydration, vitrification and vitrification-dehydration. Besides this institute only one other institute, which is under the Ministry of Agriculture, is carrying out cryopreservation work. Other institutions have only been applying slow growth methods, mostly by adding growth inhibitors, osmotic agents (usually sorbitol and mannitol), and very few of those are applying storage at low temperature. The main reason is the limited availability of human resources. Recently, the need to conduct cryopreservation has increased as researchers of several institutions came to LIPI for consultation, although no distinctive implementation has been made. In this paper, various research activities that have been conducted, ongoing and future research of Indonesian institutions are discussed. The results of the activities, problems encountered and possible solutions for increasing the quantity and quality of cryopreservation research in Indonesia are also discussed.

Cryopreservation at R&D Centre for Biotechnology – LIPI Cryopreservation research at this Centre has been focused mainly on forest tree species: during the period 1992–95 there was a project funded by UNDP/UNESCO and the Government of Indonesia on Biotechnology of Forest Tree Species. The species studied were Shorea spp., Acacia mangium, Paraserianthes falcataria, Eucalyptus urophylla, Pometia pinnata and other tree species related to this species, i.e. Euphoria longan, Nephellium lappaceum and Litchi sinensis. Besides woody species (forest and fruit trees), certain food crops 292 Cryopreservation of Tropical Plant Germplasm and horticultural crops that have been studied are maize, soyabean, cassava, garlic and Dianthus caryophyllus. In vitro conservation of plants has been one of the research topics funded by the government of Indonesia since 1993.

Forest tree and fruit tree species Attempts to preserve organs or tissues of several forest trees and fruit trees have been conducted by preserving shoot-tips, zygotic embryos, embryonic axes, and somatic embryos using either two-step freezing, encapsulation- dehydration or vitrification. Of six forest tree species and four fruit tree species, three and all four species, respectively were able to survive with different rates ranging from 10 to 80% after immersion in liquid nitrogen. The most appropriate cryopreservation technique was dependent on the species and the type of organs or tissues used. The main causes of failure for two forest tree species, namely Shorea pinanga and S. leprosula, were inappropriate water content and high production of phenolic compounds, especially after freezing, which was toxic to the tissues. Table 1 shows the results of cryopreservation of forest tree species and fruit tree species conducted at the R&D Centre for Biotechnology–LIPI. Of organs of forest tree species, four tissues/organs could be cryopreserved, namely shoot-tips (70 and 50% survival) of A. mangium (Sudarmonowati and Rosmithayani 1997) and P. falcataria, respectively, callus (50% survival) of A. mangium (unpubl.), anthers (15% survival) of P. pinnata (unpubl.), embryogenic callus and individual somatic embryos (80 and 80% survival, respectively) of P. pinnata (Sudarmonowati and Wuryan Rahayu 1998).

Cryopreservation of fruit trees Except Citrus sinensis cv. Garut, all fruit tree species tried belong to the same family as P. pinnata, i.e. Sapindaceae. The availability of fruits of this species whose seeds are considered recalcitrant was very limited because of a poor fruiting season. Of organs/tissues of fruit tree species tried, four could be cryopreserved, i.e. excised embryos (50% survival) of L. chinensis (unpubl.), embryonic axis (62.5% survival) of C. sinensis cv. Garut (Sudarmonowati et al. 1998), shoot-tips (10% survival) of N. lappaceum (unpubl.), anthers (15% survival) and embryogenic callus (30% survival) of E. longan (Sudarmonowati et al. 1998). Zygotic embryos of most fruit species tested failed to survive after immersion in liquid nitrogen. Only those of lychee were able to survive (50% survival) using vitrification technique (unpubl.). Only embryonic axes of C. sinensis cv. Garut could survive after immersion in liquid nitrogen. The highest survival rate (62.5%) was obtained using vitrification-dehydration and the vitrification solution used was a combination of 0.8M sucrose and 1.0M glycerol. Encapsulating these embryonic axes led to a reduction in survival rate (Sudarmonowati et al. 1998). Of the various dehydration periods tried, 4 h air-drying under the laminar airflow gave the best result in terms of survival rate. Current status of cryopreservation research 293

Table 1. Techniques and type of materials used for cryopreservation of forest tree and fruit tree species Species Techniques Type of materials Survival rate (%) Forest trees Acacia mangium Two-step freezing Shoot-tips 0 Encapsulation- Shoot-tips 70 dehydration Vitrification Shoot-tips 0 Paraserian Two-step freezing Shoot-tips 0 thesfalcataria Encapsulation- Shoot-tips 40 dehydration Vitrification Shoot-tips 50 Pometia pinnata Two-step freezing Zygotic embryos 0 (36.7% at –20°C) Encapsulation- Shoot-tips 0 dehydration Embryonic axes 0 Vitrification-dehydration Shoot-tips 0 Embryonic axes 0 Vitrification Embryogenic 80 callus Embryo 80 Deep freezing Somatic anthers 15 Eucalyptus Two-step freezing Shoot-tips 0 urophylla Shorea leprosula Vitrification-dehydration Zygotic embryos 0 Embryonic axes 0 Shorea pinanga Vitrification-dehydration Zygotic embryos 0 Embryonic axes 0 Fruit trees Litchi sinensis Two-step freezing Zygotic embryos 0 Embryonic axes 0 Vitrification-dehydration Zygotic embryos 50 Embryonic axes 0 Shoot-tips 0 Euphoria longan Two-step freezing Zygotic embryos 0 Embryonic axes 0 Vitrification Embryonic axes 0 Shoot-tips 0 Embryonic callus 30 Anthers 15 Nephelium Two-step freezing Seeds 0 (20% at –20°C) lappaceum Embryonic axes 0 Shoot-tips 10 Citrus sinensis Encapsulation- Embryonic axes 25 dehydration Shoot-tips 0 Vitrification Embryonic axes 62.5

Like anthers of P. pinnata, those of E. longan could also survive (15%) when they were subjected to deep freezing. The growth stage and physical conditions of the anthers were critical factors as only the earliest stage of anther development, which was indicated by a yellow colour before blooming, gave a positive response. These factors also determined the type of callus 294 Cryopreservation of Tropical Plant Germplasm regenerated from frozen anthers (Sudarmonowati et al. 1998). Embryogenic callus could be obtained from the youngest anthers that were cut in half. Other surviving anthers produced green structures or friable callus. Current status of cryopreservation research 295

Cryopreservation of horticultural crops Cryopreservation using vitrification technique was applied to two horticultural crop species, i.e. garlic (Allium sativum) and Dianthus caryophyllus. Although the latter species is not a tropical species, in vitro propagation of this species has been established (Sudarmonowati et al. 1996) so it will be used as a model for ornamental plants. Two compositions of vitrification solution were tried, i.e. PVS2 (30% glycerol+ 15% ethylene glycol + 15% DMSO) and a combination of 40% glycerol and 40% sucrose. Shoot-tips which had been precultured for 1 d were subjected to loading solution for 20 min prior to soaking in vitrification solution for 20 min. No shoot-tips could survive after immersion in liquid nitrogen. Further research is needed to obtain surviving shoot-tips by modifying the period of exposure in loading solution and vitrification solution and the composition of both solution mixtures. The two vitrification solutions used for D. caryophyllus were also used for preserving embryogenic callus of garlic (Table 2). Different exposure periods in loading solution (10 and 20 min) and in vitrification solution (5, 10 and 20 min) were tried. The highest survival (20%) was obtained from embryogenic callus soaked in loading solution for 20 min followed by soaking in PVS2 for 20 min (unpubl.).

Table 2. Cryopreservation of horticultural crops and food crops Species Techniques Type of materials Survival rate (%) Horticultural crops Allium sativum Vitrification Embryogenic callus 90 Somatic embryos 80 Dianthus caryophyllus Vitrification Shoot-tips 0 Food crops Manihot esculenta Vitrification Shoot-tips 0 Glycine max Two-step freezing Zygotic embryos 88.3 Embryonic axes 20 Zea mays Two-step freezing Zygotic embryos 76.7 Embryonic axes 26.7

Cryopreservation of food crops Attempts to preserve soyabean and maize on a long-term basis were conducted by studying the effect of the type of tissues and methods of cryopreservation. Whole seeds, excised embryos or half-part seeds with cotyledons of soyabean and maize of Indonesian genotypes were subjected to either two-step freezing or rapid freezing (Table 2). The highest survival rates (46.7% and 58.3%) were obtained when whole seeds of maize and half-part of seeds without cotyledons of soyabean, respectively, were subjected to programmable freezing at a cooling rate of 0.5°C/min down to –30°C. Lower percentages were obtained (45.0% and 26.6%) when those tissues were directly frozen in liquid nitrogen (rapid freezing) without slow cooling. 296 Cryopreservation of Tropical Plant Germplasm

Research on cryopreservation of shoot-tips of Indonesian cassava genotypes is still in progress. Vitrification has been applied by using different vitrification solutions (PVS2 or a combination of 40% glycerol and 40% sucrose) and different exposure periods to the vitrification solution. As no surviving shoot- tips could be obtained, research is under way by modifying other factors.

Research on cryopreservation at other institutions Only one institute besides the R&D Centre for Biotechnology–LIPI, namely the Research Institute of Food Crop Biotechnology, Ministry of Agriculture, has started investigating the use of cryopreservation for preserving their in vitro materials. Other institutions have only been applying slow growth techniques or routine subculture to fresh medium after a certain period of culture. Of plants that have been maintained in vitro using growth inhibitors at the Research Institute of Food Crop Biotechnology, Ministry of Agriculture, only one species (Rauvolvia serpentina) has been used for cryopreservation studies. A standard vitrification procedure, including the use of PVS2 as vitrification solution, was applied to cryopreserve axillary buds of this medicinal plant species. As no surviving buds could be obtained, research to investigate the optimal concentration of vitrification solution is in progress.

Problems faced in cryopreservation work Research and development of plant germplasm cryopreservation in Indonesia have faced some problems. They are linked, among others, to the nature of the species, human resources and research funds. Most forest and fruit tree species used contain phenolic compounds at a very high concentration which are produced excessively after freezing. Overproduction of these compounds is toxic to the tissues and causes browning, which eventually kills the tissues. Other factors related to the nature of the species are the limited availability of fruits/seeds of some species, which is caused by poor fruiting seasons, and in vitro culture of many of those species is difficult. More manpower is required for obtaining good results with cryopreservation but the development of human resources for this field has been hampered by budget and policy restrictions in most institutions. As so far research has only been funded by a government budget which was very limited, progress of the work has been very slow.

Possible solutions Holding training courses both at degree (MSc or PhD) and non-degree levels might solve the problems encountered. Short national training courses, attended by Indonesian researchers and taught by experts from abroad and from Indonesia, could be arranged if funding is available. Efforts to build strong collaborations with institutions interested in cryopreservation in Indonesia are in progress. In addition, links with institutions abroad which have developed cryopreservation techniques would be useful. To conduct more intensive research, more manpower and research funds are required. Current status of cryopreservation research 297

Conclusions The results obtained indicate that the most appropriate cryopreservation technique depends upon the species and the types of tissues used. Using other tissues or organs such as anthers, embryogenic callus and somatic embryos could solve the problems encountered in cryopreservation of organs of tropical woody species caused by the high production of phenolic compounds. Finally, several actions need to be taken to increase the quantity and quality of cryopreservation research in Indonesia.

References Sudarmonowati, E., A.S. Bachtiar and E. Yunita. 1996. Regeneration and genetic transformation via Agrobacterium tumefaciens of Dianthus caryophyllus. Proceedings of National Seminar on Ornamental Crops., Jakarta March 20, 1996. (in Indonesian) Sudarmonowati, E. and Rosmithayani. 1997. Cryopreservation of Acacia mangium shoot tips. Proceedings of Indonesian Biotechnology Conference. Jakarta, June 17–19, 1997. Sudarmonowati, E. and Wuryan Rahayu. 1998. Maturation and cryopreservation of th somatic embryos of Pometia pinnata – a tropical forest tree species. Proceedings of the 4 Asia-Pacific Conference on Agricultural Biotechnology. (P.J. Larkin, ed.). Darwin, Australia, 13–16 July 1998. Sudarmonowati, E., Rosmithayani, and E.S. Mulyaningsih. 1998. In vitro conservation of fruit tree species at –196°C. Paper presented at Second Congress and National Seminar on Biotechnology. East Java, 20–21 September 1998. 298 Cryopreservation of Tropical Plant Germplasm

Current status and future perspectives of cryopreservation in Thailand Chalermpol Kirdmanee¹ and Kanyarat Supaibulwatana² ¹ National Center for Genetic Engineering and Biotechnology, National Science and Technology Development Agency, Rajdhevee, Bangkok, Thailand ² Department of Biotechnology, Science Faculty, Mahidol University, Rajdhevee, Bangkok, Thailand

Thailand is an agricultural country. Many plant crops have to be maintained by vegetative propagation, particularly the basic plants of cultivars that are used as parent materials. Genetic resources of these crops are traditionally maintained as plants in the field. A great deal of time and labour are required for the maintenance of these field collections, and moreover, losses of plant material may occur owing to accidents or diseases. Tissue culture techniques have been employed for plant germplasm conservation. However, plants have been maintained under normal or slow growth, thus requiring periodic subcultures. Some other disadvantages associated with these conservation techniques include high costs and risks of contamination and genetic or phenotypic modification during subculturing. Cryopreservation techniques offer the possibility to save labour, space and genetic losses. A number of institutes in Thailand have started to cryopreserve rare orchids, fruit trees (mango, jackfruit, bael fruit and passion fruit), vegetables (asparagus, bamboo and strawberry), agricultural crops (cassava and potato) and medicinal plants (Table 1). The National Center for Genetic Engineering and Biotechnology and Kasetsart University can be considered to have significant activities in cryopreservation. The National Center for Genetic Engineering and Biotechnology holds the largest collection of in vitro plants in Thailand and has developed the cryopreservation technology. Kasetsart University has played an important role in cryopreservation of agricultural and horticultural crops. There has been a growing number of studies on cropreservation in Thailand. Cryopreservation techniques have been developed for a wide range of materials (cells, calluses, shoots, meristems, embryos and seeds). Examples of its very successful use can be found in the cryopreservation of cassava shoots, orchid seeds and calluses of medicinal plants. In the future, the development of simple and reliable cryopreservation techniques would allow much more widespread use.

Acknowledgements We would like to thank Biotec Officer, Miss Virapon Mongkolchaisit, for providing information. Current status of cryopreservation research 299

Table 1. Species and institutes active in cryopreservation research in Thailand Species Institute Address Endangered The Royal Chitralada Chitralada Palace, Dusit, Bangkok, species Project Thailand Cassava Kasetsart University Central Laboratory, Phahon Yothin, (Bangkang Campus) Chatuchak, Bangkok 10900, Thailand Bamboo Kasetsart University Horticultural Department, Faculty of (Bangkang Campus) Agriculture Phahon Yothin, Chatuchak, Bangkok 10900, Thailand Asparagus, Kasetsart University Central Laboratory Camphaensang, Dendrobim (Kamphaengsang Nakornphatom, Thailand Campus) Mango Kasetsart University Agricultural Department, Faculty of Agriculture, Nakornphatom, Thailand Orchid Mahidol University Department of Biology, Faculty of Science, Rama VI Road, Rajdhevee, Bangkok, Thailand Orchid Silpakorn University Faculty of Science, Prarashwangsanamjan, Meung, Nakornpathom 7300, Thailand Medicinal National Center for 73/1 Rama VI Road, Rajdhevee, species Genetic Engineering and Bangkok 10400, Thailand Biotechnology Forest species Queen Sirikit Botanical Maerim, Changmai, Thailand Garden 300 Cryopreservation of Tropical Plant Germplasm

In vitro conservation and cryopreservation in the Philippines Alfinetta B. Zamora, Cynthia N. Paet, Olivia P. Damasco and Felipe dela Cruz Institute of Plant Breeding, College of Agriculture, University of the Philippines at Los Baños, Los Baños, Laguna, Philippines

Brief overview of conservation in the Philippines The Philippines lies in the regions of diversity for many tropical crops including Musa spp. (bananas, plantains and abaca), fruit trees and root crops. Field genebanks were established by the College of Agriculture in the University of the Philippines as early as the 1950s for Musa. For the asexually propagated crops, risks associated with field conservation, particularly for Musa, had significantly reduced holdings over the years. With the perceived advantages of having an in vitro genebank, the National Plant Genetic Resources Laboratory (NPGRL), the national repository of germplasm of important and potentially useful agricultural crops, under the auspices of the Institute of Plant Breeding of the College of Agriculture in the University of the Philippines at Los Baños [UPLBCA–IPB (NPGRL)], established in vitro collections of Musa textilis (abaca or Manila hemp) and root crops in 1986 through an externally funded project of the Department of Agriculture. These crops were selected for conservation because of genetic erosion. During this time also, the Institute of Plant Breeding established an in vitro collection of bananas and plantains through a project of the Tissue Culture Section supported by the International Development and Research Center (IDRC). Likewise, breeding lines and selected clones of potato developed for the humid tropics through an intercountry programme then funded by Asian Sweet Potato and Potato Research and Development Program (ASPRAD, 1984–1994) were established in vitro. The institute also engaged in an Allium improvement project funded by the Asian Vegetable Research and Development Centre (AVRDC) and some accessions of garlic and shallots were conserved in vitro. At present, the UPLBCA–IPB continues to conserve the collections of bananas, abaca, root crops and bulb crops. It has also engaged in conservation research on bamboo. Aside from the UPLBCA–IPB, other agencies in the national research system hold in vitro collections. These include the Northern Root Crop Center of the Philippines (La Trinidad, Benguet), the National Abaca Center [Visayas State College for Agriculture (VisCA), Leyte] and the Fibers Institute Development Authority (Bicol) for Manila hemp, the National Root Crops Center ViSCA, Leyte for root crops, and the Bureau of Plant Industry–Davao National Research and Development Center in Bago Oshiro, Davao for bananas and plantain. Figure 1 shows the locations of the institutions in the country which undertake conservation of asexually propagated crops in the field as well as in vitro. Current status of cryopreservation research 301 302 Cryopreservation of Tropical Plant Germplasm

Fig. 1. Locations of the institutions in the country undertaking conservation of asexually reproduced crops. Current status of cryopreservation research 303

Utilization of in vitro conserved germplasm materials The in vitro collections served their purpose, with entries made accessible to interested researchers, and even to farmers. The field curators in Davao and in UPLBCA–IPB have drawn from the in vitro conserved banana accessions to replace “lost accessions” in the field genebank. Losses in the field were often due to the disease pressure for banana bunchy top and other systemic infections as well as to inclement weather imposed by typhoons. Farmers, through NGOs, have access to different banana accessions through the in vitro collections. Non-governmental organizations working with indigenous peoples, i.e. the Aeta communities in Zambales, the Mangyan in Mindoro and the “lumads” in Davao, have received banana accessions from the UPLBCA– IPB collection. These people were asked what bananas were available to them in the past, if there were banana cultivars they were familiar with but could not get planting material for, and if they wanted to have those bananas again. Further, the in vitro collections have been used for exchange of germplasm in cultures between laboratories with similar crops, for instance UPLBCA–IPB with FIDA for abaca, and BPI–DNRDC with UPLBCA–IPB for banana in 1996. Transport to the receiving laboratories was facilitated with tissue- cultured materials, passing through quarantine authorities of the Bureau of Plant Industry.

Needs and problems with presently used in vitro conservation strategies The in vitro collections in laboratories of the different agencies are maintained as shoot cultures on a normal growth medium or in some crops on a minimal growth medium. For some laboratories and when applicable, storage organs like microtubers in potato and in vitro bulblets in bulb crops are also used as conservation propagules. However, problems have been encountered in some cases. For potato cultured on a mannitol-supplemented medium, these problems included death of basal sections of the stems; excessive lenticel formation, which made stems look cottony; glassy vitrified shoots and albinism (Fig. 2). In banana, frequent subcultures on a normal growth medium have led to formation of a mixture of typical shoot growths and nubbins. Albino shoots may arise from nubbins. The nubbins are slower in their development, taking longer than typical proliferating shoots to regenerate plants (Fig. 3). In the greenhouse, the plantlets that arose from nubbins were also slower in growth. Somaclonal variation is a great risk with banana and abaca collections maintained on a cytokinin-supplemented culture medium. For bananas, the collection has been maintained for more than 8 years, with the earliest introductions established in 1983. We shall replace the accessions in the collection with new introductions because nubbin formation has been observed in about 50% of the collection in vitro. After the germplasm conservation meeting in Costa Rica in 1996, we shifted to a minimal medium without growth regulators for conservation, using the cytokinin medium only for initiation of cultures. However, handling of shoot 304 Cryopreservation of Tropical Plant Germplasm cultures of abaca, banana and root crops on minimal culture media once every 4–6 months or once a year for potato microtubers could still present the following problems: recurring costs, contamination and decline possibly due to systemic disease or undetected infections. Current status of cryopreservation research 305

Fig. 2. Problems associated with slow-growth medium using mannitol for potato shoots cultured in vitro include (a) death of basal stems, (b) albinism, (c) glassy/vitrified shoots, and (d) excessive lenticel formation.

Cryopreservation Cryopreservation has been recognized to be a technique which could limit costs as well as problems associated with recurring handling. IPGRI conducted a training course in November 1995 in UPLB–IPB and ViSCA that included in vitro conservation and cryopreservation. For this training, two participants from the Philippines attended; one from DNRDC–BPI (Davao) who was working on bananas and another trainee from BSU, then working on root crops. Sad to say, the trained staff had proceeded with their advanced degrees without transferring the skills they had gained from the training (Magnaye, pers. comm.). Communications with other in vitro laboratories revealed that there was a lack of knowledge on the appropriate techniques for cryopreservation, including viability testing of cryopreserved materials; hence a lack of research and adoption of this technology. There is a need to draw people from different 306 Cryopreservation of Tropical Plant Germplasm

Fig. 3. In vitro shoot cultures of banana at higher subculture cycles showing (A) typical shoot growth 4 weeks after reculture, (B) mixture of nubbins and typical shoots, (C) nubbins, (D) albino shoots arising from nubbins 8 weeks after reculture, and (E) shoots arising from nubbins after 8 weeks. research institutes for possible training on cryopreservation techniques. As perceived by its main scientist for in vitro culture in the Philippine Coconut Authority (Rillo, pers. comm.), cryopreservation is one area of R&D which can be undertaken for coconut and the cryopreserved germplasm could serve as the duplicate for the existing field genebank. For IPB, cryopreservation has been identified as a growth point in its conservation activities. However, there is a need to build up technical skills as well as facilities. Thus, it is seeking funding for cryopreservation of the in vitro collections of vegetatively propagated crops. Current status of cryopreservation research 307

Fig. 4. Somatic embryogenesis system for banana using male bud tissues: (A) male bud as source of explant, (B) 10–15 cm bell for surface-sterilization with pure bleach, (C) hands or fingers (1–7 mm) as explants, (D) embryogenic callus initiated on MS + 2,4–D + BAP, (E) somatic embryos formed 3–6 months after initiation, (F) germination/plantlet regeneration on MS + BAP + IAA, MS + zeatin, and (G) plantlet development on MS basal medium.

Ongoing cryopreservation-related research At present, we have a funded project on bananas from the Department of Science and Technology (DOST) and the Philippine Council for Agricultural Research and Development (PCARRD). Among the project components is in vitro conservation and cryopreservation. The ongoing project is not the first attempt at cryopreservation research. In 1993, a study was initiated by our group using meristems and shoot-tips excised from shoots proliferated in vitro. Among treatments tested were pregrowth of explants to allow a greater number of aseptic shoot-tips for handling, and the use of DMSO as a 308 Cryopreservation of Tropical Plant Germplasm cryoprotectant. However, our trials were short-lived with the externally funded project coming to a close. At that time, success did not find us and our assessment now in the light of more successful protocols for banana is that the banana meristems were not sufficiently cryoprotected. For the ongoing project, time was taken to develop a somatic embryogenesis system for our locally important bananas using the shoot-tip and male bud tissues (Fig 4). These procedures improved on available methodologies with respect to (i) increased percentage of compact calli induced, (ii) increased shift to somatic embryogenic calli in five accessions of banana, (iii) ready multiplication of somatic embryos by secondary somatic embryogenesis, and (iv) demonstration of plantlet regeneration in 50% of somatic embryos. The availability of an efficient somatic embryogenesis system as well as proliferating shoot culture technology allows us to use two materials for cryopreservation experiments. Trials of encapsulation of somatic embryos have been undertaken and procedures on Musa available through INIBAP and other sources shall be compared for the local bananas.

Concerns with in vitro conservation and cryopreservation At the onset of in vitro conservation, many techniques were suggested including growth regulators and sugar alcohols as supplements to the culture medium. With time, the scientific community has seen somaclonal variation as a reality in tissue-cultured materials, even with organ culture. With conservation aimed at maintaining the clones in vegetatively propagated crops, somaclonal variation arising from in vitro culture is a major problem to contend with. Minimal growth conditions through mannitol-supplemented media, a low supply of nutrients, growth inhibitors, low temperatures and other growth- limiting strategies may be selective for offtypes or shoots adapted to such culture conditions. True, the stresses imposed by these treatments may be absent during the freezing stage of cryopreservation. However, would the processes before and after the freezing be sufficiently stressful to plant cells to induce heritable changes? With susceptibility to mutation being genotype- related, there is concern for the application of cryopreservation for large numbers of accessions. Cryopreservation is promising but remains untested for the large numbers of genotypes that would be conserved. Furthermore, cryopreserved materials are several steps removed from our ability to respond to requests for plant material. Theoretically, it would be ideal to store all materials in the collection by cryopreservation and have active cultures for dissemination to field curators and researchers. Recent austerity measures of the Philippine government have caused researchers to rethink conservation strategies. Should all conservation approaches be used in view of the resources? Which strategy – should there only be one strategy for in vitro conservation? However, resource limitations could be a major factor for deciding where to go with in vitro conservation. For us, cryopreservation is still at the research level; a new growth point in the Philippines and an area of possible research collaboration with others more familiar with the technologies. Current status of cryopreservation research 309

Current status and future perspectives of plant cryopreservation in Viet Nam Nguyen Tien Thinh Laboratory of Plant Tissue Culture and In vitro Mutation Breeding, Nuclear Research Institute, Dalat, Viet Nam

To date, there has been no activity on plant cryopreservation in Viet Nam. Therefore, in this report, activities on conservation of plant genetic resources in the country are presented instead. The future perspectives of plant cryopreservation in Viet Nam will then be discussed.

Viet Nam and its biodiversity

General geography of Viet Nam Viet Nam is located on the eastern seaboard of Southeast Asia, facing China in the north, and Laos and Cambodia in the west. It occupies 329 566 km² of land area and is about 1000 km long, ranging from 8°30’N in the south to 23°N in the north. The country is S-shaped with broad deltas of the Red river in the north and the Mekong in the south, linked by a narrow central section. Viet Nam has more than 3260 km of coastline and several islands characterized by a wide range of climatic conditions.

Evaluation of biodiversity Viet Nam has a great wealth of biological diversity. This richness is expressed both through the number of species and through the proportion of those that are endemic to Viet Nam. According to the recent assessment of the World Conservation Monitoring Center (WCMC), Viet Nam was rated as the 16th most biologically diverse country in the world (WCMC 1997). Table 1 provides a general idea of the number of species in Viet Nam compared with the rest of the world.

Table 1. Comparison between the number of species in Viet Nam and the world No. of species in No. of species in Taxa Viet Nam (SV) the world (SW) SV/SW (%) Mammals 265 4000 6.8 Birds 800 9040 8.8 Reptiles 180 6300 2.9 Amphibians 80 4184 2.0 Fish 2470 19000 13.0 Plants 7000 † 220000 3.2 Mean % of global 6.2 biodiversity † Estimated to be 12 000. (Source: WCMC 1997). 310 Cryopreservation of Tropical Plant Germplasm

Threats to plant genetic resources of the country The above richness of biodiversity is unfortunately under many threats. Among these, rapid deforestation has been and currently is the most important one. It was estimated that about 30 000 ha of forest could be completely lost every year owing to logging, while another 25 000 ha were destroyed by annual fires (WCMC 1997). An extensive study by the University of Tokyo (Japan) showed that during the period 1979–91, Viet Nam was among the few countries which had lost more than 20% of its natural forest. According to WCMC (1997) a frightening total of 13 million ha or 40% of the country is currently classed as bare land. In addition to the loss of forest, many species were lost or considered endangered owing to the overexploitation (Table 2). Species of rare timbers, medicinal plants and orchid have been and remain the common targets of overcollecting. In agriculture, the monoculture of modern high-yielding varieties has also led to the extinction of various traditional and valuable (high quality but low yield) genotypes.

Table 2. Summary of plant conservation status information held at WCMC (1997) IUCN threat category Number of species Extinct 1 Endangered 7 Vulnerable 25 Rare 316 Indeterminate 15 Insufficiently known 6 No information 373

Current methods used to conserve plant genetic resources in the country The valuable plant genetic resources of the country are currently conserved using the methods described below.

Preserved areas The country has set aside about 1.1 million ha or 3% of the land area from north to south to protect the natural habitats as well as historical sites. These are organized into 87 locations, of which about 30 are sites of primarily historic or scenic interest. The forests in these preserved areas, however, are frequently under threat from illegal logging, fire and overcollecting of rare species.

Field collections Several field collections for forestry, medicinal and agricultural species have been established in different research institutes. The size of the collection fields and number of conserved accessions are often very limited because of financial difficulty. At present, with the support of INIBAP (International Network for the Improvement of Banana and Plantain) the field collection of Musa (banana) germplasm has been established and enriched through several collecting missions (Danh et al. 1998). Current status of cryopreservation research 311

Seed collections Collecting of seeds is practised for rice (more than 4000 varieties), corn (more than 400 varieties), bean (more than 320 varieties), groundnut (more than 500 varieties) and cotton (more than 200 varieties) (WCMC 1997). Commonly, collected seeds are dried and stored in cold rooms, which require costly electricity. Periodic repetition (every 1–2 years) of sowing the collected seeds, growing the derived plants and reselecting seeds for storage is costly and laborious.

Tissue culture in vitro Tissue culture in vitro is the most common plant biotechnique in the whole country. From north to south, there are about 30–40 registered tissue culture laboratories of different research institutes and universities plus many others in the private sector. However, for economic reasons, the technique is used mainly for mass propagation of economically important species. Owing to financial difficulties, research and application of tissue culture systems for the conservation of germplasm are rare. Recently, with the support of INIBAP the in vitro collection of different genotypes of Musa was started (Danh et al. 1998).

Current status and future perspectives of plant cryopreservation in Viet Nam There has been no activity on plant cryopreservation in the country. The lack of researcher/workers trained in cryopreservation and of cryopreservation equipment has hindered the application of this biotechnique. A very modest investigation on the cryopreservation of meristems of some vegetatively propagated tropical monocots (Cymbidium, Cymbopogon and ginger) has recently been carried out by this author. This rare effort was, however, a self- supported work and with the author’s own interest. Regarding the rapid loss of the country-specific and valuable species and referring to the financial difficulties to preserve plant genetic resources by the above-described systems, plant cryopreservation with its compact size and lower demand of labour appears to be a more economical and safer conservation method. Unfortunately, funding for starting the cryopreservation work is and remains the first constraint to overcome.

Conclusions Nature has given to Viet Nam a richness of plant biodiversity with considerable global significance. This richness is under threat for different reasons, of which the rapid deforestation is an important one. In agriculture, the need for cash also places tremendous pressure on various local valuable germplasm through monoculture of imported high-yielding varieties. Conservation of the country’s plant genetic resources encounters financial difficulties. Therefore, cryopreservation appears to be promising as it is more economical and requires less labour. However, funds for equipment and training in plant cryopreservation are again the first problem to solve. It is hoped that through 312 Cryopreservation of Tropical Plant Germplasm international collaboration, this difficulty can be partly solved.

References The World Conservation Monitoring Centre. 1997. The Socialist Republic of Viet Nam. WCMC, Cambridge, U.K. Danh, L.D., H.H. Nhi and R.V. Valmayor. 1998. Banana collection, characterization and conservation in Vietnam. InfoMusa 7: 10–13. Current status of cryopreservation research 313

Current status of cryopreservation in China Tie–Gang Lu, Ling–Cheng Jian and Ching–San Sun Institute of Botany, Chinese Academy of Sciences, Beijing 100093, China

Introduction Plant germplasm is endangered because of increasingly intensive human developmental activities and natural disasters. The need for the development of methodologies allowing long-term germplasm conservation has been repeatedly emphasized and has attracted close attention worldwide. On the other hand, new, specific genetic resources are being produced and accumulated as a consequence of rapid development of biotechnology and recombinant DNA technology. Among those new germplasms, some are of economic importance, difficult to reproduce and do not exist in nature. It is necessary to also develop long-term conservation methods for the “man-made” germplasm, especially for those whose effects on nature are not yet known. Cryopreservation has been confirmed, at least in some species, to be an ideal method for long-term conservation of plant germplasm. It consists of bringing the cells to a metabolically inactive state and completely arresting the growth. Seeds and pollen retain their viability and in vitro cultures retain their morphogenic potential in a number of species after storage in liquid nitrogen. Here we will give an overview on the status of plant germplasm cryopreservation in China.

Methods used for cryopreservation There are three main methods for cryopreservation: fast freezing, slow freezing and vitrification. Fast freezing is simple but not applicable to most of species tested. Jian and Sun (1987) collected willow stems growing in winter (at about -10°C) and directly plunged them into liquid nitrogen. More than 30% survived and regenerated into plants after planting in soil. Wang (1988) reported that axillary buds of Morus bombycis collected in winter (about –15°C) could survive and regenerate into plants (80% regenerated) after storage in liquid nitrogen without any cryoprotective treatment. Slow freezing allows plant samples to undergo freezing gradually in a controlled manner, followed by rapid immersion into liquid nitrogen. The Withers and King protocol (Withers and King 1980) is the most successful variant and has been used successfully with callus, cell and protoplast cultures of a number of species. Sun and Jian (1990) have studied the protocol in detail using Onobrychis viciaefolia calli, and they found that holding samples at –35°C before plunging into liquid nitrogen is critical to obtain high survival rates. Ultrastructural observation revealed that the cell endomembrane system of cryopreserved cells was severely damaged when a fast-freezing protocol was employed. Similar results were obtained when using slow-freezing protocol with programmed cooling from 0°C to –35°C at 1.0°C/min followed by direct immersion in liquid nitrogen without holding at –35°C. No survival was observed after recovery culture of cryopreserved calli in both cases. If samples were held at –35°C for 30 min, the damage to the endomembrane system of 314 Cryopreservation of Tropical Plant Germplasm cryopreserved cells was less serious than previously and mostly reversible. If samples were held at –35°C for 2 h before plunging into liquid nitrogen, the ultrastructure of the endomembrane system of cryopreserved cells was very similar to that of control (cells which did not undergo storage in liquid nitrogen). More than 60% calli survived and regenerated plants after recovery culture in the later two cases. An alternative method for performing cryopreservation is by means of vitrification, i.e. the transformation from a liquid to solid glass state. The advantages of vitrification are: (i) it does not require a sophisticated apparatus, (ii it may avoid damage of ice formation, and (iii) it may be applicable to larger samples than those usually employed with the slow-freezing method (Towill 1995). Huang et al. (1995) and Wang et al. (1996) have successfully employed this method in cryopreservation of cell cultures of rice and immature inflorescences of barley. The survival rates ranged from 50 to 100% after recovery culture; the best result was obtained when the materials were precultured for 2 d on MS medium containing 0.5M sucrose before vitrification treatments. Similar results were also obtained with cryopreservation of a homozygous dominant male sterile line of wheat, which was produced by wheat × maize crosses and subsequent chromosome elimination (Sun et al. 1998).

Summary of results on cryopreservation Table 1 presents a preliminary summary of results on cryopreservation of plant germplasm obtained in China. Research currently focuses mainly on cryopreservation of callus and cell cultures with important characters, e.g. high regeneration capacity, and high production of secondary metabolic compounds, which is important to biotechnology research and the industry. For instance, maize, rice and wheat are very important cereal crops, which may cover more than 90% of the world grain production. Cell culture and subsequent plant regeneration from some model and cultivated varieties of these crops have been achieved in many laboratories, but sometimes cell culture and plant regeneration are still difficult to achieve for a given agronomically important variety. Cryopreservation and subsequent recovery culture could provide cells suitable for further studies in a short time. Arnebia euchroma produces one type of pigment, an important component for one kind of cosmetics, e.g. lipstick. Anisodus acutangulus produces one alkaloid, which could be used as an important medicine. Cryopreservation of these cell lines with specific characters will have great significance on the biotechnology industry (Zheng et al. 1983; Li et al. 1992). Current status of cryopreservation research 315

Table 1. Summary of results obtained on cryopreservation of plant germplasm in China Plant materials used for Freezing method used for Results (survival rate and cryopreservation (Reference) cryopreservation plant regeneration) Willow – stem with axillary buds (Jian Fast freezing without 30%, plant regeneration and Sun 1987) cryoprotectants Morus bombycis – axillary buds (Wang Fast freezing without 80%, plant regeneration 1988) cryoprotectants Kiwifruit – stems (Jian and Sun 1989) Slow freezing 80%, callus formation and plant regeneration Zea mays pollen (Shi 1987) Dehydration and fast freezing 74%, seed setting after pollination calli (Sun et al. 1989) Slow freezing 24%, plant regeneration cells (Zhang et al. 1990, 1992) Slow freezing NA, plant regeneration protoplasts (Lu and Sun 1989, Slow freezing 50%, plant regeneration 1992b; Zhang et al. 1990, 1992) Oryza sativa cells (Huang et al. 1995) Vitrification 40–50%, plant regeneration protoplast-derived cells (Tang Slow freezing 32%, plant regeneration 1988) Hordeum vulgare – immature Vitrification 50–100%, plant regeneration inflorescences (Wang et al. 1996) Triticum aestivum – haploid calli Vitrification 70%, plant regeneration derived from wheat x maize crosses (Sun et al. 1998) Trititrigia – cells (T.B. Wang, pers. Slow freezing 70%, plant regeneration comm.) Panicum miliaceum – calli (Sun et al. Slow freezing 93%, plant regeneration 1988) Setaria italica calli (Lu and Sun 1992a, 1995) Slow freezing 100%, plant regeneration cells (Lu and Sun 1992a, 1995) Slow freezing 100%, plant regeneration Onobrychis viciaefolia cells (Lu et al. 1992) Slow freezing NA, plant regeneration calli (Sun and Jian1990) Slow freezing 66%, plant regeneration Fragaria x ananassa – calli (Ke 1986) Slow freezing 65%, plant regeneration Saccharum sp. – calli (Jian et al. Slow freezing 95%, plant regeneration 1987a, 1987b) Brassica campestris var. pekinensis – Slow freezing 75.4%, plant regeneration protoplast-derived cells (Luo et al. 1990) Gladiolus gandavansis – calli (Li and Slow freezing 75%, plant regeneration Wang 1989) Arnebia euchroma – calli (Li et al. Slow freezing 60% 1992) Anisodus acutangulus calli (Zheng et al. 1983) Slow freezing 90% cells (Zheng et al. 1983) Slow freezing 90% NA = data not available. 316 Cryopreservation of Tropical Plant Germplasm

Conclusions and perspectives Cryopreservation has been considered to be an ideal alternative for plant germplasm conservation. It is especially applicable to plant cell cultures with valuable characters. A cell bank funded by the Chinese Academy of Sciences (CAS) is being set up in the Institute of Botany, CAS (director: Professor Weilun Chen). More than 100 cell lines with important characters have been collected from different research organizations and universities and nearly 20 cell lines have been successfully cryopreserved using the slow-freezing and vitrification methods. The purpose of this project is not only to conserve, but also to make efficient use of those valuable resources. These conserved cell lines will be accessible to every research institute and university in China and will greatly benefit biotechnology research and industry in the country. Another programme called “biodiversity conservation” organized by the Chinese Academy of Sciences is also planning to use cryopreservation to preserve the most endangered plant species, whose list has been published in the “White Paper of Engangered Plants in China”.

References Huang, C.N., J.H. Wang, Q.S. Yan, X.Q. Zhang and Q.F. Yan. 1995. Plant regeneration from rice (Oryza sativa L.) embryogenic suspension cells cryopreserved by vitrification. Plant Cell Reports 14:730–734. Jian, L.C. and L.H. Sun. 1987. Cryopreservation of willow stems by fast freezing. Plants 5:2– 3 [in Chinese]. Jian, L.C. and L.H. Sun. 1989. Cryopreservation of stems of kiwifruit. Acta Botanica Sinica 31:66–68 [in Chinese, English summary]. Jian, L.C., D.L. Sun and L.H. Sun. 1987a. Studies on some factors, which affect efficiency of callus cryopreservation of sugarcane (Saccharam sp.). Acta Botanica Sinica 29:123–131 [in Chinese, English summary]. Jian, L.C., D.L. Sun and L.H. Sun. 1987b. Cryopreservation of sugarcane (Saccharum sp.) calli. Plant Biology 5:323–334. Ke, S.Q. 1986. Plant regeneration from cryopreserved callus cultures of stawberry (Fragaria x ananassa). Acta Botanica Wuhanica 4:231–238. [in Chinese, English summary]. Li, G.F., H.C. Ye, J.W. Dong, L.C. Jian, L.H. Sun and L.M. Gu. 1992. Cryopreservation of calli of Arnebia euchroma. Acta Botanica Sinica 34:962–964. Li, Q.S. and H.Q. Wang. 1989. Cryopreservation of callus cultures of Gladiolus gandavansis. Plant Physiology Communications 2:48–50 [in Chinese]. Lu, T.G. and C.S. Sun. 1992a. Cryopreservation of foxtail millet (Setaria italica L.). Journal of Plant Physiology 139:295–299. Lu, T.G. and C.S. Sun. 1992b. Cryopreservation of protoplasts of maize (Zea mays L.). Pp.602–604 in Agriculture Biotechnology (C.B. You and Z.L. Chen eds.). China Science and Technology Press, Beijing [in Chinese]. Lu, T.G. and C.S. Sun. 1995. Cryopreservation of foxtail millet (Setaria italica L.). Pp.236–244 in Biotechnology in Agriculture and Forestry, Vol.32 (Y.P.S. Bajaj, ed.). Springer–Verlag, Berlin. Lu, T.G., C.S. Sun, L.H. Sun and L.C. Jian. 1992. Cell division, callus formation and plant regeneration from cell-suspension derived protoplasts of Onobrychis viciaefolia Scop. Journal of Plant Physiology 140:106–109. Current status of cryopreservation research 317

Lu, T.G. and C.S. Sun. 1989. Plant regeneration from cryopreserved protoplast of maize (Zea mays L.). Chinese Journal of Botany 1:165–167. Luo, M.Z., S.Y. Jiang and H.X. Tang. 1990. Cryopreservation of suspension cells derived from protoplast culture of Brassica campestris var. pekinensis. Acta Botanica Sinica 32:432– 437 [in Chinese, English summary]. Shi, S.X. 1987. Pollination and seed setting using cryopreserved pollen of maize (Zea mays L.). Zhi Leng Xue Bao 4:56–60 [in Chinese, English summary]. Sun, C.S., T.G. Lu and H.W. Xin. 1998. Obtaining homozygous Taigu male-sterile wheat by chromosome elimination. Acta Botanica Sinica 40: (in press). Sun, D.L., Y. Wu, L.H. Sun and L.C. Jian. 1988. Cryopreservation of callus cultures of Panicum miliaceam and subsequent plant regeneration. Chinese Bulletin of Botany 5:236– 237 [in Chinese]. Sun, L.H. and L.C. Jian. 1990. Cryopreservation of sainfoin (Onobrychis viciaefolia Scop.) tissue cultures and their ultrastructural observation. Acta Botanica Sinica 32:262–267 [in Chinese, English summary]. Sun, L.H., L.C. Jian and Z.Y. Cao. 1989. Studies on cryopreservation of maize (Zea mays) callus cultures. Chinese Bulletin of Botany 6:30–32 [in Chinese]. Tang, D.T. 1988. Cryopreservation of protoplast-derived cell clusters of rice (Oryza sativa japonica). Acta Botanica Sinica 30:357–362 [in Chinese, English summary]. Towill, L.E. 1995. Cryopreservation by vitrification. Pp. 99–111 in Genetic Preservation of Plant Cells in Vitro (B. Grout, ed.), Springer–Verlag, Berlin. Wang, J.H., Q.F. Yan and C.N. Huang. 1996. Plant regeneration from barley (Hordeum vulgare) immature inflorescences cryopreserved by vitrification. Acta Botanica Sinica 38:730–734 [in Chinese, English summary]. Wang, L.M. 1988. Cryopreservation of axillary buds of mulberry (Morus bombycis) without cryoprotectant treatment and high efficient plant regeneration. Acta Agriculture Boreali– Sinica 3:103–107 [in Chinese, English summary]. Withers, L.A. and P.J. King. 1980. A simple freezing-unit and routiune cryopreservation method for plant cell cultures. Cryo–Letters 1, 213–220. Zhang, S.B., L.C. Jian, C.S. Kuo, G.P. Qu and Y.Q. Qian. 1990. Bud and root differentiation of maize protoplasts after cryopreservation. Acta Biologiae Experimentalis Sinica 23:117–121 [in Chinese, English summary]. Zhang, S.B., L.C. Jian, Y.Q. Qian and Y.P.S. Bajaj. 1992. Cryopreservation of Germplasm of Maize (Zea mays L.). Pp. 619–628 in Biotechnology in Agriculture and Forestry, Vol. 25 (Y.P.S. Bajaj, ed.). Springer–Verlag, Berlin. Zheng, G.Z., J.B. He and S.L. Wang. 1983. Studies on tissue culture of medicinal plants. An Anisodus acutangulus variant with high and stable growth rate and scopolamine content. Acta Phytophysiologia Sinica 9:129-134. 318 Cryopreservation of Tropical Plant Germplasm

Current status of cryopreservation research and future perspectives of its application in South Africa Patricia Berjak School of Life and Environmental Sciences, University of Natal, Durban, 4041 South Africa

Introduction Depredation of natural plant resources, which has received prominent attention worldwide, is exacerbated in Africa because of intensive traditional utilization of plant products. Smaller quantities are currently being traded of the commodities provided by the most popular plant species, not indicating greater conservation awareness, but the increasing scarcity of the natural resources (Mander 1998). In the case of many species, it is not only the availability of the genepools that is threatened, but also the very continuation of their existence. This is a particularly ironical situation in South Africa, considering that this country has among the highest recorded species diversity (Cowling et al. 1989). However, depending on the plant part that is used, and/or the method of harvesting, many individuals of particular species disappear on a daily basis. For example, whole plants are sacrificed when the bulbs or other subterranean parts are utilized, which occurs with many herbaceous species. In the case of trees, bark is among the most frequently utilized components. While conservative harvesting practices involve removing only localized segments at any one time, currently untrained collectors (for whom it is imperative to make a living) will strip the bark extensively, resulting in rapid death of individual trees. While there are concerted efforts to spread awareness of the desirability of non-destructive harvesting and sustainable utilization (including replanting), the 'message' is slow to spread, and is disseminated mainly only through voluntary attendance of interested traditional practitioners at workshops. Among the heavily utilized species are those characterized by seeds that are short-lived because they are recalcitrant, as well as some in which mature seeds are not normally produced, principally because of predation of the developing fruits. In such species, the rate at which they approach extinction is accelerated, as there is little or no seedling recruitment. There is the urgent need to learn as much as possible about the seed biology of the many, heavily utilized species, among a flora for which there is little such information for any species. To this end, the characteristics of mature seeds are identified whenever we are able to obtain sizeable samples of those of a particular species. Seeds that emerge as showing non-orthodox behaviour (i.e. are shed hydrated and respond adversely to dehydration), are identified as candidates for trials aimed at germplasm cryopreservation. The germplasm of any species is most conveniently conserved in the form of intact seeds. However, in most cases intact non-orthodox seeds, which cannot be stored under low RH/low temperature conditions, also can not be cryopreserved. Notwithstanding the few exceptions to this generalization, in Current status of cryopreservation research 319 most cases cryopreservation of excised zygotic axes is the most desirable alternative. However, successful cryopreservation is not implicit, and a major focus in our laboratory is the identification of the problems that might reside in the methodology and/or might be intrinsic to the axes themselves. Once identified, if the problem with a particular species is methodological, then it is potentially able to be remedied. On the other hand, problems posed by the axes themselves might dictate that an alternative form of germplasm, e.g. buds or somatic embryos, be used. While achievement of successful cryopreservation and the subsequent production of vigorous plants is our practical objective for a broad range of species (which are not exclusively in the endangered indigenous category), we seek simultaneously to understand and explain the consequences of the various manipulations of the explants concerned. Thus our investigations include analyses at the microscopical, biochemical, molecular and biophysical levels, related to vigour and viability. Basic cryopreservation research is centred at two universities in South Africa: our laboratory at the University of Natal in Durban, and at the University of the Witwatersrand in Johannesburg, where D.J. Mycock is actively involved in both complementary and some collaborative work with us. Until very recently, however, there was little impetus at the national level in the sphere of cryopreservation of plant genetic resources. Recently, the National Department of Agriculture defined a policy including the conservation of plant genetic resources, but limiting the activities to species important to agriculture. However, this category is broader than is immediately apparent, as the definition reads: "… plant genetic resources for food and agriculture are defined as including all vascular plants cultivated, used or studied by humans or impacting on their activities in one way or the other" (Loubser 1998). Although this limits the species concerned, the definition is sufficiently broadly based to include those that are utilized for traditional practices, as it goes on to state: "… Plants are also included in this definition when they are grown or managed for pharmaceutical, medicinal or industrial production in an agricultural context and when they are of direct or indirect social or economic significance". However, there is a wealth of plant species that could not be accommodated in these terms, but it is hoped that other organizations (e.g. the National Botanical Institute, a parastatal under the aegis of the Department of Environmental Affairs and Tourism) might institute similar initiatives. In terms of the activities of the National Department of Agriculture, a new Plant Genetic Resources Centre (the building of which was completed in 1998) includes facilities for the storage of base and active collections, and plans for a cryostorage facility are presently being implemented, in accordance with the stated objective: "Conservation of species with recalcitrant seeds … tissue culture or cryopreservation". This comes as a welcome development, especially in the context of the active research on cryopreservation in which we and Mycock's group are involved. 320 Cryopreservation of Tropical Plant Germplasm

An overview of selected activities Warburgia salutaris (Bertol. f.) Chiov. (= W. ugandensis) [Cannelaceae]: Warburgia salutaris is unequivocally among the most endangered, if not the most endangered species in Africa. Not only are individual trees lethally damaged as a consequence of bark-stripping, but local sources of seeds of this species are excluded because of predation. Thus our experimental material is routinely obtained from Kenya. Despite the endangered status and scarcity of W. salutaris trees in the natural environment in South Africa, an average of some 17.2 tonnes of bark of this species was recorded by Mander (1998) as being sold annually in the Durban Medicinal Trade. Elsewhere in this volume, Kioko et al. (p. 375) have described that, despite the common experience that seeds of W. salutaris are rendered non-viable by relatively slow drying, their rapid dehydration can be achieved without lethal consequences in the immediate short term. While it is not expected that these seeds would survive for useful periods under ambient or refrigerated conditions, this aspect is due to be investigated in the forthcoming season. However, we have obtained a measure of success with the cryopreservation of the rapidly dehydrated seeds of W. salutaris, which, as detailed by Kioko et al., we have tentatively related to the metabolic status. Our findings have been that it seems impossible to use excised zygotic axes of this species as explants for cryopreservation, as these are killed within a few minutes of flash-drying and also are highly infected, but lethally damaged by all the common surface-sterilization procedures. In parallel work with this species, Mycock's group has developed a protocol to produce somatic embryos, which might afford an additional form of the germplasm of this species amenable to cryopreservation. Croton sylvaticus Muell. Arg. (Euphorbiaceae): The bark and leaves of C. sylvaticus are used medicinally for respiratory ailments (Pooley 1993), and, although this species is not endangered, our work is reported here as it is another one of the few for which rapidly dehydrated seeds are amenable to cryopreservation (Berjak et al. 1996). Like those of W. salutaris, seeds of C. sylvaticus are shed at high water content, but are relatively small and contain substantial reserves, which we suggest are major factors in their retention of viability upon rapid dehydration and subsequent cryopreservation. Extrapolating from this example and that of W. salutaris, it is possible that for other non-orthodox species, small seeds might be suitable candidates for cryopreservation if they can be rapidly dehydrated and contain a sufficient volume of intracellular ergastic material such that extensive collapse does not accompany their manipulation. Another factor that might be suggested to facilitate successful dehydration and rapid cooling of such seeds is that embryo cells containing substantial reserves (i.e. lipid, protein and starch) are generally less differentiated (i.e. have relatively little intracellular membrane development). In cases, therefore, where suspected non-orthodox seeds are relatively small, preliminary microscopical examination of axis and cotyledonary tissues might give a valuable indication of the probability of their surviving rapid dehydration (after no more than minimal physical manipulation, e.g. removal of the outer coverings). Preliminary microscopical studies are also recommended when excised Current status of cryopreservation research 321 zygotic axes are identified as the most likely explants to be used for cryopreservation. During the course of our studies on seeds of Trichilia dregeana Sond. (Meliaceae), virtually every possibility was explored to achieve successful axis cryopreservation, yet none was successful (Kioko et al. 1998). The best result obtainable after freezing was the formation of callus by some of the embryonic axes. We ultimately carried out light and transmission electron microscopical studies on these axes, which revealed that a large proportion of the cells were highly vacuolated and lacked much by way of stored reserves. A primary cause of the lack of success in obtaining axes that would germinate (rather than forming callus) was identified by microscopical analysis as collapse of the vacuolated root cap cells on dehydration down to the narrow window that would facilitate safe cryopreservation, and consequent damage to the root apex proper (Berjak et al. 1999). In this species, therefore, there is the urgent need to develop alternative forms of the germplasm, e.g. somatic embryos, or to use other explant material, e.g. apical or axillary buds, in an attempt to achieve successful cryopreservation. In other cases, however, we have had encouraging success with the cryopreservation of excised zygotic axes of exploited indigenous species. We are currently working with Ekebergia capensis Sparrm. (Meliaceae), the bark and roots of which are utilized in traditional medicine. While the seeds of E. capensis are relatively small and, after endocarp removal, dehydrate sufficiently rapidly to suggest the potential for whole-seed cryopreservation, they do not survive freezing. However, if the axes are excised with small segments of each cotyledon attached, they will survive ultra-rapid freezing (Wesley–Smith et al. 1999) following fast flash-drying. However, a persistent problem with these axes and generally those of other species is that following rapid thawing in either distilled water or liquid medium, no shoot development will occur. While our practical objective in all the cryopreservation studies is the development of successful protocols, we adopt a focused, analytical approach in order to ascertain the cause(s) of failure as well as the basis of success(es). In the case of the axes of E. capensis, following a breakthrough made with those of Quercus robur (Berjak et al. 1999), we currently have achieved success in 2+ 2+ germinating the axes and obtaining seedlings after thawing in a Ca /Mg solution that was calculated to optimize reconstitution of the cyto- and nucleoskeletons. We are also working on recalcitrant seeds that have large, hypertrophied, yet undeveloped axes, which can neither be contemplated as explants for cryopreservation, nor present defined apical meristems that could serve this purpose. In such cases, it is necessary to develop alternative explants for cryopreservation, and our studies thus far have been concentrated on the production of adventitious buds or embryogenic callus from segments of the hypertrophied axes. We have worked on two species: Barringtonia racemosa (L.) Roxb. (Lecythidaceae) and Garcinia livingstonei T. Anders (= G. angolensis) [Clusiaceae (= Guttiferae)]. While plant parts of both species are used medicinally, our primary interest in these is their relationship to economically important species from elsewhere in the world. Seeds of B. racemosa share properties with those of Brazil nut (also Lecythidaceae) and G. livingstonei is 322 Cryopreservation of Tropical Plant Germplasm related to the mangosteen, G. mangostana, of tropical Asia. For B. racemosa, we have so far been successful in producing embryogenic callus, while adventitious buds that developed into plantlets have been produced from seed segments of G. livingstonei. Studies on both these species, however, are far from complete, and presently callus induction from material other than the hypertrophied axes is also being investigated. Other current research foci include investigations on the fidelity of the genome following dehydration, cryopreservation and, in those cases where recourse must be made to other explants, during in vitro procedures that will yield somatic embryos. The inter-relationship between water content and cooling (freezing) rate, and the effects of the latter parameter itself, also receive considerable attention, as has been recently described by Wesley–Smith et al. (1999). This overview describes some of the activities aimed at cryopreservation of non-orthodox germplasm in which we are involved. Other species are presently under active investigation, and our quest to characterize seed behaviour from a wide spectrum of locally important species is ongoing.

References Berjak, P., C. Calistru, A. de Roquemaurel and A. Chalvin. 1996. Preliminary studies on the storage behaviour of seeds of Croton sylvaticus. Proceedings of the Microscopy Society of Southern Africa 26: 59. Berjak, P., J.I. Kioko, M. Norris, D.J. Mycock, J. Wesley–Smith and N.W. Pammenter. 1999. Cryopreservation – an elusive goal? in Recalcitrant Seeds. IUFRO, (in press). Cowling, R.M., G.E. Gibbs–Russell, M.T. Hoffman and C. Hilton–Taylor. 1989. Patterns of plant species diversity in southern Africa. Pp. 19–50 in Biotic diversity in Southern Africa. Concepts and conservation. B.J. Huntley (ed.) Oxford, Cape Town, South Africa. Kioko, J., P. Berjak, N.W. Pammenter, M.P. Watt and J. Wesley–Smith. 1998. Desiccation and cryopreservation of embryonic axes of Trichilia dregeana. Cryo–Letters 19: 5–14. Loubser, W. 1998. Proposal of a departmental policy for the conservation, management and sustainable use of plant genetic resources for food and agriculture. National Department of Agriculture, Pretoria, South Africa. Mander, M. 1998. Marketing of indigenous medicinal plants in South Africa. A case study in Kwazulu–Natal. FAO, Rome. Pooley, E. 1993. Trees of Natal, Zululand and Transkei. Natal Flora Publications Trust, Durban, South Africa. Wesley–Smith, J., C. Walters, P. Berjak and N.W. Pammenter. 1999. A method for the cryopreservation of embryonic axes at ultra-rapid cooling rates. in Recalcitrant Seeds. IUFRO, (in press). Current status of cryopreservation research 323

The application of cryopreservation techniques to national programmes: Nigeria's perspective P.M. Kyesmu Department of Botany and Microbiology, University of Jos, Jos, Nigeria

Introduction Nigeria is a large country situated between longitude 2°45'E to 14°30'E and latitude 4°15'N to 13°45'N. Its main vegetation can be easily divided into two groups: the tropical rain forest zone in the south and the savanna in the north. The forest belt produces most of Nigeria's tree crops (for example oil-palm, rubber, hardwood) as well as banana, plantain, pineapple and cacao. The heavy rains in the south also encourage the growth of root and tuber crops such as yams, cassava and cocoyam (taro) and a wide range of vegetables. Cereals such as millet, sorghum and maize are the staples of the northern economy with groundnuts, bambara nut and cotton also important. A variety of other crops are produced on a minor scale.

Agricultural activities Agricultural activities in Nigeria are shared between Federal and State governments. The Federal government sets basic policies and national priorities, while the states implement the programmes. The Federal government sets its policies through the Ministry of Agriculture. The Ministry is divided into nine departments headed by directors for effective programme implementation. The departments are land resources, agriculture, fisheries, forestry, cooperatives, personnel, finance, agricultural sciences, livestock and pest control services. Agricultural sciences department oversees activities of 18 national research institutes (NARI) in the country. Eleven of the NARIs are directly involved in plant research activities. These are Cocoa Research Institute of Nigeria (CRIN), Forest Research Institute of Nigeria (FRIN), Institute of Agricultural Research (IAR), Institute of Agricultural Research and Technology (IAR and T), Lake Chad Research Institute (LCRI), National Cereal Research Institute (NCRI), National Horticultural Research Institute (NIHORT), National Root Crops Research Institute (NRCRI), National Institute for Oil-palm Research (NIFOR), National Stored Product Research Institute (NSPRI) and Rubber Research Institute of Nigeria (RRIN). Five of these 11 institutes coordinate the activities of agricultural development programmes within the 36 states of Nigeria. They also have a clear mandate on crops produced within their zones. Whereas NRCRI, Umudike, handles all research activities on root and tuber crops and NCRI, Badeggi, on sugarcane, all the other institutes are concerned with seed-producing crop plants. The NARIs, as part of their mandate, develop strategies for the conservation of their mandate crops, which in most cases are traditional methods that provide only short- to medium-term storage. 324 Cryopreservation of Tropical Plant Germplasm

Cultivated genetic resources and their conservation in Nigeria The agro-ecological regions of Nigeria have sustained a rich germplasm of crop plants in the country. In addition to the varied environments, continuous selection by farmers and maintenance of promising types over a long period of time have resulted in a large number of agro-ecotypes which are adapted to the various agro-ecological regions and growing conditions. Wild and cultivated species of yams, for example, are found in various ecological niches in most parts of the country. The genetic resources unit (GRU) of IITA, Nigeria has collected some of these. Other crop plants grown in different agro-climatic regions are listed in Table 1. Roots and tuber crops occupy a significant position in agricultural production in general and in the country's food economy in particular. The volume of root and tuber crops produced during the year 1993 was 61.6% of crops. In terms of land use, the amount devoted to the cultivation of the crops is not significant. Only about 5.8 million hectares, representing about 15.8% of land devoted to food crops in Nigeria, was devoted to root and tuber crops (Federal Ministry of Agriculture 1993). This implies that root crops, especially yams and cassava, have high relative value per unit of land used when compared with other crops, especially cereals. The storage or conservation of the germplasm of these crops in the light of their significance to the country's economy cannot be overemphasized. Presently, all root and tuber crops are conserved by traditional methods. The methods depend on the volume and duration of storage desired and type of crops involved. Yams and cassava are stored in the field and later as 'tuber seeds' in racks. Traditional methods in grain storage are more varied. These include the use of small huts where they are occasionally smoked and storage in earthen pots, house ceilings, underground pits and granaries made of dried mud and plant materials. Grains are less susceptible to damage during storage than root crops and traditional storage techniques are therefore more efficient as only 4% of these crops are lost. In contrast, the damage done to vegetatively propagated crops under storage is higher. Root and tuber crops are, therefore, excellent targets for long-term conservation strategies since they can not be stored for much longer periods.

Cryopreservation: a technique for the long-term conservation of plant germplasm Research activities on crops in Nigerian research institutes as regards conservation rely heavily on the development of low-cost technologies; hence the technologies developed are based on traditional conservation methods. The method where crops are stored in the field or as 'tuber seed' exposes the crop plants not only to climatic uncertainties, rodents, pathogens and pests, but also to the management of large field collections, which often lead to identification errors. In vitro germplasm maintenance that is relatively efficient in terms of space and security has been investigated as a useful method of germplasm conservation for some crops (Jarret and Florkowski 1990; Towill 1991). With Current status of cryopreservation research 325 the exception of IITA in Nigeria which has developed in vitro normal and minimal 326 Cryopreservation of Tropical Plant Germplasm

Table 1. Cultivated resources of Nigeria, area and production Production Common name Scientific name Area ('000 ha) '000 t per ha Root crops Yams Dioscorea spp. 1986.68 21633.16 10.89 Cassava Manihot spp. 3029.23 30128.93 9.95 Cocoyams (Taro) Colocassia spp. 614.57 4605.58 7.49 Sweet potato Ipomoea batatas 80 311.12 3.91 Irish potato Solanum tuberosum 97 759 7.83 Cereals & pulses Maize Zea mays 5102.41 6290.78 1.23 Guinea corn Sorghum vulgare 6016.28 6050.70 1.01 Millet Pennisetum spp. 4950.67 4601.91 0.93 Rice Oryza spp. 1328.85 2413.29 1.82 Wheat Triticum aestivum 125.33 107.05 0.86 Cowpea Vigna spp. 430.34 1580.33 3.67 Pigeon pea Cajanus cajan 15.63 14.77 0.95 Oil seeds Groundnut Arachis hypogaea 1837.29 1415.93 0.77 Beniseed Polygala butyracea 137.51 72.531 0.53 Soyabeans Glycine max 194.57 318.6 1.64 Melon (Egusi) Colocynthis citrullus 320.83 312.85 0.98 Tree crops Cacao Theobroma cacao 842.56 106.12 0.13 Kolanut Cola spp. 114.2 153.1 1.34 Rubber Hevea brasiliensis 312 156 0.5 Oil-palm Elaeis guineensis 637 2834 4.45 Mango Mangifera indica 361.45 1158.97 3.21 Guava Psidium spp. 91.31 50.21 0.55 Cashew/nut Anacardium spp. 264.25 167.49 0.63 Citrus Citrus spp. 472.17 948.57 2.01 Coffee Coffea arabica 157 29 0.19 Industrial crops Cotton Gossypium spp. 323.49 215.38 0.67 Sugarcane Saccharum officinarum 218 303 1.39 Vegetables Tomatoes Lycopersicum spp. 1448.78 951.36 0.66 Pepper Piper spp. 454.62 526.62 1.16 Onions Allium cepa 122.45 467.91 3.82 Okro Hibiscus esculentus 523.69 285.64 0.55 Ginger Zingiber spp. 97 96 0.99 Leafy vegetables 1463.25 534.43 0.37 Others Pineapple Ananas comosus 26.31 221.32 8.41 Pawpaw Carica papaya 168.02 103.05 0.61 Banana Musa sapientum 391 1057.00 2.7 Plantain Musa paradisica 1943.57 2216.60 1.14 Source: Annual Agricultural Statistics 1993. growth strategies for yams (Ng and Ng 1991) and cassava (Ng and Ng 1997) followed by NRCRI, no research institute in the country carries out similar Current status of cryopreservation research 327 activities. NRCRI relies heavily on IITA in times of power failure. IITA has further initiated research activities on the development of cryopreservation protocols for cassava and yam shoot-tips. Reluctance to adopt cryopreservation technique on a broader scale by NARIs has largely been due to government policies and priorities, the cost of adopting such strategies and lack of specific protocols available as well as skilled personnel for in vitro maintenance of these and other crops. However, with emerging technologies, protocols for the cryopreservation of some vegetatively propagated tropical crops have been developed, e.g. banana (Thinh and Takagi, unpubl.), cassava (Escobar et al. 1997), taro (Takagi et al. 1997) and yams (Mandal et al. 1996; Kyesmu et al. 1997). Some of these protocols, however, require further evaluation and improvement before their adoption. With the current status of cryopreservation research activities on these and other crops, it is expected that the NARIs would (i) establish laboratories for advanced technologies in order to either initiate research activities, or (ii) adopt protocols currently developed for banana, cassava, taro and yams on a broader scale.

Conclusions The world contains about 300 000 species of higher plants, of which approximately two-thirds are found in the tropics. Humans use only about 3000 species (1%). From the 3000 only 200 species are under cultivation and about 30 of these species supply nearly all the food consumed by the entire human population (Chang 1985). About three-quarters of the food is being provided by only eight cereal species (wheat, rice, maize, barley, oats, sorghum, millet and rye). In Nigeria, with the exception of oats and rye, all the grain crops are cultivated on either large or small scale (Table 1). Grain crops are by far the most convenient plants for storage. Their seeds are less susceptible to damage during storage than are root and tuber crops. It is relatively much easier to conserve the germplasm of grain crops (once the water content of the seeds and suitable storage temperature are determined) than for vegetatively propagated crops. Vegetatively propagated crops such as sugarcane, root crops, banana and plantain, however, require other techniques for their conservation, hence the need to strongly develop or adopt already developed cryopreservation protocols for their conservation. The IITA has already initiated programmes on the development of protocols for the long-term conservation of germplasm of cassava and yams. Unless the national government and the international community reverse the trend in germplasm conservation of vegetatively propagated tropical crops over the next few decades, the genetic erosion of Nigeria's and indeed other developing nations' biological legacy will continue to accelerate. Conservation efforts on a global basis that can build on efforts at the national level should be matters of high priority. Progress in all these efforts, however, is hindered by government policies and priorities, lack of financing, infrastructure and dearth 328 Cryopreservation of Tropical Plant Germplasm of scientists trained for systematic and conservation studies in tropical countries including Nigeria. The insufficient number of adequately trained scientists makes the preparation of even simple inventories very difficult. In situations where trained scientists are available the absence or lack of infrastructural facilities and funding hamper any progress. In view of the above, there is an urgent need for the international community to assist developing countries to develop strategies for the long- term conservation of their wild and cultivated resources by strengthening institutional capacity. Unless developing countries build their own teams of competent researchers and develop solid institutions in which they can work, effective research on the conservation of plant germplasm will not be accomplished. In addition, the cardinal objective of the first Global Plan of Action for the conservation and sustainable utilization of plant genetic resources agreed upon by 150 countries at the International Technical Conference on plant genetic resources, in Leipzig, Germany (in June 1996) will, therefore, not be achieved.

Acknowledgements The author wishes to thank Professor N.E. Gomwalk, the Vice Chancellor of University of Jos, for the express permission and assistance given to enable him to attend the workshop.

References Chang, T.T. 1985. Principles of genetic conservation. Iowa State Journal of Research 59: 325– 348. Escobar, R., G. Mafla and W. Roca. 1997. A methodology for recovering cassava plants from shoot tips maintained in liquid nitrogen. Plant Cell Reports 16:474–478. Federal Ministry of Agriculture. 1993. Annual agricultural statistics. Department of Planning, Research and Statistics, Federal Ministry of Agriculture, Abuja, Nigeria. Jarret, R.L. and W. Florkowski. 1990. In vitro active vs field gene bank maintenance of sweet potato germplasm: major costs and other considerations. HortScience 25: 141–146 Kyesmu, P.M., H. Takagi and S. Yashima. 1997. Cryopreservation of shoot apices of white th yams (Dioscorea rotundata Poir.) by vitrification. Abstract of the 15 meeting of the Japanese Society of Plant Cell and Molecular Biology, 2D–13:162 Mandal, B.B., K.P.S. Chandel and S. Dwivedi. 1996. Cryopreservation of yam (Dioscorea spp.) shoot apices by encapsulation-dehydration. Cryo–Letters 17: 165–174 Ng, S.Y.C. and N.Q. Ng. 1991. Reduced-growth storage of germplasm. Pp. 11–339 in In vitro methods for conservation of plant genetic resources, (J.H.Dodds, ed.). Chapman and Hall, London. Ng, S.Y.C. and N.Q. Ng. 1997. Cassava in vitro germplasm management at the International Institute of Tropical Agriculture. African Journal of Root and Tuber Crops 2: 232–234 Takagi, H., N.T. Thinh, O.M. Islam, T. Senboku and A. Sakai. 1997. Cryopreservation of in vitro grown shoot tips of taro (Colocasia esculenta (L.) Schott) by vitrification: 1. Investigation of basic conditions of the vitrification procedure. Plant Cell Reports 16:594– 599. Towill, L.E. 1991. Cryopreservation. Pp. 41-70 in In vitro methods for conservation of plant genetic resources (J.H. Dodds, ed.). Chapman and Hall, London. Current status of cryopreservation research 329

Current status of cryopreservation research and future perspectives for its application in Costa Rica Ana Abdelnour–Esquivel Biotechnology Research Center, Costa Rica Institute of Technology, Cartago, Costa Rica

Introduction In Costa Rica, the development of cryopreservation research is recent. It started in 1990 with a project at CATIE (Center for Agronomic Research and Training) supported by IBPGR. The general objective was to develop an efficient method to cryopreserve the germplasm of bananas and plantains (Musa spp.). In 1992, under the support of USAID, CATIE and the University of Florida worked on cryopreservation of coffee (Coffea spp.). Graduate student projects on cryopreservation of cacao (Teobroma cacao) and the bacteria Pasteuria penetrans (a nematode biocontroller) also were conducted. In 1995, the Costa Rica Institute of Technology initiated work on cryopreservation, with a project on rescue, multiplication and cryopreservation of endangered species of orchids under the support of the Italian goverment. In 1998, a project on cryopreservation and molecular characterization of chayote (Sechium edule) with collaboration from IPGRI was initiated, and it is currently being developed. At present, no collection is maintained under cryopreservation; but at least three institutions (Costa Rica Institute of Technology, CATIE and INBIO, the National Institute of Biodiversity) are making efforts to establish long-term banks for native species of economic and ecological importance.

Methods and results In Costa Rica, several methods have been tested for the cryopreservation of tropical species including Musa spp., Coffea spp., Theobroma cacao, Bactris gasipaes, Lycaste bradeorum, Sechium edule and the bacterium Pasteuria penetrans. Methods utilized and the results obtained are briefly described.

Musa spp. For the production of fertile diploid bananas and plantains, Musa acuminata (AA) and Musa balbisiana (BB), the method of embryo isolation, air-desiccation and rapid freezing in liquid nitrogen (LN) was evaluated (Normah et al. 1986; Chin et al. 1989). This study reported successful cryopreservation of zygotic embryos. Mature embryos were highly resistant to desiccation and the survival rate of freezing in LN was fairly high (83 and 94% for the two cultivars, respectively) at water content around 15%; but even at lower water content (9%) survival was achieved. Regenerated plants were later planted in the field and compared with plants regenerated from non-frozen embryos; no morphological differences were observed (Abdelnour–Esquivel et al. 1992a). Since somatic embryos are highly useful for micropropagation, genetic transformation and germplasm conservation, cryopreservation was also tested 330 Cryopreservation of Tropical Plant Germplasm with somatic embryos and small aggregates of three to four embryos of banana, Musa AAA cv. Grande Naine, induced from male flowers according to the methodology described by Escalant et al. (1994). The experimental material was cultivated for 1–3 d on a medium with increasing concentrations of sucrose (0.3 to 1M) and 1 hour before freezing, the embryos were incubated in liquid medium with 5% DMSO. Freezing was carried out slowly, at 1ºC/min to –40ºC, then embryos were rapidly immersed in LN. Survival was obtained by direct germination of frozen embryos (40%) and by callus production (35%). However, embryos surviving as callus rapidly produced somatic embryos. Embryos germinated normally and were transferred to the greenhouse for aclimation (Abdelnour–Esquivel and Escalant 1994).

Coffea spp. Various cryopreservation protocols were tested with zygotic and somatic embryos, and apical meristems of coffee. For apical shoots of C. arabica cv. Catimor, the vitrification protocol described by Towill and Jarret (1992) was tested. Incubation of shoots in a medium with 0.4M sucrose and 20% PVS2 for 20 min prior to rapid freezing in LN was the only tested treatment that allowed 28% survival of the shoots; recovery was through callusing only (unpubl. data). Regarding zygotic embryos, we performed experiments with three genotypes, Coffea arabica cv. Caturra, C. canephora cv. robusta, and the hybrid Arabusta (C. arabica x C. canephora), using two embryo maturity stages (green fruits, 2 months before harvest, and yellow fruits, 4 d before harvest). The process of air desiccation and rapid freezing in LN was used since it is characterized by its simplicity and efficiency in several species. Higher survival rates were obtained with more mature embryos, i.e. those extracted from yellow fruits (96% survival for C. arabica at 16% water content), compared with those extracted from green fruits (50% survival at 21% water content).

However, the addition of GA3, which is known to promote the growth of immature embryos, in the recovery medium resulted in an increase in the survival rate of immature embryos of C. arabica to a level comparable to that of more mature ones (83% survival) (Abdelnour–Esquivel et al. 1992b). For somatic embryos, two genotypes were used: C. arabica cv. Catimor and C. canephora cv. robusta. All embryogenic cultures were initiated by placing 1.5-cm² leaf sections on induction medium (Yasuda et al. 1985). The protocol reported by Bertrand–Desbrunais et al. (1988) which utilizes a pretreatment with increasing concentrations of sucrose up to 0.75M, followed by incubation with 5% DMSO and slow freezing at 0.5ºC/min to –40ºC before storing in LN, was utilized. The average rate of recovery for robusta was 61%; however, recovery rate for Catimor was very low, reaching 9% at the best. Lack of success with C. arabica may be due to inadequacy of both somatic embryogenesis and recovery media. During the process of somatic embryogenesis, many abnormal embryos were produced from this line and the germination percentage was very low (Abdelnour–Esquivel et al. 1993). Current status of cryopreservation research 331

Pasteuria penetrans Since cryopreservation is a useful technique to conserve not only plant material but also biological control agents, in this line of research we experimented with the bacterium Pasteuria penetrans, a parasite of nematodes like Meloidogine incognita and M. arabicida, two serious pests of coffee. The procedure consisted of incubation of bacteria homogenates in DMSO (0–30%) during 0–60 min. Rapid freezing in LN was used in all cases. All treatments allowed bacteria survival. The highest survival percentage (87%) was obtained when DMSO at 20% for 20 min was used as cryoprotectant; however, comparable results were obtained when 5% DMSO for 30 min was utilized (82%). Survival was evaluated based on percentage of bacteria attached to nematodes (Rojas and Abdelnour, unpubl.).

Theobroma cacao Recovery of zygotic and somatic embryos was possible for cacao. When immature zygotic embryos were incubated on increasing concentrations of sucrose up to 0.6M and frozen rapidly in LN, 70% of the embryos survived as callus. When immature embryos were cultivated 3 d on MS media, plus 1 g/L casein hydrolyzate and 10% coconut water and incubated for 12 h in medium with 1M sucrose before freezing (0.5ºC/min to –40°C followed by rapid immersion in LN), 14% of embryos were recovered through secondary embryogenesis. On the other hand, when somatic embryos were incubated for 24 h on medium with 0.75M sucrose, and frozen either slowly (0.5ºC/min to -40°C + LN) or rapidly (directly into LN), survival reached 20% (Cisne 1992).

Bactris (Guilielma) gasipaes Pejibaye (Bactris gasipaes) is a palm native of the Amazonia and the lower parts of Central America. It has a high agro-industrial potential for wet areas of the tropics. Commercialization of its fruits and palm hearts is increasing rapidly; therefore germplasm collecting, evaluation and conservation are getting more attention. We conducted some preliminary tests on cryopreservation of zygotic embryos and even though some survival was obtained, lack of financial support did not allow the continuation of the research.

Lycaste bradeorum The orchid family is the largest and most diverse of the angiosperms. According to conservation studies, Costa Rica, with an area of 52 000 km², possesses 1416 species grouped in 179 genera varying from the climbing, large vanilla to the tiny moss orchids; however, many of these native orchids are endangered due to deforestation, urbanization and overcollecting for commercialization (Mora–Retana and Garcia 1992). In order to collaborate with the rescue, multiplication and conservation of these materials, a research project using tissue culture techniques was developed during a 2-year period. Cryopreservation of shoots using vitrification solutions (PVS2) was tested on Lycaste bradeorum, one of the most precious orchids of the country. Incubation with PVS2 for 30, 45 and 60 min reduced survival to 67, 67 and 20%, respectively; however it was possible to obtain organized regrowth after 332 Cryopreservation of Tropical Plant Germplasm freezing in LN (14, 33 and 3%, respectively). Much effort should be dedicated to orchids, and these initial results indicate the potential of cryopreservation for their long-term conservation (Abdelnour and Muñoz, unpubl.).

Sechium edule Chayote, Sechium edule (Cucurbitaceae), is an important staple food in Mexico, Central America, the West Indies and Tropical South America. Every part of the plant is useful. However, its main use is as a table vegetable of which fruits, tuberous roots, tender leaves and shoots are all edible. This plant also has medicinal properties. Commercial production and export of chayote fruits have been increasing in recent decades, and for Costa Rica, the leading exporter, it represents an important source of revenue. During the last 30 years, the severe selection of chayote phenotypes for commercialization and export have rapidly increased the genetic erosion of the species and conservation programmes are urgently needed. Since the species cannot be stored by seeds (it is viviparous) and seed is damaged upon extraction and killed by drying (testa slightly lignified or totally unlignified) (Lira 1996), methods of conservation recommended for recalcitrant species must be used. A project on micropropagation, cryopreservation and characterization of chayote germplasm was initiated this year with the support of IPGRI. The development of a methodology of micropropagation from shoots, production of virus-free material and some experiments on cryopreservation of shoots by vitrification are being carried out (Abdelnour and Engelmann, unpubl.).

Conclusions Small projects and reduced financial support without continuity have characterized cryopreservation research in Costa Rica. Among the main limiting factors contributing to the slow progress in this field and its non- application in Costa Rica and Central America, are the lack of a significant number of trained personnel and financial support from the goverment and international organizations responsible for conservation of genetic resourses. In addition, the establishment of micropropagation protocols for native tropical species prior to cryopreservation is a must for many species, and therefore future research projects should consider this. To alleviate the first problem, a series of courses has been given in Costa Rica to professionals from Mexico, Central and South America. CATIE and the Costa Rica Institute of Technology have been reponsible for the courses; technical and financial support were received from IPGRI, RELAB (Latin American Network of Botany) and the WWF (World Wildlife Fund for Central America). In addition, courses on plant genetic resources and conservation methods have been included in the curricula of a new biotechnology programme offered by the Costa Rica Institute of Technology. Regarding the use of cryopreservation to maintain long-term collections in Costa Rica, research directed toward development of efficient protocols for native species (for food and precious woods) of ecological and economical interest for Central America, mainly those that are not under the scope of International Centres, is highly recommended. International, regional and Current status of cryopreservation research 333 national institutions, organized groups of producers and related businesses should be motivated to collaborate on this task.

References Abdelnour–Esquivel, A., F. Engelmann, C. Hibjan, V. Villalobos, D. Gray and M. Dufour. 1993. Zygotic and somatic embryo cryopreservation in coffee (Coffea arabica, C. canephora th and arabusta). In 15 International Scientific Colloquium on Coffee, Montpellier, France, 6–11 June 1993. Abdelnour–Esquivel, A. and J.V. Escalant. 1994. Crioconservacion de embriones somaticos de Musa Gran Enano (AAA). In Abstracts XI Meeting ACORBAT, San Jose, Costa Rica, 13–18 February 1994. Abdelnour–Esquivel, A., A. Mora and V. Villalobos. 1992a. Cryopreservation of zygotic embryos of Musa acuminata (AA) and Musa balbisiana (BB). Cryo–Letters 13:159–164. Abdelnour–Esquivel, A., V. Villalobos and F. Engelmann. 1992b. Cryopreservation of zygotic embryos of Coffea spp. Cryo–Letters 13: 297–302. Bertrand–Desbrunais, A., J. Fabre, F. Engelmann, J. Dereuddre and A. Charrier, 1988. Reprise de l'embryogenèse adventive à partir d'embryons somatiques de caféier (Coffea arabica L.) après leur congélation dans l'azote liquide. Comptes Rendus de l’Académie des Sciences Paris 307, Sér. III: 795–801. Chin, H.F., B. Krishnapillay and Y.L. Hor. 1989. A note on the cryopreservation of embryos of young coconuts (Cocos nucifera var. MAWA). Pertanika 12: 183–186. Cisne, J.D. 1992. Crioconservación de embriones cigóticos y somáticos de cacao (Theobroma cacao). Thesis Magister Scientiae, CATIE, Turrialba, Costa Rica. Escalant, J.V., C. Teisson and F. Cote. 1994. Amplified somatic embryogenesis from male flowers of triploid banana and plantain cultivars (Musa sp.). In Vitro Cellular and Developmental Biology 30P: 181-186. Lira, R. 1996. Chayote. Sechium edule (Jacq.) SW. Promoting the conservation and use of underutilized and neglected crops. 8. Institute of Plant Genetics an Crop Plant Research, Gatersleben/International Plant Genetic Resources Institute, Rome, Italy. Mora–Retana, D.E. and J. Garcia. 1992. Lista actualizada de las orquídeas de Costa Rica. Bremesia 37: 70–124. Normah, M.N., H.F. Chin and Y.L. Hor. 1986. Desiccation and cryopreservation of embryogenic axes of Hevea brasiliensis Muell. Arg. Pertanika 9: 299–303. Towill, L.E. and R.L. Jarret. 1992. Cryopreservation of sweet potato (Ipomoea batatas (L) Lam.) shoot tips by vitrification. Plant Cell Reports 11:175–178. Yasuda, T., Y. Fujii and T. Yamaguchi. 1985. Embryogenic callus induction from Coffea arabica leaf explants by benzyladenine. Plant Cell Physiology 26:595-597. 334 Cryopreservation of Tropical Plant Germplasm

Current status of cryopreservation research and future perspectives of its application in the South Pacific Mary Taylor¹ and Takako Murikami² ¹ TaroGen, SPC, Suva, Fiji ² Takako Murikami, 140-1, Shimoota, Shimokawara, Morioka-shi 020, Japan

Present conservation strategies Genetic diversity is as important to agricultural productivity in the Pacific Island Countries (PICs) as it is anywhere in the world. Ongoing crop-based projects in the region are currently looking at selecting and/or breeding cultivars that are resistant to serious diseases; a broad genetic base is obviously of significance to the success of these projects. However, throughout the PICs, genetic erosion is occurring at an alarming rate. Plant genetic resources are generally maintained in field genebanks. Sadly, these genebanks are proving to be a very inefficient and unreliable means of conserving germplasm and many collections have been completely lost. Information is not readily available on many of the crop species but Table 1 illustrates the situation with the two major food crops (taro and yam) between 1985 and 1994. With some of these collections the situation has worsened since 1994; for example, in Papua New Guinea all the taro accessions have been lost. Losses in genetic resources from field collections have not only occurred with taro and yam. There is also concern about sweet potato, cassava and banana collections. The reasons stated for these losses are cost of maintenance of plants that require frequent harvesting and replanting, inadequate storage facilities for yams, extreme climatic conditions (cyclones and drought), lethal diseases and poor management, e.g. mislabelling of accessions.

Table 1. Collections of taro and yams in Pacific Island Countries Accessions 1985 1994 Country Taro Yam Taro Yam Cook Island 57 8 0 3 Fiji 72 106 78 93 Niue 52 24 0 0 PNG (Laloki) 135 254 0 250 PNG (Bubia) 120 0 400 0 Solomon Islands 31 414 2 138 Tonga 0 0 21 0 Vanuatu 138 345 0 0 Samoa (MAFFM) 20 0 17 0 Samoa (USP) 28 23 0 7 Source: Jackson 1994. Current status of cryopreservation research 335

In addition there is evidence that general genetic erosion is occurring with crops such as taro and yam. Before diseases like taro leaf blight (Phytophthora colocasiae), taro and yams were the main crops grown in the Solomon Islands; in recent years sweet potato cultivation has received priority. It has been suggested by Kesevan and Aburu (1982) that taro production in Papua New Guinea has declined as a result of population pressure, plant diseases and the introduction of higher-yielding crops such as Xanthosoma, sweet potato and cassava. In Samoa, the introduction of taro leaf blight has led to the demise of taro cultivation, with Xanthosoma and Alocasia species replacing taro. In Micronesia, declining taro production has been attributed to pests and diseases, and ranks behind breadfruit, yam, bananas and imported rice as a staple food (Raynor and Silbenus 1992). Most of these crops require a high labour input for the efficient maintenance of field genebanks. This is not often available in a region where resources as a whole are generally limited. It is fairly common to find that a staff member responsible for a field collection also has numerous other duties. In addition, wages are low and so staff turnover is high and continuity a problem. Land availability too can be limited with much of the land being custom land; ownership disputes have occurred over land on which field collections are being maintained. These problems, combined with the more documented ones such as management, pest and disease outbreaks, render field genebanks a vulnerable system for conservation in this region.

In vitro conservation strategies There is obviously an urgent need to address this issue and to establish more secure systems of conservation for these crops. Using in vitro methodology for conservation can offer some distinct advantages over alternative strategies, especially within PICs. These include: (i) material can be maintained in a pathogen-tested state, (ii) pathogen-tested material allows for safer distribution across borders compared with vegetative material, (iii) cultures are not subject to environmental disturbances such as cyclones, pest and disease outbreaks, etc., and (iv) this methodology is less labour-intensive. Within this region, both regional and national tissue culture laboratories have been established mainly for the distribution of pathogen-tested material and for the provision of planting material to growers through rapid multiplication. Within the regional laboratories, collections of taro, sweet potato, cassava, yam and banana are maintained under standard in vitro conditions. Even with these relatively small collections the maintenance of this material is demanding of labour, especially if contamination becomes a problem. It is likely in the near future that these collections will expand, especially with some of the major root and tuber crops such as taro and yam. It is not feasible to maintain relatively large collections under standard in vitro conditions. Low-temperature storage could be a possibility. Work by Bessembinder et al. (1993) showed that under conditions of 9°C and total darkness, Colocasia esculenta could be stored for more than 8 years with transfer intervals of approximately 3 years. However, within the Pacific Island region the cost of electric power is high, thus making low- temperature storage not a realistic option for countries where funding is often 336 Cryopreservation of Tropical Plant Germplasm limited. Cryopreservation is being recognized as an effective tool for long-term storage of vegetatively propagated plant material, offering a technology which requires minimum space, minimum maintenance and genetic stability. Classical cryopreservation techniques involve slow cooling down to a defined prefreezing temperature, followed by rapid immersion in liquid nitrogen, and as such are dependent on a programmable freezer. The same techniques also proved to be more successful with culture systems consisting of small units of relatively uniform morphology, and have generally not been suitable for the cryopreservation of larger units comprising a mixture of cell sizes and types such as apices and embryos. The newer cryopreservation techniques are less complex than these methods, and do not require a programmable freezer. These techniques are far more suited for use in basic tissue culture laboratories where relatively complicated equipment should be avoided. With the advent of these ‘new’ cryogenic procedures such as vitrification (Langis et al. 1990; Sakai et al. 1990; Yamada et al. 1991), encapsulation-vitrification (Matsumoto et al. 1995) and encapsulation-dehydration (Dereuddre et al. 1990; Fabre and Dereuddre 1990), the number of species that can be cryopreserved has increased significantly in recent years. Cryopreservation of tropical or subtropical species has been less extensively investigated than that of cold-hardy species. However, as previously stated there has been an increase in the number of species successfully cryopreserved using the non-classical techniques. Several species of Dioscorea were successfully cryopreserved through encapsulation-dehydration of the shoot apices (Mandal et al. 1996). Using vitrification protocols, cryopreservation of sweet potato (Ipomoea batatas) shoot-tips was achieved, although shoot regeneration from the cryopreserved material varied widely among replications (Towill and Jarret 1992). Some survival was also reported with banana after proliferated meristem clumps were cryopreserved using the encapsulation- dehydration methodology (Panis 1995). A high rate of recovery (approximately 80%) of taro (Colocasia esculenta) shoot-tips after cryopreservation using the vitrification protocol was reported (Takagi et al. 1997), indicating that this technique could be very promising and could have significant implications for the long-term conservation of taro germplasm. From the literature it would appear therefore that the non-classical, low-technology cryopreservation techniques have potential for many of the important food crops in the PICs. Some funding has been provided by the International Plant Genetic Resources Institute (IPGRI) and the United Nations Educational, Scientific and Cultural Organization (UNESCO) for investigating the potential of cryopreservation for conservation of taro germplasm. Some preliminary experiments were carried out in the regional laboratory at the University of the South Pacific (USP), Samoa; further work will be continued at the regional laboratory attached to the Secretariat of the Pacific Community (SPC), Fiji. The preliminary experiments carried out at USP studied the encapsulation- dehydration technique using shoot-tips of taro (Colocasia esculenta var. esculenta) excised from pathogen-tested in vitro material from the genebank. In all experiments the basal medium (BM) was Murashige and Skoog (1962) Current status of cryopreservation research 337 supplemented with inositol (100 mg/L), thiamine (0.04 mg/L) and sucrose (3%). All experiments were cultured at a temperature of 24°C under a 12-h -2 -1 light/12-h dark photoperiod and at a light intensity of 40 µmol m s . Only three criteria were investigated: regeneration times from encapsulated and non-encapsulated shoot-tips, optimum preculture treatment, and optimum dehydration time. There was no significant effect on the regeneration of the shoot-tips after encapsulation in the alginate bead; however, it was noted that if the explant was positioned in the centre of the bead, regeneration was problematic. The optimum preculture treatment was 24 h on BM containing 0.25M sucrose; other treatments investigated were combinations of 24-h and 48-h precultures with media containing 0.25M, 0.5M and 0.75M sucrose. Finally there was no difference in the percentage of explants regenerating after dehydration for either 4 h or 6 h.

Future applications of cryopreservation Further work will continue in this area with the focus, initially, on taro germplasm. Research into cryopreservation of taro germplasm is a major component of an AusAID-funded project, Taro Genetic Resources: Conservation and Utilization. Taro throughout the region is being collected and characterized using both morphological characters and genetic markers, and it is anticipated that eventually accessions will be identified for maintenance in the Regional Germplasm Centre at SPC, Fiji. As already stated it will not be possible to maintain a relatively large collection under standard in vitro conditions, and so it is hoped that cryopreservation will provide a sustainable strategy for long-term conservation. The Regional Germplasm Centre at SPC will be responsible for the region’s germplasm. As the plant material being conserved will be genebank material, the clonal integrity of this germplasm is a major consideration. Because of this and the need to reduce reliance on technology, the less classical cryopreservation techniques will be investigated: vitrification, encapsulation-dehydration and droplet freezing.

References Bessembinder, J.J.E., G. Staritsky and E.A. Zandvoort. 1993. Long-term in vitro storage of Colocasia esculenta under minimal growth conditions. Plant Cell, Tissue and Organ Culture 33, 121–127. Dereuddre, J., C. Scottez, Y. Arnaud and M. Duron. 1990. Resistance of alginate-coated axillary shoot tips of pear tree (Pyrus communis L. cv. Beurré Hardy) in vitro plantlets to dehydration and subsequent freezing in liquid nitrogen: effects of previous cold hardening. Comptes Rendus de l’Académie des Sciences Paris, 310 Ser 111:317–323. Fabre, J. and J. Dereuddre. 1990. Encapsulation-dehydration: a new approach to cryopreservation of Solanum shoot-tips. Cryo–Letters 11:413–426. Jackson, G.V.H. 1994. Taro and yam genetic resources in the Pacific and Asia. Report prepared for ACIAR and IPGRI. Anutech Pty Ltd. Kesevan, V. and K. Aburu. 1982. Conservation of plant genetic resources. Pp. 379–384 in Traditional Conservation in Papua New Guinea: Implications for Today. (L. Morautu, J. Pernatta and W. Heaney, eds.) PNG. Langis, R., B. Schneibel Preikstas, E.D. Earle and P.L. Steponkus. 1990. Cryopreservation of carnation shoot tips by vitrification. Cryobiology 27:657-658. Mandal, B.B., K.P.S. Chandel and S. Dwivedi. 1996. Cryopreservation of yam (Dioscorea 338 Cryopreservation of Tropical Plant Germplasm

spp.) shoot apices by encapsulation-dehydration. Cryo–Letters 11:165–174. Matsumoto, T., A. Sakai, C. Takahashi and K. Yamada. 1995. Cryopreservation of in vitro- grown apical meristems of wasabi (Wasabia japonica) by encapsulation-vitrification method. Cryo–Letters 16:189–196. Murashige, T. and F. Skoog. 1962. A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiologia Plantarum 15:473–497. Panis, B. 1995. Cryopreservation of banana germplasm. Ph.D. Thesis. No.207. Catholic University, Leuven, Belgium. Raynor, B. and S. Silbanus. 1992. Ecology of Colocasia taro production on Pohnpei. Pp 20– 24 in Proceedings of the Sustainable Taro Culture for the Pacific Conference. (L. Ferentinos, ed.) University of Hawaii. Sakai, A., S. Kobayashi and I. Oiyama. 1990. Cryopreservation of nucellar cells of navel orange (Citrus sinensis Osb. var. ‘brasiliensis Tanaka’) by vitrification. Plant Cell Reports 9:30–33. Takagi, H., N.T. Thinh, O.M. Islam. T. Senboku and A. Sakai. 1997. Cryopreservation of in vitro-grown shoot tips of taro (Colocasia esculenta (L.) Schott) by vitrification. 1. Investigation of basic conditions of the vitrification procedure. Plant Cell Reports 16:594– 599. Towill, L.E. and R.L. Jarret. 1992. Cryopreservation of sweet potato (Ipomoea batatas (L) Lam.) shoot tips by vitrification. Plant Cell Reports 11:175–178. Yamada, T., A. Sakai. T. Matsumura and S. Higuchi. 1991. Cryopreservation of apical meristems of white clover (Trifolium repens L.) by vitrification. Plant Science 78:81–87. 338 Cryopreservation of Tropical Plant Germplasm

Cryostorage of in vitro-induced dormant buds of primula after desiccation Satoshi Kato¹, Masaya Ishikawa², Miwako Ito¹ and Tatsuo Matsumoto¹ ¹ Saitama Ornamental Plant Research Center, Fukaya, Saitama 366-0815, Japan ² National Institute of Agrobiological Resources, Tsukuba, Ibaraki 305-0856, Japan

Introduction Primula (Primula sieboldii), known as Sakurasou in Japan, is a 10-cm tall herbaceous plant which has pale green oval leaves with prominent rounded serration. It is a winter-hardy perennial and the flower bud is dormant in winter and blooms in spring. Primula sieboldii requires a humus-rich, well- drained soil. Many P. sieboldii native colonies are endangered owing to environmental problems, but some of them are conserved by organizations and local governments. Tajimagahara in Saitama Prefecture is a unique national conservation field of wild P. sieboldii. Primula sieboldii has many cultivars because it has been cultivated as one of the oldest ornamental plants in Japan. The flower colour ranges from white, red to purple and the flower form from cup-shaped to deeply fringed petals. Saitama Ornamental Plant Research Center has a rich collection of more than 300 cultivars (including wild varieties) of P. sieboldii, which are mostly maintained as pot-cultures and by in vitro cultures. To safely preserve a number of varieties for the medium term, we have attempted the storage of in vitro-induced dormant buds under various conditions. The cryostorage of dormant buds, if successful, is expected to be a useful tool for safely preserving Primula genetic resources for the long term. Dormant buds are natural organs produced by many plant species to overcome cold seasons. The use of such organs is an important approach for ex situ conservation of plant genetic resources. Winter buds of various woody species have been used for cryopreservation. However, there have been few attempts to cryopreserve dormant buds of herbaceous plants and to artificially induce dormant buds in vitro in both woody and herbaceous plants. Here we report cryopreservation following desiccation of in vitro-induced dormant buds of vegetatively propagated P. sieboldii.

Materials and methods Dormant buds were induced by placing 5-leaved in vitro plants (cv. Shiratama) on a semi-solid medium (hormone free, 3% sucrose) at 21°C in the dark for 60 d or more. The upper parts (7–10 mm) of the buds formed (2 cm long) were serially precultured on ½ MS media containing increasing concentrations of sucrose (0.3, 0.5, 0.7M for 2 d each and 0.9M for 1 d). The tips (2–3 mm), excised from the precultured buds, were dried in a laminar flow chamber for 0–11 hours without encapsulation, then immersed in liquid nitrogen (LN). Following rapid thawing in water, the tips were transferred to regrowth medium containing 1 ppm GA3 for breaking dormancy. Posters 339

The tips dissected from the precultured dormant buds (hereafter referred to as bud tips or tips) were also used for cryopreservation using vitrification and slow prefreezing methods. For vitrification, the tips were either gradually (by three-step loading) or directly placed in the vitrification solution L (Ishikawa and Tandon, unpubl.) and incubated for 0–60 min. The samples were then immersed in LN. Following 1 h storage and rapid rewarming in water at 40°C, the vitrification solution was diluted with 1.2M sucrose. For slow prefreezing, the tips were directly soaked in a cryoprotectant CSP1 (Ishikawa et al. 1996) for 1 h. Then they were ice-inoculated at –8°C and further cooled at 0.3°C/min to –30°C prior to immersion in LN. Following rewarming in water at 40°C, the cryoprotectant was diluted slowly with 3% sucrose over 15 min. The specimens cryopreserved with these two methods were transferred to regrowth medium as described above.

Results and discussion Using the desiccation method described above, we could successfully cryopreserve the bud tips derived from in vitro-induced dormant buds with a fairly high subsequent plant regeneration rate. The highest survival (80%) following cryopreservation was obtained when the tips were desiccated in the laminar flow chamber for 11 h (Table 1). Shorter desiccation periods resulted in progressively reduced survival following cryopreservation. Desiccation for up to 11 h alone was not harmful to the bud tips as they retained 90–100% regeneration rates following desiccation and reculture. The tips withstood desiccation and subsequent cryopreservation. They resumed regrowth within a month and grew into plants without callus formation.

Table 1. Survival (plant regeneration rates) following cryopreservation using a desiccation method of Primula buds tips derived from precultured in vitro-induced dormant buds Survival (%) Desiccation period (h) Desiccated Desiccated and cryopreserved 0 90 0 4.5 100 20 6.5 90 40 8 90 50 9 90 60 11 80 80

By contrast, the slow prefreezing and vitrification procedures allowed only low survival (0–10%) of the bud tips dissected from the precultured in vitro- dormant buds. In the vitrification methods using both three-step loading and direct soaking, the incubation for up to 60 min in the vitrification solution L alone was not harmful to the bud tips. However, we could obtain only 10% regeneration when the tips were either incubated in L for 20 and 30 min using the three-step loading method or for 10 min using the direct soaking method. The slow prefreezing method using CSP1 allowed no survival of the bud tips following cryopreservation, whereas incubation in CSP1 alone was not harmful to the specimens. 340 Cryopreservation of Tropical Plant Germplasm

Cryopreservation of desiccated in vitro-induced dormant buds seems promising for long-term conservation of Primula and its application to other cultivars is required. The results are preliminary but are suggestive for other plants which can form dormant buds in vitro. To our knowledge, this is probably the first report on cryopreservation of Primula and on the use of in vitro-induced dormant buds for cryopreservation.

Reference Ishikawa, M., P. Tandon, M. Suzuki and A. Yamaguishi–Ciampi. 1996. Cryopreservation of bromegrass (Bromus inermis Leyss) suspension cultured cells using slow prefreezing and vitrification procedures. Plant Science 120: 81–88. Posters 341

Cryopreservation of oil-palm embryogenic suspensions Nathalie Chabrillange¹, F. Aberlenc–Bertossi¹, M. Noirot¹, Y. Duval¹ and Florent Engelmann² ¹ GeneTrop, ORSTOM, BP 5045, 911 avenue Agropolis, 34032 Montpellier cedex 01, France ² IPGRI, 00145 Rome, Italy

Introduction Oil-palm (Elaeis guineensis Jacq.) embryogenic cell suspensions produced from leaf calli were cultivated on liquid medium supplemented with 2,4– dichlorophenoxyacetic acid (2,4–D) (de Touchet et al. 1991; Duval et al. 1995). In this work, a cryopreservation protocol was developed to conserve the embryogenic capacities of the suspensions and to limit the risks of losing the material due to contamination (Engelmann 1997). A classical freezing was employed for freezing these oil-palm embryogenic cell suspensions. The effects of glucose and dimethylsulfoxide on cell survival were investigated and the optimal duration of post-treatment was determined.

Materials and methods Four different cell lines (Nos. 121, 123, 221 and 341) were sampled during their proliferation phase. The pretreatment was carried out by incubating 0.3 ml of suspension per ml of cryoprotective medium for 1 h at 4°C. The cryoprotective media tested contained a range of four concentrations of glucose (0.1, 0.5, 1, 1.5M) combined with four concentrations of DMSO (0, 5, 10, 15%). After the pretreatment, cells were cooled at 0.5°C/min to –40°C, then immersed in liquid nitrogen. After rapid thawing (2 min at 40°C), the viability was estimated using staining with triphenyl tetrazolium. Results were expressed as percentage of the untreated control. Cells were then placed on semi-solid medium for regrowth before their transfer to liquid medium. The influence of the duration of post-treatment on semi-solid medium was tested from 0 to 28 d, and evaluated by measurement of subsequent growth rate of the cultures in liquid medium. Three replicates were used for each treatment. Results were analyzed by one-way ANOVA. Newman and Keul’s tests were applied when significant effects were observed. The relative importance of each effect was then measured.

Results

Pretreatment The effect of glucose and DMSO on survival after cryopreservation varied with the cell line (Fig. 1). Clone effects represented 36% of the total variance (data not shown). For all cell lines, DMSO had the main effect on survival after cryopreservation compared with glucose. DMSO explained 38–81% of the total variance (Table 1). Glucose had a significant effect on three of the four cell lines (Table 1). Statistical analyses allowed identification of the optimal concentration of DMSO as being 10% and for glucose, as 1M. Though glucose- 342 Cryopreservation of Tropical Plant Germplasm

DMSO interaction was found to be significant in two lines, its importance was always lower than that of the separate effects of each compound alone.

Post-treatment After a post-treatment of 14 d on semi-solid medium, the growth rate of cryopreserved cell suspensions was 70% during the first subculture in liquid medium, and 240% during the second subculture. Extending the post- treatment duration did not significantly improve the growth rate of cryopreserved cells (Fig. 2). Regeneration of somatic embryos from all cryopreserved cell lines was successful (data not shown). Thus, the cryopreservation protocol has no effect on embryogenic capacities.

50% 50% 121 123

40% 40%

30% 30%

20% 20%

10% 10%

0% 0% 0 5 10 15 0 5 10 15 DMSO (%) DMSO (%)

50% 50% 221 341

40% 40%

30% 30%

20% 20%

10% 10%

0% 0% 0 5 10 15 0 5 10 15 DMSO (%) DMSO (%)

Fig. 1. Effect of the composition of cryoprotective medium on survival of four different cryopreserved cell lines. Cryprotectant was a combination of four concentrations of DMSO and four concentrations of glucose (0.1, 0.5, 1, 1.5M – bars, left to right).

Table 1. Relative importance of glucose and DMSO effects on survival after liquid nitrogen treatment for four cell lines. Values represent % of the total variance. Cell line number 121 123 221 341 Glucose 13.68 17.54 NS 44.01 DMSO 78.88 81.45 58.14 38.16 Interaction 7.44 NS NS 17.83 Posters 343

400% B

300% B B

200%

Growth rate b A 100% ab ab a A a a 0% 0 1 7 14 21 28 Duration of post-treatment on semi-solid medium (days)

Fig. 2. Post-treatment of cryopreserved cells. Growth rate during the first ( ) and the second ( ) subculture in liquid medium. Same letters (small letters for the 1st cycle and capital letters for the 2nd cycle) indicate results not significantly different at the 0.05 level according to the Newman and Keul’s tests.

Conclusion A protocol including pretreatment of cell suspensions with 10% DMSO and 1M glucose, two-step freezing, rapid rewarming and a 14-d post-treatment on semi-solid medium is now routinely employed in our laboratory for the cryopreservation of oil-palm embryogenic cell lines.

References de Touchet, B., Y. Duval and C. Pannetier. 1991. Plant regeneration from embryogenic suspension cultures of oil palm (Elaeis guineensis Jacq). Plant Cell Reports 10, 529–532. Duval, Y., F. Aberlenc and B. de Touchet. 1995. Use of embryogenic suspensions for oil palm micropropagation. ISOPB International Symposium on Recent Development in Oil Palm Tissue Culture and Biotechnology. 24–25th Sept. 1993, Kuala Lumpur, Malaysia. Engelmann, F. 1997. In vitro conservation methods. Pp. 119–162 in Biotechnology and Plant Genetic Resources: Conservation and Use. Ford–Lloyd B.V., H.J. Newbury and J.A. Callow (eds.). CABI, Wallingford, UK. 344 Cryopreservation of Tropical Plant Germplasm

In vitro cryopreservation of cacao genetic resources Bruno Florin, Eric Brulard and Vincent Pétiard Centre de Recherche Nestlé – Tours, Tours cedex 02, France

Introduction Theobroma cacao L. is one of the major cash crops for a number of developing countries. Cacao seeds are unsuitable for long-term storage owing to their lack of dormancy and sensitivity to desiccation. Genetic resources are currently preserv-ed by growing trees in field collections but their future seems to be more and more problematic (cost and risk for genetic, phytosanitary and political reasons). Owing to recent progress in cacao somatic embryogenesis, we have established a protocol for the long-term storage of cacao germplasm in liquid nitrogen.

Materials and methods Embryogenic strains were initiated from flower buds of trees cultivated in a greenhouse at NRC–Tours (France). These trees were developed from seeds harvested from National Ecuadorian clones. The embryogenic strains were established according to the procedure developed by Lopez–Baez et al. (1991). They were subcultured every 3 weeks on a multiplication medium at a density of 0.5 g fresh weight per 50 ml of solid medium containing equal concentrations of sucrose and maltose (20 g/L). These strains were cultured in the dark at 26–27°C. Owing to the slow growth of cacao cultures, their sensitivity to stress and their constitution (mixture of cells and embryos at different stages), we had to induce freezing tolerance using a hardening treatment with sucrose, as described in Table 1. At the end of the incubation period in 1M-sucrose liquid medium, the tissues were collected and placed in cryotubes for drying. The hardened-off tissues were then placed in an atmosphere of 43% RH. This RH was fixed by the addition of an oversaturated salt solution of potassium carbonate (K2CO3) in the containers. Freezing was carried out by direct immersion in liquid nitrogen of cryotubes containing control, hardened-off and/or pre-dried tissues. For regrowth, thawed samples were placed on a high sucrose concentration medium, then rehydrated using a stepwise decrease (0.25M/d) of the sugar content in the medium, from 0.75M to 0.1M. Viability is expressed by the percentage of reproliferating and regenerating calli observed 4 to 6 weeks after rehydration.

Results The freezing tolerance of cacao embryogenic tissues was improved using a hardening-off treatment on a medium supplemented with a high concentration of sucrose. Table 1 shows that the highest survival rates were obtained with the longer treatments and a progressive increase of the sucrose content of the medium (93% of reproliferating calli after freezing in liquid nitrogen with treatment V). Posters 345

Table 1. Viability of cacao embryogenic calli after cryopreservation according to the hardening-off treatment Sucrose content Hardening treatment of culture media successively used I II III IV V VI Maturation 100 µM ABA – – – – – 21d Pretreatment 0.25M solid – 1d – 7d 7d 6d 0.4M solid – – 7d – – – 0.50M solid – 1d – 7d 7d 7d 0.75M solid – 1d – 3d 7d 6d 1.0M solid – 1d – – 7d – 1.0M liquid 3d 3d 3d 2d 3d 3d Total duration of the 3d 7d 10d 21d 31d 21d+22d hardening treatment (43d)

Water content Hardened-off 239 226 219 232 182 234

(g H2O/100g dry wt)

% Regrowing calli 37 42 14 58 93 96 after LN

The hardening-off treatment induced a large water loss (about 65% of the freezable tissue’s water); however, it also modified the drying rate by decreasing the water loss rate during the drying phase. Freezing tolerance was shown only for hardened-off tissues, which reached a residual water content ranging from 0.8 to 0.2 g/g dry weight, depending on the strain and the drying duration. It required at least 60 h of drying under the controlled conditions described above. In addition, excessive dehydration was responsible for a decrease in the survival rate before and after freezing (Fig. 1), most likely due to some tissue alterations. The addition of ABA (treatmentVI) enhanced the desiccation tolerance of tissues, which led to definition of a more flexible protocol in terms of dehydration duration. This protocol has been successfully applied to embryogenic strains from different cacao genotypes. It has been shown that the cryopreserved strains maintain their ability to regenerate somatic embryos after freezing in liquid nitrogen (Table 2). In vitro plantlets were produced from cryopreserved strains, and transferred to the greenhouse for acclimatization. No difference was observed in the development of plantlets from frozen and unfrozen explants. The development of these plantlets looked similar to that of the control and they will soon be transferred to the nursery and fields for conformity trials.

Conclusion For the first time, the viability of cryopreservation of cacao embryogenic strain has been shown. Moreover, cacao plantlets could be developed from cryopreserved tissues and are now growing in the greenhouse. The method developed is reliable and appears to be applicable for the long-term preservation and exchange of cacao germplasm. 346 Cryopreservation of Tropical Plant Germplasm

Growth Rate Survival rate Before freezing in LN

500 120

450 A 100 400

350 80 300

250 60

200 40

150 Regrowing calli (%) Regrowth rate A (%)

100 20 50

0 0 Control hardened-off Dried 52h Dried 66h Dried 89h

450 100

After freezing in LN 400 B 90

80 350

70 300 60 250 50 200 40

150 Regrowing calli (%) Regrowth rate A (%) 30

100 20

50 10

0 0 Control hardened-off Dried 52h Dried 66h Dried 89h Type of culture

Fig. 1. Survival and regrowth rates of control and hardened-off cacao strains (A) before and (B) after freezing in liquid nitrogen.

Table 2. Production of somatic embryos by control, hardened-off and/or dried, cryopreserved strains CC260 (5) strain Embryo production (Emb/g fresh wt.) Control 926 ± 245 Pretreated 1029 ± 151 Dried (66 h) 694 ± 125 Cryopreserved 518 ± 80 Estimations were performed 8 weeks after thawing for regrowing tissues after LN. Posters 347

Finally, using an adequate pretreatment, cacao embryogenic tissues appear to withstand cryopreservation and they showed a similar behaviour to that of other species considered as less recalcitrant to storage methods.

Acknowledgement This work was partially supported by a grant from the American Cocoa Research Institute (ACRI).

Reference Lopez–Baez, O., H. Bollon, A. Heskes and V. Pétiard. 1993. Somatic embryogenesis and plant regeneration from flower parts of cocoa Theobroma cacao L. Comptes Rendus de l’Académie des Sciences, Paris, 316: 579–584. 348 Cryopreservation of Tropical Plant Germplasm

Development of a simplified method for the routine cryopreservation of coffee germplasm collection Bruno Florin, Eric Brulard, Jean Paul Ducos, Hervé Tessereau and Vincent Pétiard Centre de Recherche Nestlé – Tours, Tours cedex 02, France

Introduction Coffee has great economic importance for many countries in the world. In vitro techniques are applied for multiplication and conservation of this crop with varying degrees of success; however, they are of real interest in the development of breeding programmes and for the preservation of genetic resources of this tropical species. Coffee produces semi-recalcitrant seeds, which are unsuitable for long-term storage. Cryopreservation of embryogenic cells may be used for germplasm storage and exchange, but also to ensure a constant supply of embryogenic cells for plant propagation or genetic modification experiments. Because of the decline in their embryogenic potential with the increase in the number of subcultures, cryopreservation appears to be the best alternative to avoid the loss of their regeneration ability. A simple method comprising a sucrose pretreatment and a two-step cooling protocol has been optimized with the aim of routine use in preservation of embryogenic cell cultures.

Materials and methods The embryogenic strains used in this study originated from Coffea canephora (P.) and Coffea arabica (L.) clones. They were cultured either in Yasuda medium (Yasuda et al. 1985) or MS medium, depending on the strain. Cultures were -2 -1 maintained at +26°C under a light intensity of 50 µE m s with a photoperiod of 16-h light/8-h dark. The first set of experiments was performed to adapt coffee embryogenic cell suspensions cultivated in liquid media to the simplified freezing process that we had previously developed in our laboratory (Lecouteux et al. 1991). Using four clones of Coffea canephora, a two-step pretreatment was optimized, comprising a first incubation in 0.4M sucrose liquid medium followed by a second incubation in a 1.0M sucrose liquid medium. Cells were transferred to cryotubes which were placed in a freezing container (Nalgene Cat. No. 5100– 0001). The two-step freezing method consists of prefreezing the samples by placing the container in a standard freezer at –25°C for about 20 h followed by direct immersion of the samples in liquid nitrogen. A second set of experiments was performed to extend the application of this method to a large collection including 27 embryogenic calli strains cultivated on solid media. Finally, we verified both the embryo production and the conformity of plants from cryopreserved strains in the field. Viability was estimated after 6 weeks of culture either by the percentage of reproliferating calli or by the growth rate in the case of cell suspensions. Posters 349

Results Three sucrose pretreatments were tested on four clones. Table 1 shows that first, for the four tested genotypes, regrowth was observed whatever the pretreatment. Second, the B-pretreated tissues showed the highest TTC values, regrowth of cryopreserved samples, and the best growth rate for three of the four clones tested. Therefore, the freezing tolerance of embryogenic cell suspensions requires a stepwise sucrose pretreatment for a progressive osmotic dehydration ending by a short incubation period of 1 d in a 1M sucrose medium. However, some variations in sensitivity to freezing exist. Clone G200 required a longer pretreatment duration than the other clones. Clone 126B appeared more sensitive to the freezing process (only 2 of 3 samples regrew after the A–type treatment). Finally, viability expressed by the TTC values after thawing can be used to predict experimental conditions showing the highest growth rate recorded after 6 weeks of culture (Table 1). The optimization of the prefreezing temperature led to use of a freezer at -25°C without control of the cooling rate. The temperature of –25°C determines the threshold of cell dehydration which is reached after a minimum of 7 h after the start of the prefreezing process. This method has been also successfully adapted to coffee embryogenic calli strains cultivated on solid medium. With calli, the pretreatment phase is longer than for cell suspensions (10 d in 0.4M sucrose followed by 3 d in 1.0M sucrose). This method has already been applied to 27 embryogenic strains. Figure 1 shows that no strain appears to be recalcitrant to freezing, and that 59% of strains regrow with a reproliferating rate higher than 60%. These embryogenic strains were preserved for their ability to regenerate plants. Table 2 gives some estimations of the embryo production from control and cryopreserved strains of various Coffea canephora and Coffea arabica varieties. In all cases, the ability to regenerate somatic embryos was preserved. Even if some slight differences may exist between control and cryopreserved cells, depending on the strain, there was no irremediable loss of embryogenic potential. The embryos developed normally into in vitro plantlets. About 4000 plants from cryopreserved embryogenic strains are already in the nursery, ready to be transferred to the field for conformity studies.

Conclusion Embryogenic cell suspension and calli strains of coffee can be cryopreserved in liquid nitrogen using a simple method. Thus, cryopreservation appears a reliable method for the preservation of coffee genetic resources and the supply of embryogenic inoculum for propagation and genetic modification purposes. 350 Cryopreservation of Tropical Plant Germplasm

Table 1. Freezing tolerance induced by various sucrose pretreatments applied to embryogenic cell strains from four Coffea canephora genotypes Type of pretreatments † ‡ § A B C TTC Growth TTC Growth TTC Growth b,c of the b of the b of the Coffee (AU triplicat Growth (AU triplicat Growth (AU triplicat Growth genotype ) e rate a,d ) e rate a ) e rate a G200 0.17 +/+/+ 326 0.37 +/+/+ 414 0.57 +/+/+ 533 126B 0.08 –/+/+ 202 0.22 +/+/+ 409 0.17 +/+/+ 246 J13C 0.16 +/+/+ 76 0.38 +/+/+ 1164 0.32 +/+/+ 319 197C 0.07 +/+/+ 83 0.26 +/+/+ 354 0.15 +/+/+ 215 † Pretreatment A = 1.0M for 1 d. ‡ Pretreatment B = 0.4M for 3 d followed by 1.0M for 1 d. § Pretreatment C = 0.4M for 3 d followed by 1.0M for 3 d. a Growth rate = [ (F. wt. Final – F.wt. Init )/ F. wt. Final ] x 100. b TTC TEST = 2,3,5–tri–phenyltetrazolium chlorite test: optic density estimated at 485 nm (see Towill and Mazur 1975). c Assessment of viability using TTC test has been made just after thawing and is expressed as DO/mg dry wt. d Estimation of regrowth has been made after 6 weeks of culture in standard conditions.

Fig. 1. Freezing tolerance of various embryogenic tissues strains of Coffea canephora. Posters 351

Table 2. Production of somatic embryos by control and cryopreserved strains Number of embryos produced x 103/g FW inoculated† Embryogenic strains Control Cryopreserved Coffea canephora P. Callus 126 43.7 ± 1.7 14.7 ± 2.9 Cell suspension AP97 81.1 ± 6.9 144.9 ± 11.1 197 110.8 ± 15.3 98.1 ± 13.9 Coffea arabica L. Cell suspension CAT8661 50.8 ± 8.2 44.1 ± 7.2 DH97 189.5 ± 72.2 169.8 ± 46.1 † Inoculation density = 1 g/L of expression medium; somatic embryos were produced using the method previously described by Zamarripa et al. 1991; duration of culture = 70 d.

References Lecouteux, C., B. Florin, H. Tessereau, H. Bollon and V. Pétiard. 1991. Cryopreservation of carrot somatic embryos using a simplified freezing process. Cryo–Letters 12: 319–328. Towill, L.E. and P. Mazur. 1975. Studies on the reduction of 2,3,5–triphenyltetrazolium chloride as a viability assay for plant tissue cultures. Canadian Journal of Botany 20: 567– 573. Yasuda, T., Y. Fujii and T. Yamaguchi. 1985. Embryogenic callus induction from Coffea arabica leaf explants by benzyladenine. Plant and Cell Physiology 26 : 595–597. Zamarripa, A., J.P. Ducos, H. Bollon, M. Dufour and V. Pétiard. 1991. Production of somatic embryos of coffee in liquid medium : effects of inoculation density and renewal of the medium. Café, Cacao, Thé 35: 233–244. 352 Cryopreservation of Tropical Plant Germplasm

Incorporation of antibodies into tobacco cells treated with solutions similar to vitrification solutions 4 Pramod Tandon¹, Masaya Ishikawa², Atsushi Komanine³ and Hiroo Fukuda ¹ Dept. of Botany, North-Eastern Hill University, Shillong 793 022, India ² Dept. of Genetic Resources, National Institute of Agrobiological Resources, Tsukuba 305-8602, Japan ³ The Research Institute of Evolutionary Biology, Kamiyoga 2-4-28, Setagaya-ku, Tokyo 1 5 8-0098, Japan 4 Dept. of Biological Sciences, Graduate School of Sciences, Tokyo University, Hongo 7-3-1. Tokyo 1 13-0033, Japan

Introduction The plasmalemma and cell wall of plant cells control the transport and entry of large molecules. Transient and reversible permeabilization of this barrier, if possible, would offer several useful applications such as locating macromolecules and blocking receptors using specific antibodies. Permeabilization of plant cells has been attempted using chemical and physical treatments (Felix 1990, 1992). Some organic solvents successfully permeabilized cells but cell viability tended to be rather low (Felix 1990). Methods that could permeabilize cells only transiently without hindering cell viability and growth are ideal. In the process of developing new cryoprotective solutions for vitrification methods, we found that these solutions made cells considerably leaky, which hinted that they could be used for cell permeabilization. We have recently found that solutions similar to vitrification solutions in composition can permeabilize suspension-cultured cells (Tandon et al. 1999). Here we report the essence of the work: a transient cell permeabilization procedure for high incorporation of fluorescein isothiocyanate-conjugated anti-mouse immunoglobulin G (FITC–IgG) by the use of cocktails comprising glycerol, sucrose, ethylene glycol and DMSO and indicate the potential for a similar phenomenon happening during treatment with vitrification solutions made up of similar ingredients.

Materials and methods The tobacco (Nicotiana tabacum) BY–2 cell line was harvested on the 4th day of weekly subculture and used as detailed elsewhere (Tandon et al. 1999). About 10 mg cells were permeabillzed using 100 µl of cell permeabilization solutions (CPS) as detailed in Table 1, containing 1 µl of FITC–IgG (FITC anti- mouse IgG; 150 kDa) for 15 s at room temperature and then immediately washed twice with 1 ml of ice-cold 1M sucrose solution containing 10 mM

CaCl2 and then once with 1 ml of modified LS medium. The cells were pelleted by centrifugation at 3000 rpm for 5 min at 0°C in between the washings. As varying factors, different permeabilization solutions, duration of permeabilization, addition of CaCl2 in CPS, cell age and different diluting conditions were tested for their effects on incorporation of FITC–IgG into Posters 353 tobacco cells and cell viability. 354 Cryopreservation of Tropical Plant Germplasm

Cell viability was determined by staining with fluorescein diacetate (FDA). For both viability measurements and incorporation of FITC–IgG, about 5000 cells were observed for each treatment under UV-microscopy. The control cells showed only marginal autofluorescence during the 7-d culture period.

Results and discussion Of the permeabilization solutions used, a fine balance of glycerol, sucrose, ethylene glycol and DMSO (20:5:20:5 w/v%) present in CPS–3 was most effective in incorporating high molecular weight FITC–IgG and at the same time retaining cell viability. From Table 1, it is apparent that the presence of both ethylene glycol and DMSO in the permeabilization solution was essential for better uptake and cell survival. The optimal duration of permeabilization for both higher incorporation of FITC–IgG and survival of the cells was obtained using a 15-s incubation. The addition of 10 mM CaCl2 to CPS–3 resulted in improved uptake of FITC–IgG and cell survival. The highest incorporation of FITC–IgG and cell survival were observed with cells harvested on the 4th day of culture. Following the permeabilization and washing procedures described above, the morphology of the cells remained intact and there was no lysis of cells or destruction of subcellular organization. The permeabilized cells showed fluorescence from FITC–IgG in the peripheral and nuclear regions as well as in cytoplasmic strands. It was confirmed that fluorescence was located only in the cytoplasm and not in the wall and that plasma membrane impermeability to osmoticum (1M sucrose) was recovered showing plasmolysis similar to untreated control cells. The permeabilized cells retained high viability, as determined by FDA staining (data not shown).

Table 1. Incorporation of FITC–IgG into tobacco cells and cell survival using different cell permeabilization solutions: comprising glycerol (Gly), sucrose (Suc), ethylene glycol (EG), dimethyl sulfoxide (DMSO) and 40 mM CaCl2. About 10 mg of cells were permeabilized using 100 µl of CPS containing I µl of FITC–IgG for 15 s at room temperature and then immediately washed twice with 1 ml of ice-cold 1M sucrose solution containing 10 mM

CaCl2 and then once with 1 ml of modified LS medium. The data are the mean ± SE (n=50). Concentrations (w/v%) Cells showing Cell survival assessed from Permeabilization FITC–IgG FDA staining solution Gly Suc EG DMSO incorporation (%) (%) Control † – – – – 0 98.0 ± 4.1 CPS-1 20 5 30 10 93.9 ± 2.8 21.4 ± 1.7 CPS-2 20 5 25 5 87.4 ± 3.4 37.0 ±1.5 CPS-3 20 5 20 5 76.4 ± 2.6 78.2 ± 2.5 CPS-4 20 10 10 5 58.3 ± 1.9 56.8 ± 3.2 CPS-5 20 5 20 0 49.6 ± 1.6 76.0 ± 2.9 CPS-6 20 10 0 10 65.7 ± 1.8 68.1 ± 4.3 CPS-7 20 5 0 5 60.0 ± 2.0 77.3 ± 1.4 † Control cells were suspended in modified LS medium with FITC–IgG and washed with the medium. Posters 355

Regrowth of the permeabilized cells was 75–80% of untreated control cells after 21 d of culture. A weak fluorescence from incorporated FITC–IgG was noticeable in the dividing cultured cells for about 7 d, which faded later (data not shown). The permeabilization shown here may involve permeabilization of the cell wall and plasmalemma resulting in pore formation in the membrane, followed by the uptake of FITC–IgG through hypertonic and/or hypotonic shock. While glycerol and sucrose in CPS may provide needed osmotic stress, ethylene glycol and DMSO bring about permeation of the cell wall and plasma membranes. The organization of the pectic substances is a major controlling element in defining the sieving properties of the cell wall (Baron–Epel et al. 1988). DMSO is often used for solubilization of polysaccharides such as pectin from the cell wall and is also known to increase membrane fluidity. The presence of 10% glycerol has been reported to completely inhibit the release of cellular materials and enzymes (Felix 1992). In conclusion, the procedure described herein is a reversible type of cell permeabilization that allows incorporation of antibodies and possibly even larger molecules. Various applications may be expected such as locating macro- molecules and blocking receptors with specific antibodies. We expect similar cell permeabilization in cells treated with vitrification solutions such as PVS2 (Sakai et al. 1990) as the composition is similar to the solutions used in this study.

References Baron–Epel, O., P.K. Gharyal and M. Schindler. 1988. Pectins as mediators of wall porosity in soybean cells. Planta 175: 389–395. Felix, H. 1990. Bioconversions in permeabilized cells. Pp. 259–278 in Extractive Bioconversions, (B. Mattiasson and O. Holst, eds.). Marcel Dekker, New York. Felix, H. 1992. Permeabilized cells. Analytical Biochemistry 120: 21 1–234. Sakai, A., S. Kobayashi and I. Oiyama. 1990. Cryopreservation of nucellar cells of navel orange (Citrus sinensis Osb. var. brasiliensis Tanaka) by vitrification. Plant Cell Reports 9: 30–33. Tandon, P., M. Ishikawa, A. Komamine and H. Fukuda. 1999. Incorporation of fluorescein- conjugated anti-mouse immunoglobulin G into permeabilized Nicotiana tabacum BY-2 cells. Plant Science 140: 63–69. 356 Cryopreservation of Tropical Plant Germplasm

Cryopreservation of Doritaenopsis cells in suspension culture by vitrification H. Tsukazaki, Keiko Ishikawa and M. Mii Plant Cell Technology Laboratory, Faculty of Horticulture, Chiba University, Matsudo-City, Chiba 271-8510, Japan

Introduction Doritaenopsis (Phalaenopsis × Doritis ) is a tropical orchid that is popular for pot plants or cut flowers. Applying micropropagation methods to Doritaenopsis, green Protocorm-Like Bodies (PLB) and, in a few cases, callus with high multiplication capacity could be obtained. Calluses are useful for protoplast isolation, genetic transformation, and also for biological analysis. So, conservation of these cells without somaclonal variation is necessary. In this paper, we describe cryopreservation of Doritaenopsis callus by the vitrification method and the formation of PLB from cryopreserved cells. We show suitable conditions for the preculture period, including sucrose and ABA concentrations in the preculture medium.

Material and methods Suspension cultures of Doritaenopsis cv. New Toyohashi maintained in liquid ND medium (New Dogashima medium; Tokuhara and Mii 1993) containing 0.1 mg/L NAA, 1.0 mg/L BA and 0.056M sucrose were used as plant material in this study. Cells were precultured for 3–14 d in liquid ND medium containing 0.056–0.4M sucrose and 0–5.0 mg/L ABA. After preculture, cells were treated with a loading solution (2M glycerol and 0.4M sucrose) at room temperature for 15 min, then with PVS2 solution (Sakai et al. 1990) at 0°C for 1–3 h, and plunged into liquid nitrogen. After rewarming in hot water (35°C), cells were washed with ND medium containing 1.2M sucrose for 1–2 h. The survival of vitrified and cryopreserved cells was assessed using TTC reduction and regrowth. TTC stainability (%) was estimated by the percentage of the number of stained cells divided by the number of total cells, multiplied by 100. After washing, cells were cultured on solidified ND medium supplemented with 0.2M sucrose at 25°C in the dark for 2 d, then transferred on ND medium supplemented with 0.1 mg/L NAA, 1.0 mg/L BA and 0.056M sucrose under -2 -1 continuous white fluorescent light (36 mmol m s ) at 25°C. After 1 month, cells were transferred onto 1/2 ND medium (half strength of macro- and microelements) supplemented with 1% maltose and 3 g/L Gelrite for PLB formation. To confirm genetic stability, RAPD analysis was performed using Operon 10-mer Kits (Operon, USA). Total DNA was extracted from the non- cryopreserved cells and PLB obtained from cryopreserved cells. Posters 357

Results and discussion At first, we confirmed that survival rates of cryopreserved cells estimated by TTC reduction assay and by regrowth method had high correlation (R=0.79). Non-precultured cells treated by PVS2 at 0°C for 30 min to 2 h prior to immersion in LN showed very low (6.7%) TTC stainability. By contrast, cells precultured in liquid ND medium with 0.1M sucrose for 1 week, then loaded and vitrified with PVS2 solution at 0°C for 1 h, displayed about 50% TTC stainability. Based on these results, cells were precultured in liquid ND medium supple- mented with 0.056–0.4M sucrose for 1 week. TTC stainability of cryopreserved cells was enhanced when 0.1M sucrose was present in the preculture medium (50%). On the other hand, TTC stainability of vitrified cells decreased when the cells were precultured on ND medium containing more than 0.2M sucrose. In other experiments, cells were transferred to other preculture media, consisting of liquid ND medium supplemented with 0–5.0 mg/L ABA and 0.1M sucrose for 1 week. TTC stainability of vitrified (89%) and cryopreserved cells (68%) was increased when 1.0 mg/L ABA was present in the preculture medium. It has been reported that sucrose and ABA could induce the accumulation of proline in cells which would become tolerant to desiccation and freezing (Eberhardt and Wegmann 1989; Panis et al. 1996). Our results are in agreement with these previous reports and ABA may act by inducing freezing tolerance in Doritaenopsis cells. Furthermore, both solution 1 and PVS2 were necessary as cryoprotectants for cryopreservation of Doritaenopsis cells. There was no significant difference among the incubation periods from 1 to 3 h with PVS2. Toxic effects caused by PVS2 were not observed. When cells were incubated in PVS2 for 30 min, TTC stainability of cryopreserved cells was only 15%. For cryopreservation of embryos of Bletilla striata, more than 1 h incubation of PVS2 was also necessary (Ishikawa et al. 1997). When cryopreserved cells were cultured on solidified ND medium, PLB were regenerated on the surface of the calluses about 3 months after cryopreservation. To confirm genetic stability, RAPD analysis was performed using 40 primers (10-mers). Total DNA was extracted from non-cryopreserved cells and PLB regenerated from cryopreserved cells. Genetic variation was not detected using 40 primers in material after preculture, cryopreservation and regrowth.

References Eberhardt, H. and K. Wegmann. 1989. Effects of abscisic acid and proline on adaptation of tobacco callus to salinity and osmotic shock. Physiologia Plantarum 76:283–288. Ishikawa, K., K. Harata, M. Mii, A. Sakai, K. Yoshimatsu and K. Shimomura. 1997. Cryopreservation of zygotic embryos of a Japanese terrestrial orchid (Bletilla striata) by vitrification. Plant Cell Reports 16:754–757 Panis, B., T. Totte, K. Van Nimmen, L.A. Withers and R. Swennen. 1996. Cryopreservation of banana (Musa spp.) meristem cultures after preculture on sucrose. Plant Science 121:95–106. Sakai, A., S. Kobayashi and I. Oiyama. 1990. Cryopreservation of nucellar cells of navel 358 Cryopreservation of Tropical Plant Germplasm

orange (Citrus sinensis Osb. Var. brasiliensis Tanaka) by vitrification. Plant Cell Reports 9:30–33. Tokuhara, K. and M. Mii. 1993. Micropropagation of Phalaenopsis and Doritaenopsis by culturing shoot tips of flower stalk buds. Plant Cell Reports 13:7–11. Posters 359

A cryopreservation protocol for strawberry cell suspension cultures Yongjie Wu¹,², Florent Engelmann³, Andrea Frattarelli² and Carmine Damiano² ¹ Changli Institute of Pomology, Hebei Academy of Agricultural and Forestry Sciences, Changli Town 066600, Qin Huang Dao City, Hebei Province, P.R. China ² Istituto Sperimentale per la Frutticoltura, 00040 Ciampino Aeroporto, Rome, Italy ³ IPGRI, 00145 Rome, Italy

Introduction Cryopreservation techniques have been developed for long-term conservation of more than 100 plant species cultured in vitro as protoplasts, cell suspensions, calluses, shoot apices, or somatic and zygotic embryos (Kartha and Engelmann 1994; Engelmann 1997). Cryopreservation is employed for conservation of plant genetic resources, but it is also applied to the conservation of biotechnological products, including metabolite-producing cultures and genetically engineered cell strains. Although cell suspensions are a material of choice for genetic transformation experiments, so far leaf disks and calluses only have been transformed in the case of strawberry (James et al. 1990; Nehra et al. 1990; El Mansouri et al. 1996). Transformation from single cells is highly desirable, since this pathway overcomes the possibility of regenerating chimeric plants. Owing to the difficulty of obtaining transformed material, it is important to develop cryopreservation protocols that allow conservation of the transformed cell lines. This paper summarizes the protocol developed for the cryopreservation of strawberry cell suspensions (Wu et al. 1997) and highlights the effect of several parameters within the freezing process on the viability of the cell suspension cultures.

Materials and methods Cell suspensions were obtained from callus induced on leaf explants of strawberry (Blando et al. 1993) and cultured on a rotary shaker (90 rpm) at 25°C under a photoperiod of 16-h light/8-h dark, and a light intensity of 37 -2 -1 µmol m s . For cryopreservation, cells were sampled 5, 10 or 15 d after the last transfer to new culture medium and pretreated at 0°C for 60–180 min in PVS3 vitrification solution consisting of 50% (w/v) and 50% (w/v) glycerol (Nishizawa et al. 1993), with or without the addition of DMSO at 5%, then cooled at 0.5°C/min down to temperatures ranging between –35 and –45°C and immersed rapidly in liquid nitrogen. Thawing was performed either at room temperature or in a water-bath set at controlled temperatures between 20 and 40°C. Cells were then plated on standard solid medium and observed for regrowth.

Results 360 Cryopreservation of Tropical Plant Germplasm

In the case of strawberry cell suspensions, pretreatment with various combinations of sucrose and DMSO did not ensure survival after cryopreservation (data not shown) and high survival rates (around 80% of unfrozen control) were achieved when using the PVS3 vitrification solution, modified, or not, by the addition of 5% DMSO. The influence on cell survival of two parameters, the effect of thawing regime and the effect of the duration of the last subculture was studied. No viability was observed when rewarming was carried out in air at room temperature or in a water-bath at 40°C (Table 1). However, thawing at 20 or 30°C permitted high levels of viability with both cryoprotective solutions employed.

Table 1. Effect of thawing regime (in air at room temperature (RT) or in a water-bath thermostated at 20, 30 or 40°C) and of cryoprotective solution employed during pretreatment (MPVS3: PVS3 + 5% DMSO) on the viability (%) of cryopreserved strawberry cell suspensions. Cells were pretreated at 0°C for 1 h, then cooled at 0.5°C/min down to – 40°C before rapid immersion in LN. (Adapted from Wu et al. 1997, with permission). Thawing regime Pretreatment RT 20°C 30°C 40°C PVS3 0 87 83 2 MPVS3 0 89 87 2

The viability of pretreated, prefrozen and cryopreserved cells decreased drastically with increasing duration of culture after the last transfer into new medium (Table 2).

Table 2. Effect of culture period (days) after last subculture on the viability (%) of pretreated, prefrozen (–LN) and cryopreserved (+LN) strawberry cell suspensions. Cells were pretreated with MPVS3, cooled at 0.5°C/min down to –40°C before immersion in LN, then thawed in a water-bath thermostated at 30°C. (Adapted from Wu et al. 1997, with permission). Days after subculture Pretreatment 5 10 15 pretreated 100 87 27 –LN 91 60 1.3 +LN 77 53 0.1

Discussion/conclusion This study represents the first report of successful cryopreservation of cell suspension cultures using pretreatment with vitrification solutions followed by slow, controlled cooling. The experiment on the effect of thawing procedure generated some interesting results. Total loss of viability after thawing samples in air at room temperature was probably due to recrystallization and ice crystal growth during the ensuing low rewarming rates. However, such dramatic differences in viability after thawing cell suspensions at temperatures in the range tested Posters 361 here have not been reported previously. A precise analysis of thawing rates and thermic events taking place during rewarming of the strawberry cell suspensions should be performed. 362 Cryopreservation of Tropical Plant Germplasm

The importance of using cells during their exponential growth phase to achieve high viability, as notably demonstrated by Withers (1985), was re- emphasized during this study. In conclusion, additional experiments will be necessary to determine optimal conditions for regrowth of cryopreserved cells and reinitiation of cell suspensions.

Acknowledgements Yongjie Wu gratefully acknowledges the support provided by a Special Skills Training Award within the Italian Government funded training scheme of IPGRI. The authors thank the Istituto Sperimentale per la Frutticoltura for providing the research facilities.

References Blando, F., A. Niglio, A. Frattarelli, S. Speranza and C. Damiano. 1993. Cell suspension cultures in strawberry: growth characterization and variability. Acta Horticulturae ( In Vitro Culture) 336: 257–262. El Mansouri, I., J.A. Mercado, V. Valpuesta, J.M. Lopez–Aranda, F. Pliego–Alfaro and M.A. Quesada. 1996. Shoot regeneration and Agrobacterium-mediated transformation of Fragaria vesca L. Plant Cell Reports 15: 642–646. Engelmann, F. 1997. In vitro conservation methods. Pp. 119–162 in Biotechnology and Plant Genetic Resources. Conservation and Use. B.V. Ford–Lloyd, H.J. Newburry and J.A. Callow (eds.). CABI, Wallingford, UK. James, D.J., A.J. Passey and D.J. Barbara. 1990. Agrobacterium-mediated transformation of the cultivated strawberry (Fragaria x ananassa Duch.) using disarmed binary vectors. Plant Science 69,:79–94. Kartha, K.K. and F. Engelmann. 1994. Cryopreservation and germplasm storage. Pp. 195– 230 in Plant Cell and Tissue Culture, I.K. Vasil and T.A. Thorpe (eds.). Kluwer Academic Publishers, Dordrecht. Nehra, N.S., R.N. Chibbar, K.K. Kartha, R.S.S. Datla, W.L. Crosby and C. Stushnoff. 1990. Agrobacterium-transformation of strawberry calli and recovery of transgenic plants. Plant Cell Reports 9, 10–13. Nishizawa, S., A. Sakai, Y., Amano and T. Matsuzawa. 1993. Cryopreservation of asparagus (Asparagus officinalis L.) embryogenic suspension cells and subsequent plant regeneration by vitrification. Plant Science 91: 67–73. Withers, L.A. 1985. Cryopreservation of cultured plant cells and protoplasts. Pp. 243–267 in Cryopreservation of Plant Cells and Organs. K.K. Kartha (ed.). CRC Press, Boca Raton, Florida. Wu, Y., F. Engelmann, A. Frattarelli, C. Damiano and L.A. Withers. 1997. Cryopreservation of strawberry cell suspension cultures. Cryo–Letters 18: 317–324. Posters 363

Current status of pollen cryopreservation research: relevance to tropical horticulture S. Ganeshan and R.K. Rajashekaran Pollen Storage Laboratory, Plant Genetic Resources Section, Indian Institute of Horticultural Research, Bangalore 560 089, India

Introduction Conservation of nuclear genetic diversity (NGD) using pollen is desirable in horticultural species for a variety of reasons. Cryopreserved pollen can be a major access point for pre-breeding germplasm lines, hybrid seed production, biotechnological and other basic studies. In the case of tree species, germplasm can be easily received and exchanged through pollen, eliminating a long juvenile phase. The objective of a useful pollen cryostorage protocol is to collect mature pollen from a plant and treat it so as to retain its normal function, ultimately assessed by its ability to germinate in vivo and effect normal fertilization (Hanna and Towill 1995). Alexander and Ganeshan (1993) reviewed the work on pollen storage in fruit crops. Hoekstra (1995) has assessed the merits and demerits of pollen as a genetic resource. Ganeshan and Rajasekharan (1995) reviewed work on ornamental crop pollen storage. Grout (1995) detailed the methodology for pollen cryopreservation. Recently, Barnabas and Kovacs (1997) and Berthaud (1997) stressed the importance and need for pollen conservation. The present paper aims to discuss the current status of pollen cryopreservation research and its relevance to tropical horticulture.

Materials and methods Protocols for pollen collection, viability assessment (pre- and post-storage), processing for cryopreservation, retrieval and fertility assessment followed at IIHR have been described in detail for fruit (Alexander and Ganeshan 1993; Ganeshan 1998), vegetables (Rajasekharan and Ganeshan 1994) and ornamental crops (Ganeshan and Rajasekharan 1995).

Results Responses to cryopreservation experiments obtained with pollen of 45 species belonging to 15 families are presented in Table 1. In some of the recently cryostored pollen, only feasibility tests were carried out. With most species, protocols are optimized for establishing pollen cryobanks.

Discussion Besides the existing role of pollen banks in breeding, there are many promising applications which have come to focus with the recent advances in allied bioscientific areas. Some of the practical utilities are discussed below. 364 Cryopreservation of Tropical Plant Germplasm

Redesigning crop breeding strategies Crossing desirable genotypes involves multiple and staggered plantings in order to synchronize flowering. This can be avoided when cryopreserved viable pollen is available, facilitating hybrids between genera, species and genotypes. This could effectively conserve field and greenhouse space. A pollen cryobank for a given crop can provide a constant supply of viable and fertile pollen and can also allow supplementary pollinations for improving seed set. The variability due to daily pollen collection can be nullified (Barnabas and Kovcas 1997). Male sterile populations can be perpetuated by cryostored maintainer pollen, thus avoiding frequent planting of maintainer line. Large-scale consolidation of potential pollen from male parents will ensure an uninterrupted supply of the male gametophyte for hybrid seed production at a given location, and pollen can be transported to different locations where seed parents are grown for crossing (Ganeshan and Rajasekharan 1995). Cryogenic technology applied to pollen conservation facilitates integration of conventional breeding methods with modern biotechnological practices. For example, prolonged pollen viability conferred because of cryogenic storage enables a pollen biotechnologist to carry out basic studies over extended durations on viable and fertile pollen. These studies could include genetic characterization of pollen, which allows more discrete use of pollen stocks for breeding. Another area of study could be to introduce known sequences of alien DNA in pollen with extended viability and fertility which could have an impact on the understanding of its integration with the gametophyte prior to use in breeding. Thus, where viable pollen is a limiting factor in such studies, cryopreservation can be a panacea.

Pollen as a component of genepool for international exchange of germplasm The international transfer of germplasm in the form of dry pollen is not generally restricted (Hoekstra 1995). Moreover, this will eliminate the need for growing plant populations to produce pollen. Pollen is subjected to less stringent quarantine restrictions. So, it can be easily shipped and used. Through exchange of pollen, desired crosses can be made directly on the seed parent, allowing introgression of characters at a much faster rate. This would find favour especially in breeding of tree species with a long juvenile phase.

Role of cryobiology in nuclear genepool management Conservation and management of NGD in plant species calls for constant cryobiological inputs in terms of material needed. These include cryobiological containers, constant supply of liquid nitrogen, its periodical replenishment, minimizing loss under ambience and above all, secure personnel safety. Stacking pollen samples in cryobiological systems needs to be done in sealed laminated poly pouches, either in gelatin capsules or in butter paper packets. It is important to maintain a proper inventory of pollen samples under cryopreservation. Improper pouch sealing may lead to bursting of the sample packet, leading to loss of pollen. Unless otherwise known, periodic viability checks need to be Posters 365

Table 1 – 2 landscape pages

[P-Table 1.doc] 366 Cryopreservation of Tropical Plant Germplasm Posters 367 carried out. It is preferable to cryopreserve samples with a high initial viability profile (Ganeshan 1998). To sum up, the science of cryobiology is the lifeline of a pollen cryobank, which conserves NGD of plant species (Grout 1995).

Extent of value addition contributed by cryopreserved pollen in genetic enhancement The availability of pollen with good quality in a pollen cryobank will provide a constant supply of the same for extended durations. Pollen in such a state can be termed "value added" by virtue of its potential extended life, for having been able to be kept viable and fertile for extended durations to perform its natural function of fertilization, leading to formation of fruit and seed set, e.g. pollen used in hybrid seed production. The process of genetic characterization further enhances the value of pollen, especially when it is established that it contains specific DNA sequences which are attributable to specific traits, e.g. pollen with marker genes (Ganeshan and Rajasekharan 1995).

Need for protection and grant of IPR to cryopreserved pollen IPR issues for value-added (cryopreserved, characterized) pollen achieve prominence since such pollen can be carried everywhere for use in crop breeding and genetic enhancement programmes. Gene-rich countries must take necessary steps to enact legislations and consider pollen material as a national bioresource, which could prevent illegal dealings like piracy, poaching, etc. once it is established that characterized pollen can be transported and used across political barriers. Such legislations should allow restricted accessibility and sustainable use of value-added pollen, giving due consideration to aspects such as sovereignity and process patents. Necessary initiatives are to monitor, survey, collect, conserve and characterize the NGD of economically important plant species endemic to their regions (Ganeshan 1998).

Acknowledgement The second author would like to thank IPGRI, Rome for the financial support for presenting the work at the International Workshop held in Japan, particularly Dr F. Engelmann, In Vitro Conservation Officer.

References Alexander, M.P. and S. Ganeshan. 1993. Pollen storage. Pp. 481–96 in Advances in Horticulture Vol.1 (Fruit crops Part 1), K.L. Chadha and O.P. Pareek (eds.). Malhotra Publishing House, New Delhi. Barnabas, B. and G. Kovacs. 1997. Storage of pollen. Pp. 293–314 in Pollen Biotechnology for crop Production and Improvement, K.R. Shivanna and V.K. Sawney (eds.). Cambridge University Press, UK. Berthaud, J. 1997. Strategies for conservation of genetic resources in relation with their utilization. Euphytica 96:1–12. Ganeshan, S. and P.E. Rajasekharan. 1995. Genetic conservation through pollen storage in ornamental crops. Pp.87–108 in Advances in Horticulture Part-1 Ornamental Crops, K.L. Chadha and S.K. Bhattacharjee (eds.). Malhotra Publishing House, New Delhi. 368 Cryopreservation of Tropical Plant Germplasm

Ganeshan, S. 1998. Pollen storage in tropical fruits. Pp.120–126 in Tropical Fruits in Asia: Diversity, Maintenance, Conservation and Use. Proceedings of the IPGRI–ICAR–UTFANET Regional Training Course on the Conservation and Use of Germplasm of Tropical Fruits in Asia, R.K. Arora and V.R. Rao (eds.). IIHR, 18–31 May 1997 Bangalore, India. Ganeshan, S. 1998. IPR for crop pollen diversity in India: Implications in seed trade and commerce. Journal of Palynology (in press.) Grout, B.W.W. (ed.) 1995. Genetic Preservation of Plant Cells In Vitro. Springer Verlag, Berlin. Hanna, W.W. and L.E. Towill. 1995. Long-term pollen storage. Pp.179–207 In Plant Breeding Reviews, J. Janick, (ed.). John Wiley and Sons, Chester, USA. Hoekstra, F.A. 1995. Collecting pollen for genetic resource conservation Pp. 527–550 in Collecting Plant Genetic Diversity. Technical Guidelines, L.Guarino, V. Ramanatha Rao and R. Reid (eds.). CAB International, Wallingford, U.K. Kozaki, I. 1975. Storage methods of pollen. Pp. 29–94 in JIBP Synthesis V Gene Conservation – Exploration, Collection, Preservation and Utilization, T. Matsuo, (ed.). Tokyo University Press, Tokyo. Rajasekharan, P.E. and S. Ganeshan. 1994. Pollen cryopreservation in vegetables as a th possible aid to heterosis breeding. Pp. 185–186 in Proceedings of 8 Kerala Science Congress, 27–29 January 1994, Thiruvanathapuram, India. Posters 369

Cryopreservation of maize (Zea mays L.) pollen Eiji Yamaguchi¹ and Masaya Ishikawa² ¹ National Center for Seeds and Seedlings,Minami Takaki, Nagasaki 859-1211, Japan ² National Institute of Agrobiological Resources, Tsukuba, Ibaraki 305-0856, Japan

Preservation of pollen is useful for pollination between cultivars that differ in the flowering period. Pollen of Gramineae crops, however, has a short viability and high sensitivity to desiccation, therefore they are difficult to conserve. Maize pollen is no exception. Here we attempted to cryopreserve pollen of three maize cultivars (CI64, B14×CI64, Colorado) using controlled desiccation. In the first series of experiments, two maize cultivars grown in the field were used as experimental materials. Fresh pollen shed from the anthers was dried under 50, 60, 70, 80 and 90% relative humidities (RH) at 6–7°C for 1 d. In the later experiments three maize cultivars grown in the field and in the glasshouse (GH, not air-conditioned) were used as experimental materials. Fresh pollen was dried under 70, 75 and 80% RH at 6–7°C for 1 d. Ependorf tubes (0.25 ml) about 1/4-filled with the dried pollen were sealed in vinyl bags using a hot sealer. Then the samples were immersed in liquid nitrogen (LN). Following 1 h of storage, the samples were rapidly rewarmed in water. The moisture content (MC) of pollen was determined by oven-drying at 104°C for 1 d and MC of pollen was calculated on a fresh weight basis. The viability of pollen was examined by observing germination on artificial media of 400–500 pollen grains per sample. The fertility of stored pollen was tested by determining the ability to set seeds as compared with that of fresh pollen. In the first series of experiments, we attempted to determine the optimum drying RH and MC for LN storage. Pollen of CI64 desiccated under 70 and 80% RH with MC of 14 and 17%, respectively, had the highest germinability of 24 and 53% following LN storage (Table 1). Desiccation to 70–80% RH also gave the highest survival following LN storage with pollen of B14×CI64. In the meantime, pollen desiccated to 50–60% RH had a MC of 10–12% but had reduced germinability (desiccation alone without LN storage). Pollen desiccated to 90% RH had a MC of 22–27%, retained high germination rate as fresh pollen, but had reduced germinability after LN storage. In further experiments on seed-setting using three cultivars, desiccation of pollen at 70–80% RH was employed. MC of pollen dried at 70–80% RH was in the range of 12.1–19.6%, depending on the cultivar and time and/or place (growing in the field and GH) of collection. Pollen with MC of 12.1–16.5% retained high germinability on artificial medium after LN storage, while the highest seed-setting rates following LN storage tended to be seen in pollen with MC around 16%. These results suggest that maize pollen can be successfully cryopreserved when dried at 6–7°C to suitable MC (14–17%) under appropriate RH (70–80%) depending on the cultivar and/or time and place of collection. Cryopreserved pollen retains fertility, as indicated by its ability to set seeds. 370 Cryopreservation of Tropical Plant Germplasm

Table 1. Germination and seed-setting rates of maize pollen collected from two sources and dried at different relative humidities Seed-setting rate Drying Moisture Germin- after LN storage cond- content ation rate (%, compared Place of ition (% after drying after LN with fresh collection Cultivar RH) (%) storage (%) pollen) Field CI64 70 14.6 5.8 8.3 B14 × CI64 75 16.5 19.8 25.5 Colorado 80 19.6 2.8 0.3 CI64 70 12.1 44.8 3.5 B14 × CI64 75 15.0 21.6 25.1 Colorado 80 16.7 10.5 25.7 CI64 70 13.3 4.1 1.3 B14 × CI64 75 14.5 15.7 0.9 Colorado 80 15.7 0.7 0.0 Glasshous CI64 70 15.9 20.5 34.6 e B14 × CI64 75 16.2 24.8 61.5 Colorado 80 18.6 20.8 10.8 CI64 70 14.0 15.6 8.5 B14 × CI64 75 15.7 21.9 26.4 Colorado 80 18.5 18.1 18.9 CI64 70 16.1 5.7 156.3 B14 × CI64 75 15.9 4.0 177.7 Colorado 80 17.3 4.2 13.4 Posters 371

Cryopreservation of somatic embryos of sweet potato by slow prefreezing method Kei Shimonishi¹, Minoru Karube¹ and Masaya Ishikawa² ¹ Kagoshima Biotechnology Institute, Kushira, Kagoshima, 893-1601 Japan ² National Institute of Agrobiological Resources, Tsukuba, 305-8602 Japan

Introduction Maintenance of plant genetic resources in field collections not only requires a lot of labour and large space but also has risks of viability loss due to pests and natural disasters. Cryopreservation of regenerative tissue, if successful, could be an effective tool for long-term preservation, especially with vegetatively propagated crops such as sweet potato (Ipomoea batatas), because it could reduce costs and risks. With sweet potato, which is an important root crop especially in southeastern Japan, a wide range of germplasm is preserved in the field to develop various types of cultivar at breeding stations. There are several reports of sweet potato cryopreservation using shoot-tips frozen by vitrification (Towill and Jarrett 1992), somatic embryos (Yoshinaga and Yamakawa 1994) and embryogenic tissue (Blakesley et al. 1996) frozen by the desiccation method. In this paper, we report cryopreservation of somatic embryos of sweet potato using a slow prefreezing method which enables high-viability storage in LN.

Materials and methods Somatic embryos of sweet potato (cultivar Kokei-14) were induced from apical meristems on MS medium with 3% sucrose containing 1 mg/L 2–4 dichlorophenoxyacetic acid (2,4–D) in the dark at 25°C. Masses of induced somatic embryo (SEMs) at early stages were divided into 3-mm masses prior to cryopreservation. In the first experiment, they were precultured with the following additives to determine which additive was effective to induce freezing tolerance; 10 mg/ml abscisic acid (ABA); 0.25M or 0.5M proline; 0.2M/0.7M sucrose. In the second experiment, SEMs were precultured with 5 mg/L ABA for 3 or 6 d to ensure the effect of ABA. Lastly, we compared the culture conditions after induction of somatic embryos including liquid media with 1 mg/L 2,4–D and/or 1–5 mg/L ABA prior to prefreezing. The precultured SEMs were treated with two cryoprotectant mixtures: · A: 10% (v/v) DMSO and 10% (w/v) sucrose, or · B: mixture (A) with 5% (w/v) glycerol (Ishikawa et al. 1991). Following ice inoculation, the specimens were cooled to –30°C at a cooling rate of 0.3°C/min using a programmable freezer, then plunged into liquid nitrogen. Following 1 d of storage, they were thawed rapidly in a 40°C water- bath and washed with 10% (w/v) and 3% sucrose solution, or placed on a filter paper to remove cryoprotectants prior to plating onto regeneration medium. Viability was determined by regrowth after reculture and the value was calculated based on the number of surviving SEMs versus the total number of 372 Cryopreservation of Tropical Plant Germplasm cryopreserved SEMs. Posters 373

Results and discussion No additives other than ABA were effective in achieving survival after storage in LN, and there was no clear difference between a 3-d and 6-d preculture duration. Plantlets were recovered by transferring surviving embryos onto regeneration medium, and they were morphologically normal in culture pots. Cryopreserved SEMs turned green within a few days of reculture at the earliest and embryos germinated, which indicated that tissues survived cropreservation as a whole, and microscopic observation supported this expectation. In this regard, it differed from the case of embryogenic callus of taro (Colocasia esculenta), for which regrowth after storage was very slow because only fragments of the tissues withstood cryopreservation (Shimonishi et al. 1993). When precultured with 5 or 10 mg/L ABA, the viability rate of somatic embryos was over 80% in most cases, and high viability was achieved in the case where somatic embryos were cultured in liquid medium containing 1 mg/L 2,4–D and 5 mg/L ABA as well (Fig. 1). This is similar to the report by Yoshinaga and Yamakawa (1994), in which ABA was used only at the somatic embryo-induction stage and no extra ABA treatments were performed prior to cryopreservation by the desiccation method. With sweet potato, ABA is often used to induce somatic embryos from embryogenic callus. Therefore, somatic embryos induced with ABA are considered to have already obtained freezing tolerance during induction, which means that no extra treatment is required to achieve freezing tolerance. Thus, somatic embryos of sweet potato could be cryopreserved with a high frequency of regeneration using a slow prefreezing method.

Germinated Regenerated Not Regenerated

ABA5+D1(L) D1(L) ABA5+D1(L)->ABA5 ABA1+D1(L)->ABA5 D1(L)->ABA5 D1(G)->ABA5 D1(G)

0 20 40 60 80 100

Viability / Regenerated and Germination rate (%)

Fig. 1. Effect of preculture on the viability and regeneration of cryopreserved somatic embryo masses (SEMs) of sweet potato. Total length of the bars represents the viability based on the number of masses. (G) and (L) stand for gellan gum medium and liquid medium with 1 mg/L 2,4–D, respectively. “–>ABA5” means preculture on a plate with 5 mg/L ABA. Viability of the SEMs precultured with ABA (–>ABA5) or cultured in a liquid medium with ABA were over 80%. 374 Cryopreservation of Tropical Plant Germplasm

References Blakesley, D., S.Al Mazrooei, M. H. Bhatti and G. G. Henshaw. 1996. Cryopreservation of non-encapsulated embryogenic tissue of sweet potato. Plant Cell Reports 15: 873–876. Ishikawa, M., P. Tandon, A.C. Yamaguishi and S. Miyazaki. 1991. Cryopreservation of bromegrass cells with slow prefreezing and rapid prefreezing methods. Proceedings of the Annual Meeting of Japanese Plant Physiologists, p. 96. Shimonishi, K., M. Karube and M. Ishikawa. 1993. Cryopreservation of taro (Colocasia esculenta) embryogenic callus by slow prefreezing. Japanese Journal of Breeding 43 (Supplement 2): 187. Towill, L.E. and R.L. Jarret. 1992. Cryopreservation of sweet potato (Ipomoea batatas (L.) Lam.) shoot tips by vitrification. Plant Cell Reports 11:175–178. Yoshinaga, M. and O. Yamakawa. 1994. Cryopreservation of sweet potato somatic embryos involving a desiccation step. Japanese Journal of Breeding 44 (Supplement 4): 290. Posters 375

Seeds of the African pepper bark (Warburgia salutaris) can be cryopreserved after rapid dehydration in silica gel Joseph Kioko¹, Patricia Berjak¹, Hugh Pritchard² and Matthew Daws² ¹ Plant Cell Biology Research Unit, School of Life and Environmental Sciences, University of Natal, Durban 4041 South Africa ² Royal Botanic Gardens Kew, Ardingly, West Sussex RH17 6TN, UK

Introduction Warburgia salutaris (Bertol.f.) Chiov. (= W. breyeri, W. ugandensis), a member of the family Canallaceae and commonly known as the pepper bark tree, is one of the most highly utilized medicinal plants in tropical and subtropical Africa. The bark has been shown to yield at least five sesquiterpenoid dialdehydes, including warburganal and polygodial (Hutchings 1996). That author reports that warburganal has broad antimicrobial activity against various yeasts and moulds and is a potent antifeedant against Spodoptera eempta, the African armyworm, while polygodial has been shown to enhance the antibiotic activities of actinomycin D, rifampicin and maesanin significantly. Bark from roots and stems is used by local communities as an expectorant, smoked for colds and coughs, or used for relief of gastro-intestinal disorders and skin complaints, among other ethnobotanical applications. The high demand (over 15 000 kg of bark per year in one market alone) and attendant overexploitation have made the species highly endangered and consequently almost extinct in the wild (Cunningham 1988). While seed storage would offer an efficient method for the conservation of this species, the seeds are parasitized by fungi and insects, and predated by birds and primates to such an extent that in some countries (e.g. South Africa) mature seed production in the wild is virtually unknown. There is also little, and inconclusive, available information on the post-shedding physiology and storability of the seeds (e.g. Albrecht 1993; Hutchings 1996). However, our preliminary results (unpublished) indicate that the seeds are non-orthodox, and therefore the conventional seed storage methods (e.g. storage at low water content and temperature) are not applicable to the conservation of the germplasm of W. saluratis. For such species, cryopreservation (storage at low temperatures, such as in liquid nitrogen at -196°C) is currently considered to be the only viable method. Cryopreservation immobilizes metabolic activities, thus suspending ageing and genetic variations (Kartha 1985), and also has low maintenance costs, providing a cost-effective means for the potential long-term storage of non- orthodox seed germplasm. However, most non-orthodox seeds are so large that they would cool far too slowly for successful cryopreservation (Berjak et al. 1996). This restricts cryopreservation to excised embryonic axes, and therefore necessitates the development of suitable techniques for the in vitro culture of embryonic axes, and the investigation of the response of embryonic axes to dehydration and cryopreservation (e.g. Kioko et al. 1998). 376 Cryopreservation of Tropical Plant Germplasm

Thus, the present study was aimed at establishing the response of embryonic axes and whole seeds of Warburgia salutaris to dehydration, and developing a method for cryopreservation of the germplasm of this species.

Materials and methods Fruits were harvested in Kenya during 1998 and stored at 16°C for up to 4 weeks, as they were still green on receipt (by airfreight) in Durban. Seeds were then extracted from the fruits, cleaned and subjected to the following treatments: · Rapid drying: Embryonic axes were excised from the seeds and placed on dry filter paper in a laminar airflow for up to 2 h. · Slower drying: Whole seeds were buried in activated silica gel at 25°C for up to 72 h. · Cryopreservation: Seeds were enclosed in 1-ml cryotubes (5 seeds per cryotube), and plunged into liquid nitrogen. They were maintained at -196°C for 1 h, after which the cryotubes were rapidly plunged into a water-bath at 35°C for 2 min to effect thawing of the seeds. · Sampling: At intervals, seeds or embryonic axes were sampled and tested for: (i) viability – germination (for whole seeds) and tetrazolium staining (for embryonic axes)

(ii) axis water content – determined in terms of g H2O per g dry matter (g/g) (iii) ultrastructural studies (transmission electron microscopy) (iv) freezing in liquid nitrogen (cryopreservation).

Results and discussion

The effect of dehydration on seed germination and ultrastructure of embryonic axes Fresh seeds had an average axis water content of about 2.4 g/g, and maintained viability to a water content as low as 0.1 g/g following drying in silica gel (Table 1). Ultrastructurally, there was no discernible damage in cells of the root meristems of embryonic axes from these seeds (Fig. 2A). However, as indicated in Figure 1, performance was less than optimal at the lower water contents. The rate of germination, represented in this figure by the slope of the curve, generally increased directly with the degree of dehydration, reaching a maximum at a water content of 0.3 g/g and dropping thereafter. In contrast to the response of seeds to slower drying, rapid dehydration of embryonic axes resulted in viability loss at relatively high water contents (Table 2). Thus, whereas seeds dried to 0.3 g/g over 48 h maintained a high germination percentage achieved more rapidly than fresh non-dried seeds, embryonic axes dehydrated to a similar water content over 20 min exhibited only 10% viability. Further rapid dehydration of the axes (over 90 min) to a similar water content to that attained by axes of seeds dried slowly over 72 h, resulted not only in total loss of viability, but also in complete ultrastructural damage (Fig. 2B). Posters 377 378 Cryopreservation of Tropical Plant Germplasm

Table 1. Changes in the water contents and germination of whole seeds of W. salutaris following drying in silica gel at 25°C Drying time (h) Axis water content (g/g) % germination 0 2.38 90 6 1.07 85 10 0.88 80 18 0.43 90 24 0.59 80 48 0.26 100 72 0.11 80

100

90

80

70

60

50 % germination 40

30

20

10

0 1 2 3 4 5 6 7 8 9 10 11 12 Weeks after sowing 2 g/g 0.5g/g 0.3g/g 0.1g/g

Fig. 1. Changes in percentage germination, over time, of seeds sown at different axis water contents achieved after drying in silica gel.

These results indicate that seeds of W. salutaris may possess mechanisms which modulate against desiccation damage, but that during rapid axis dehydration, there is insufficient time for these mechanisms to come into operation. Tolerance to slow, rather than rapid, dehydration is atypical of recalcitrant seeds (Potts et al. 1997; Kioko et al. 1998; Pammenter et al. 1998), but is observed in developing orthodox seed-embryos that have acquired the ability to withstand dehydration (Bochicchio et al. 1994). This response, coupled with the ability to withstand desiccation to 0.1 g/g, might imply that W. salutaris seeds are fully desiccation-tolerant, but, as optimal performance of these Posters 379 seeds occurred at 0.3 380 Cryopreservation of Tropical Plant Germplasm g/g, they are most likely not typically orthodox. It is possible that these seeds undergo indeterminate development, in which the degree of desiccation tolerance depends on the developmental stage at which the seeds are shed (Ellis and Hong 1996; Finch–Savage and Blake 1994). This aspect is the subject of further studies on this species.

Fig. 2. Ultrastructure of meristematic cells of: A. Embryonic axes from freshly harvested seeds dehydrated slowly (over 72 h) to a water content of 0.1 g/g. Cells had conspicuously abundant lipid bodies (L) and organelles appeared well constituted (n, nucleus; nu, nucleolus; ne, nuclear envelope). There was no apparent damage caused by dehydration. B. Embryonic axes rapidly dehydrated (over 90 min) to the same water content as in A. There was extensive subcellular derangement, with no organellar organization remaining. C. Embryonic axes from seeds dehydrated slowly to 0.1 g/g, cryopreserved in liquid nitrogen, and placed on germination medium for 10 d. Cells exhibited a high metabolic state, with frequent Golgi bodies (Gb), mitochondria with well-developed cristae (m), and profiles of rough endoplasmic reticulum (er).

Fig. 3. Germinating seeds of W. salutaris. The seeds were sown when either (A) freshly harvested and fully dehydrated, or (B) dehydrated slowly (over 72 h) to a water content of 0.1 g/g, or (C) dehydrated (slowly to 0.1 g/g) and cryopreserved in liquid nitrogen. Posters 381

Table 2. Changes in the water contents and viability of excised embryonic axes of W. salutaris following drying in a laminar airflow. The percentage of axes staining red with tetrazolium chloride was taken to indicate viability. Drying time (min) Water content (g/g) % viability 0 2.4 100 5 1.25 80 10 0.77 65 15 0.54 50 20 0.28 10 30 0.19 0 45 0.15 0 60 0.20 0 90 0.15 0 120 0.13 0

Cryopreservation of whole seeds The fact emerging from this study, that whole seeds could withstand more extreme dehydration than excised embryonic axes, implied that the seeds would be the more suitable materials for cryopreservation, as there would be less water available for ice formation during freezing. The use of whole seeds would also avert the problems associated with cryopreservation of excised embryonic axes, such as the development of appropriate embryo excision methods and in vitro culture techniques (Chin et al. 1988). Furthermore, work in this laboratory (unpublished) shows that the embryonic axes of W. salutaris are highly contaminated by fungi and bacteria, as is typical of non-orthodox seeds (Mycock and Berjak 1990; Berjak 1996), and that the axes are killed by all common surface-sterilants and antimicrobial agents at the concentrations which would eliminate the contaminants. Thus, cryopreservation trials were carried out only on whole seeds. Only seeds dehydrated to a water content of 0.1 g/g could withstand cryopreservation in liquid nitrogen (Table 3). After thawing and being placed in a germination medium (vermiculite) for 10 d, the ultrastructure of these seeds indicated a high metabolic rate, with frequent Golgi bodies, mitochondria with well-developed cristae, and profiles of rough endoplasmic reticulum (Fig. 2C). The seeds germinated within 6 weeks to a give final germination of 30%. The resultant seedlings appeared normal and similar to those from fresh non- frozen seeds (Fig. 3). The survival of only 30% of the seeds after freezing may be due to the variability in the maturity stages of the seeds at harvest, which was observed by ultrastructural studies. It is presently hypothesized that ongoing development not only increases the frequency of subcellular organelles, but also depletes lipid reserves. Both these factors might contribute to the increasing vulnerability of the more developed seeds to cryopreservation. 382 Cryopreservation of Tropical Plant Germplasm

Table 3. Survival of whole seeds, dried in silica gel to different axis water contents, after cryopreservation in liquid nitrogen % Germination after: Dehydration Water content Dehydration + period (h) (g/g) Dehydration alone cryopreservation 0 2.3 90 0 6 1.0 85 0 10 0.9 80 0 18 0.4 90 0 24 0.5 80 0 48 0.3 100 0 72 0.1 80 30

It is noteworthy that, despite optimal germination performance of seeds dried to 0.3 g/g, none survived cryopreservation at this water content. Thus, for effective conservation of the germplasm of W. salutaris as cryopreserved whole seeds, it seems that the water content must be below this presently indicated optimal of 0.3 g/g. Therefore the range of water contents between 0.3 g/g and 0.1 g/g needs to be explored to ascertain that which will optimize survival after cryopreservation.

Conclusion Seeds of Warburgia salutaris can withstand dehydration and freezing, so far with a recovery of 30% being attained. Since the ability to withstand desiccation (and, subsequently, cryopreservation) may depend on the stage of development at which the seeds are shed, further studies are being conducted to investigate the effect of both desiccation and cryopreservation on seeds harvested at different maturity stages. Secondly, because of the nearly extinct status of the species, there is an urgent need to develop appropriate seed storage methods. Thus, there are also ongoing investigations to ascertain whether the seeds can be stored at lower moisture contents, using conventional seed storage methods.

Acknowledgements This study was supported by the International Plant Genetic Resources Institute (IPGRI), and by the Foundation for Research Development (South Africa).

References Albrecht, J. (ed). 1993. Tree Seed Handbook of Kenya. GTZ Forestry Seed Centre, Muguga, Kenya. Berjak, P. 1996. The role of micro-organisms in deterioration during storage of recalcitrant and intermediate seeds. Pp. 121–126 in Intermediate / Recalcitrant Tropical Forest Tree Seeds. Proceedings of a workshop on Improved Methods for Handling and Storage of Intermediate / Recalcitrant Tropical Forest Tree Seeds, 8–10 June 1995, Humlebaek, Denmark, A.S. Ouédraogo, K. Poulsen and F. Stubsgaard (eds). IPGRI, Rome and DANIDA Forest Seed Centre, Humlebaek, Denmark. Posters 383

Berjak, P., D.J. Mycock, J. Wesley–Smith, D. Dumet and P.M. Watt. 1996. Strategies for in vitro conservation of hydrated germplasm. Pp. 19–52 in In Vitro Conservation of Plant Genetic Resources, M.N. Normah, M.K. Narimah and M.M. Clyde (eds.). Percetakan Watan Sdn.Bhd., Kuala Lumpur, Malaysia. Bochicchio, A., C. Rizzi, P. Vernieri and C. Vazzana. 1994. Sucrose and raffinose contents and acquisition of desiccation tolerance in immature maize embryos Seed Science Research 4: 123–126. Chin, H.F., B. Krishnapillay and Z.C. Alang. 1988. Media for embryo culture of some recalcitrant species. Pertanika 11: 357–363. Cunningham, A.B. 1988. Over-exploitation of medicinal plants in Natal/ Kwazulu: Root causes. Veld and Flora, September 1988: 85–87. Ellis, R.H. and H.T. Hong. 1996. Induction of desiccation tolerance in seeds. Pp. 127–135 in Intermediate / Recalcitrant Tropical Forest Tree Seeds.Proceedings of a workshop on Improved Methods for Handling and Storage of Intermediate / Recalcitrant Tropical Forest Tree Seeds, 8– 10 June 1995, Humlebaek, Denmark, A.S. Ouédraogo, K. Poulsen and F. Stubsgaard (eds). IPGRI, Rome and DANIDA Forest Seed Centre, Humlebaek, Denmark. Finch–Savage, W.E. and P.S. Blake. 1994. Indeterminate development in desiccation- sensitive seeds of Quercus robur L. Seed Science Research 4: 127–133. Hutchings, A. 1996. Zulu medicinal plants: an inventory. University of Natal Press, Pietermaritzburg, South Africa. Kartha, K.K. 1985. Meristem culture and germplasm preservation. Pp. 115–158 in Cryopreservation of plant cells and organs, KK Kartha (ed). CRC Press Inc., Boca Raton, Florida. Kioko, J., P. Berjak, N.W. Pammenter, M.P. Watt and J. Wesley–Smith. 1998. Desiccation and cryopreservation of excised embryonic axes of Trichilia dregeana. Cryo–Letters 19: 5–13. Mycock, D. and P. Berjak. 1990. Fungal contaminants associated with several homoiohydrous (recalcitrant) seed species. Phytophylactica 22: 413–418. Pammenter, N.W., V. Greggains, J.I. Kioko, J. Wesley–Smith, P. Berjak and W.E. Finch– Savage. 1998. Seed Science Research 8: 463–471. Potts, S.E. and T.A. Lumpkin. 1997. Cryopreservation of Wasabia spp. seeds. Cryo–Letters 18: 185–190. 384 Cryopreservation of Tropical Plant Germplasm

Effects of moisture content on Passiflora seed viability after immersion in liquid nitrogen J.A. Ospina¹, C.L. Guevara¹, L.E. Caicedo¹ and V. Barney² ¹ Genetic Resources Unit, CIAT, Cali, Colombia ² CIRAD–FLHOR/ IPGRI, A.A. 6713, Cali, Colombia

Introduction Liquid nitrogen (LN) storage is a very promising technique for long-term conservation of germplasm, and its advantages (e.g. low maintenance costs, suspension of aging and postponing regeneration) have motivated the development of cryopreservation protocols for seeds of many economically important species (Stanwood and Roos 1979; Stanwood 1985). The seed moisture content (MC) is probably the most critical factor in the definition of a successful cryopreservation protocol. As it relates to the composition of the seed, its storage behaviour and desiccation sensitivity imply precise levels of desiccation and tolerance to LN by species. Although several species of Passiflora (Passifloraceae; passion fruit) are important for fruit consumption, little research has been done to define seed storage behaviour and much less on cryopreservation (Ellis et al. 1985). The objectives of this study were to define ranges of tolerance and critical points for seed content through different methods of drying and to compare the effect of cryopreservation on seed viability of Passiflora edulis and P. ligularis at different seed moisture contents.

Materials and methods

Seed source and conditioning Mature fruits from Passiflora edulis Sims f. flavicarpa Degener and P. ligularis Juss. were obtained from a farmer and selected for similar size and weight. At the laboratory, seeds were conditioned by washing after aril extraction, then selected by density in water; the light fraction was discarded.

Desiccation and moisture content Four moisture content levels were obtained by drying under the following conditions: · 8 h under laboratory conditions (22°C and 40–50% air RH) · 5 d under laboratory conditions (22°C and 40–50% RH) · 5 d in the drying room (22°C and 20–30% RH) · 2 d over silica gel (22°C and 5–13% RH). Seeds were placed over a small screen in a sealed plastic tray containing 80 g of silica gel. After each desiccation period, seeds were removed for evaluation of germination, moisture content and cryoexposure. Moisture content was determinated gravimetrically in an oven at 130°C for 1 h (ISTA 1993) and expressed as percentage of fresh weight basis (f.w.b) with three replicates of 50 seeds. Posters 385

Cryoexposure of Passiflora seeds Seeds at different moisture contents were packed in vacuum-sealed trilaminar foil bags and directly dipped in LN held in a styrofoam cooler for 1 h (fast cooling). After cooling and before planting for germination, the seeds were thawed by two different methods: · Slow thawing (STHAWING) – seeds were rewarmed at room temperature · Fast thawing (FTHAWING) – sealed bags with seed were immersed for 1 min in a water-bath set at 37°C.

Evaluation

Germination test Germination tests were undertaken on three replicates of 100 seeds after applying treatments to break dormancy (Ospina, unpubl.). In the case of

P. ligularis, seeds were soaked in a GA3 solution at 500 ppm (24 h). The solution was then removed and the seeds left for 48 h before soaking them in sterile water for 24 h. Seeds were then placed in rolls of standard germination paper at laboratory temperature (22°C). For P. edulis, seeds were soaked in sterile water for 24 h, then the rolls were incubated in a germinator at 35°C/25°C (8- h/16-h), with a photoperiod of 16-h light/8-h dark. At the beginning of the test, the paper was wetted with a fungicide solution of Banrot 1% to avoid growth of saprophytic fungi. Evaluations were recorded for 4 weeks.

TTC test Before examining the seeds from the different treatments and since there were no standard patterns for testing viability with 2,3,5–Triphenyl–2H–tetrazolium chloride (TTC) on Passiflora seed, those were defined (Ospina, unpubl.). Seed was preconditioned for the test by nicking a small piece of seed coat from the distal embryo region, fracturing the seed coat with the aid of pliers and detaching at least 50% of it. Seeds were then immersed in the 0.5% (w/v) TTC solution for 24 h and finally rinsed and evaluated according to the pattern. Since dormancy was not fully overcome at the end of the germination test, the viability test was done on recovered seeds (non-germinated seeds). Thus, the evaluation of all treatments was made using the criteria of total viability (GERM+TTC) by adding to the number of normal seedlings the number of non- germinated but viable seeds according to the TTC test.

Results and discussion General results for moisture content and total viability of seeds of P. edulis and P. ligularis are given in Table 1.

Effects of drying After seed extraction and conditioning, the initial MC for P. edulis and P. ligularis was 21 and 30%, respectively. These values decreased, depending on the drying treatment and the species. After 8 h in the laboratory, the MC for P. 386 Cryopreservation of Tropical Plant Germplasm edulis and P. ligularis was 14.4 and 16.7%; after 5 d, MC was 7.7 and 8.8%, respectively. In the drying room, seed MC reached 5.8 and 5.9% for P. edulis and P. ligularis, respectively, while seeds which were exposed to the silica gel showed larger differences in their MC, with 2.6 and 4.0%, for P. edulis and P. ligularis, respectively (Table 1). Independently of the drying conditions, seeds of P. ligularis, being more hygroscopic, reached slightly higher MC values than P. edulis, thus suggesting differences in the seed composition between these species (Table 2).

Table 1. Effect of the drying conditions and thawing procedure on the total viability of cryopreserved P. edulis and P. ligularis seeds Total viability (%) Drying condition Cryo Cryo Species (time) † MC (%) No Cryo Sthawing Fthawing Passiflora edulis LC (8 h) 14.4 94.3 0.0 0.0 LC (5 d) 7.7 79.6 72.0 51.0 DR (5 d) 5.8 77.6 60.0 67.3 SG (2 d) 2.6 57.0 50.6 55.3 Passiflora ligularis LC (8 h) 16.8 97.6 0.0 0.0 LC (5 d) 8.0 89.6 79.3 89.3 DR (5 d) 5.9 86.0 53.6 60.0 SG (2 d) 4.0 81.3 56.0 68.0 † LC= Laboratory conditions, DR= Drying room, SG= Silica gel.

Table 2. Analysis of variance procedure Source of variation MC (%) Total viability (%) Species * ** Drying condition ** ** Method of thawing – NS Species x Drying condition NS ** Species x Method of thawing – ** Drying condition x Method of thawing – ** Species x Drying condition x Method of thawing – ** ** = highly significant (P<0.01), * = significant (0.010.05).

In relation to tolerance of drying, the evolution of total viability showed that both species were affected by a decrease in their MC. For P. edulis, the total viability decreased from 94.3 to 79.0% when MC dropped from 14.4 to 7.7% and increased to 57.0% for a MC of 2.5%. The total viability of seeds of P. ligularis varied from 97.6 to 89.0% when seed MC dropped from 16.7 to 7.9% and increased to 81.3% if MC was 4.0% (Table 1). The drying sensitivity observed with seeds of P. edulis confirms its intermediate behaviour, as suggested by Hong et al. (1996), while the lower sensitivity displayed by seeds of P. ligularis suggests a more orthodox behaviour. Posters 387 388 Cryopreservation of Tropical Plant Germplasm

Effect of cryopreservation Independently of the thawing method and for MC of 14.4 and 16.8% for P. edulis and P. ligularis, respectively, lethal values (0%) of germination and total viability were reported (Table 1). This suggests that these moisture contents are well above any high moisture freezing limit (HMFL) for these species. On the other hand, the higher total viability values (72 and 89.3%) obtained for P. edulis and P. ligularis respectively at 7.7 and 8.0% MC, imply the existence of a safe freezing MC for both species. Considering the MC at which higher values were observed, it is suggested that P. edulis has a safe freezing MC around 11%, while that of P. ligularis is around 9.0%. These differences also relate to differences in their drying sensitivity: P. edulis, being more sensitive to drying, showed a higher safe MC than P. ligularis. For the two species and with the majority of moisture content levels tested, the fast-thawing method resulted in better results than the slow thawing.

Conclusion Data suggest that the Passiflora seeds tested can tolerate desiccation to about 10.0% MC (f.w.b). However, each species has different levels of tolerance. The sensitivity of P. edulis seeds to drying confirms their intermediate behaviour, as suggested by Hong et al. (1996); to a lesser extent, the sensitivity of P. ligularis seeds suggests a more orthodox behaviour. As expected, seeds of none of the species tested were able to withstand immersion in LN at high moisture levels (15.0%). The higher total viability values obtained were around 8% for both species and a progressive decrease with lower MC values. It is also suggested that the safe critical value for P. ligularis is about 9.0% MC, while it is about 11.0% for P. edulis. For species with dormant seeds such as those used in this experiment, it is necessary to carry out some preliminary work to define dormancy breaking and germination pretreatments as well as patterns of viability using TTC.

References Ellis, R.H., T.D. Hong and E.H. Roberts. 1985. Handbook of seed technology for genebanks. Volume II. Compendium of specific germination information and tets recommendations. International Board for Plant Genetic Resources, Rome. Hong, T.D., S. Linington and R.H. Ellis. 1996. Seed storage behaviour: a Compendium. International Plant Genetic Resources Institute, Rome, Italy. ISTA (International Rules for Seed Testing). 1993. Seed Science and Technology 21, Supplement. Stanwood, P.L. 1985. Cryopreservation of seed germplasm for genetic conservation. Pp. 199–236 in Cryopreservation of Plant Cell and Organs, K.K. Kartha (ed.). CRC Press, Boca Raton, Florida. Stanwood, P.L. and E.E. Roos. 1979. Seed storage of several horticultural species in liquid nitrogen (–196°C). Hortscience 14: 625–630. Posters 389

Cold acclimation improves the cryopreservation of in vitro-grown Pyrus and Rubus meristems Yongjian Chang¹ and Barbara M. Reed² ¹ Department of Horticulture, Oregon State University, Corvallis, OR 97331, USA ² USDA–ARS National Clonal Germplasm Repository, Corvallis, OR 97333-2521, USA

Introduction Cold acclimatization (CA) is effective in improving survival of some cryopreserved tissues (Reed 1988, 1990). The improvement in cold tolerance is probably due to a variety of factors. There are many changes in the plasma membrane and cytoplasmic components such as sugars and proteins during cold acclimation which increase the freezing tolerance of plants (Guy 1990). Recent studies of cold acclimatization have shown that slow vegetative growth at low temperature is an important trait for winter survival of wheat plants in the field (Chun et al. 1998). There are no reports on how much CA is required for plants to reach a maximum freezing tolerance for cryopreservation (Reed and Chang 1997). Our objective was to determine if prolonged periods of CA would improve freezing tolerance and thus recovery from cryopreservation for several diverse Pyrus and Rubus genotypes.

Materials and methods Micropropagated shoots of eight Pyrus and two Rubus accessions were cultured on NCGR–PYR medium (Reed 1990) and NCGR–RUB medium (Reed 1993). Plantlets were given 0 to 15 weeks of cold acclimatization (CA) in an incubator -2 -1 with 22°C, 8-h days (3 µmol m s) and –1°C, 16-h nights before cryopreservation. Using the method developed by Reed (1990), excised meristems were pretreated for 2 d in the CA incubator on medium with 5% DMSO and additional Gelrite (0.3 g/L), then transferred to 0.25 ml liquid medium in 1.2-ml plastic cryotubes. One ml of the cryoprotectant PGD [10% each polyethylene glycol (MW 8000), glucose and DMSO in liquid medium] was added over a half-hour period (Finkle and Ulrich 1979). Samples were allowed to equilibrate at 4°C for 30 min after which the cryoprotectant was drawn down to 1 ml; the Pyrus were frozen at 0.1°C/min to -40°C and the Rubus at 0.5°C/min to –35°C and then plunged into LN. Samples were thawed for 1 min in 45°C water, then 1 min in 22°C water, rinsed in liquid medium and plated on recovery medium. Survival was assessed as growth of shoots at 6 weeks.

Results and discussion Shoot formation from cryopreserved meristems of R. parviflorus Nutt. and R. caesius L. significantly increased with increasing CA (Table 1). Rubus parviflorus fully acclimatized in 2 weeks and produced shoots on 82% of cryopreserved meristems. Rubus caesius required 6–10 weeks CA for shoot regrowth to reach 80%. Earlier, 1 week of CA was shown to double the 390 Cryopreservation of Tropical Plant Germplasm recovery of Rubus meristems from cryopreservation (Reed and Lagerstedt 1987; Reed 1988). Our new data indicate that genotypes which respond poorly or not at all to 1 week of CA can be further acclimatized and produce high recovery following cryopreservation. Posters 391

Table 1. Effects of cold acclimatization duration on cryopreservation of in vitro-grown Pyrus and Rubus meristems % meristem regrowth (mean ± S.D.) Cold acclimatization duration (weeks) Genotypes 0 1 2 3 4 6 10 15 Pyrus cordata 0 0 10 – 10±4 22±3 – 100 P. pashia 17±3 30±13 30±9 – 65±13 73±13 – – Rubus caesius 0 0 8±3 22±6 – 65±20 80±17 – R. parviflorus 0 22±3 82±8 73±13 – 93±12 90±5 – Cold acclimatization duration conditions: 22°C, 8-h days (3 µmol m-2 s-1) and –1°C, 16-h nights. Means of three or more replications.

One week of cold acclimatization significantly increased shoot formation of cryopreserved Pyrus koehnei Schneider, P. communis L. cvs. Beurré d’Amanlis Panachée and Monchallard, and P. calleryana Decne. from <20% to >70%. This is similar to results published earlier for other Pyrus genotypes (Reed 1990). Further CA of these genotypes did not result in improved recovery following LN exposure. Regrowth of meristems of several pear genotypes following cryopreservation remained below 40%. Increasing the length of CA to 4–6 weeks significantly improved the regrowth rates of P. pashia Buch.–Ham. ex D. Don, and P. communis cv. Beurré Bosc and a hybrid P. communis x P. pyrifolia (Burm. f.) Nakai from below 20% with no CA to >75% after 4 or 6 weeks CA (Table 1). Pyrus cordata Desv. improved from 0 to 20% with 6 weeks CA, but lengthening the cold period to 15 weeks resulted in 100% regrowth of cryopreserved meristems.

Conclusions Storage of living tissues of plants in liquid nitrogen may be improved by several techniques. Cold acclimatization of plants before cryopreservation involves growing the plant under conditions of short cool days and long cold nights prior to freezing. Both pears and blackberries had improved survival following cryopreservation when cold acclimatized. Some pear cultivars were greatly improved by 1–15 weeks of cold acclimatization while others were improved after 1 week, but no improvement was noted for further cold treatment. Blackberries required 2–10 weeks of acclimatization, but 4 weeks provided adequate improvement for most genotypes.

References Chun, J.U., X.M. Yu and M. Griffith. 1998. Genetic studies of antifreeze proteins and their correlation with winter survival in wheat. Euphytica 102:219–226. Guy, C.L. 1990. Cold acclimation and freezing stress tolerance: role of protein metabolism. Annual Review Plant Physiology and Plant Molecular Biology 41:187–233. Finkle, B.J. and J.M. Ulrich. 1979. Effects of cryoprotectants in combination on the survival of frozen sugarcane cells. Plant Physiology 63:598–604. Reed, B.M. 1988. Cold acclimation as a method to improve survival of cryopreserved Rubus meristems. Cryo–Letters 9:166–171. 392 Cryopreservation of Tropical Plant Germplasm

Reed, B.M. 1990. Survival of in vitro-grown apical meristems of Pyrus following cryopreservation. HortScience 25:111–113. Reed, B.M. 1993. Responses to ABA and cold acclimation are genotype dependent for cryopreserved blackberry and raspberry meristems. Cryobiology 30:179–184. Reed, B.M. and Y. Chang. 1997. Medium- and Long-Term Storage of In Vitro Cultures of Temperate Fruit and Nut Crops. Pp. 67–105 in Conservation of Plant Genetic Resources In Vitro, Vol. 1. M.K. Razdan and E.C. Cocking (eds.). Science Publishers, Inc., Enfield, NH, USA. Reed, B.M. and H.B. Lagerstedt. 1987. Freeze preservation of apical meristems of Rubus in liquid nitrogen. HortScience 22:302–303. Posters 393

Replacement of cold acclimatization with high sucrose pretreatment in black currant cryopreservation Dominique Dumet¹, Yongjian Chang², Barbara M. Reed³ and Erica E. Benson¹ ¹ Plant Conservation Biotechnology Group, Division of Molecular and Life Sciences, School of Sciences and Engineering, University of Abertay-Dundee, Dundee DD1 1HG, Scotland ² Department of Horticulture, Oregon State University, Corvallis OR 97331, USA ³ United States Department of Agriculture, Agricultural Research Service, National Clonal Germplasm Repository, Corvallis, OR 97333-2521, USA

Introduction Successful cryopreservation of black currant meristems can be achieved after either vitrification (with or without controlled freezing rate) or encapsulation- desiccation (Benson et al. 1996). Whatever the method used, the very first step of the protocol is a cold acclimation of the 3 to 4-week-old plantlets. Such treatment requires the presence in the laboratory of a cold acclimation chamber which can be a limiting factor for the cryopreservation protocol development. In some instances, it has been shown that soluble sugars (sucrose + raffinose) imbibition of plant tissues can replace the effect of the cold period, increase cold-hardiness and impart desiccation tolerance (Stushnoff et al. 1998). In this study we investigated whether or not a high sucrose conditioning of apical shoots can mimic the effect of the cold acclimatization of plantlets when black currant meristems are cryopreserved via encapsulation-desiccation.

Materials and methods Black currant plantlets were subcultured at monthly intervals on a modified Murashige and Skoog (1962) medium (Rib MS) containing one-third of the original ammonium and potassium nitrate concentration, 20 g/L glucose, 0.1 mg/L N6–benzyladenine, 0.2 mg/L gibberellic acid and 6 g/L agar. The standard growth conditions were a photoperiod of 16-h light/8-h dark and a temperature of 25°C. Cold-hardening was applied to the whole plantlets by transferring them to a controlled environment chamber with 8-h light at 22°C/16-h dark at –1°C. For high sucrose conditioning, apical shoots showing a few leaves were subcultured on solid 0.75M sucrose Rib MS medium under standard conditions. After a 7-d cold- or sucrose-conditioning period, meristems were encapsulated in medium with 3% alginate and 0.75M sucrose, then transferred to 0.75M liquid Rib MS medium for 20 to 22 h before desiccation for 4 h under the laminar flow hood. Desiccated beads were introduced in cryovials and plunged directly into liquid nitrogen. After slow thawing at ambient temperature, the encapsulated meristems were transferred to standard Rib MS medium for recovery assessment. Survival after desiccation or desiccation + cryopreservation was tested on two different cultivars, Ben Lomond and Ojebyn (5–20 meristems per condition, three replicates per cultivar). Three to 4-week-old cultures were used in this experiment. 394 Cryopreservation of Tropical Plant Germplasm

Results and discussion Very high survival rates were obtained after desiccation with both cultivars, ranging from 93 to 100% (Table 1). Survival after cryopreservation was equally high; indeed, in the three replicates performed with cultivar Ben Lomond, all the desiccated meristems survived after cryopreservation when they were excised from high sucrose conditioned shoots. Survival rate was slightly lower (95%) when meristems were sampled on cold-hardened plantlets. With cultivar Ojebyn, survival after cryopreservation was high (90–91%) whatever the preconditioning treatment. A Tukey’s pairwise comparison test showed no significant effect of either the treatment or the cultivar and all the survivors regenerated shoots.

Table 1. Survival (%) after desiccation or desiccation+cryopreservation of meristems excised from cold-hardened or sucrose-conditioned plant tissues of two black currant cultivars, Ben Lomond and Ojebyn Survival (%) after: Sucrose conditioning † Cold conditioning Cultivar DH DH + LN DH DH + LN Ben Lomond 100 100 100 95±8 Ojebyn 93±11 91±10 100±0 90±5 † DH = desiccation only, LN = cryopreservation.

This experiment showed that when cryopreservation of black current meristems is performed using encapsulation-desiccation, cold-hardening treatment of plantlets may be replaced by high sucrose conditioning of the shoots. Such replacement may be very useful in laboratories which do not possess a specialized cold-acclimation room. It would be interesting to test the sucrose-conditioning effect on other plants that are traditionally cold-hardened before cryopreservation. It is well known that cold-hardening results in endogenous changes in the plant tissues, which contribute to improvement of their freezing tolerance (Stushnoff et al. 1998). Further experiments must be performed to determine whether the improvement of tolerance to cryopreservation induced by high sucrose conditioning of shoots is due to a massive absorption of sucrose within the meristem or is the result of the osmotic stress developed by the high-sucrose medium. Indeed, sucrose is well known for its ability to improve survival of plant tissues to desiccation (Koster and Leopold 1988) and cryopreservation (Monnier and Leddet 1978; Dumet et al. 1994). The osmotic stress due to the high sucrose concentration could also result in endogenous changes similar to those induced by the cold-hardening treatment.

References Benson, E.E., B.M. Reed, R.M. Brennan, K.A. Clacher and D.A. Ross. 1995. Use of thermal analysis in the evaluation of cryopreservation protocols for Ribes nigrum L. germplasm. Cryo–Letters 17:347–362. Dumet, D., F. Engelmann, N. Chabrillange, S. Dussert and Y. Duval. 1994. Effect of various sugars and poly-ols on the tolerance to desiccation and freezing of oil palm Posters 395

polyembryonic cultures. Seed Science Research 4:307–313. Koster, K.L. and A.C. Leopold. 1988. Sugars and desiccation tolerance in seeds. Plant Physiology 88:829–832. Monnier, M. and C. Leddet. 1978. Sur l’acquisition de la résistance au froid des embryons immatures de Capsella bursa–pastoris. Comptes Rendus de l’Académie des Sciences, Paris 287:615–618. Murashige, T. and F. Skoog. 1962. A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiologia Plantarum 15:473–497. Pannetier, C., P. Arthuis and D. Liévoux. 1981. Néoformations de jeunes plantes d’Elaeis guineensis à partir de cals primaires obtenus sur fragments foliaires cultivés in vitro. Oléagineux 36:119–122. Stushnoff, C., M.J. Seufferrheld and T. Creegan. 1998. Oligosaccharides as endogenous cryoprotectants in woody plants. Pp. 301–309 in Plant Cold Hardiness, Li and Chen (eds.). Plenum Press, New York. 396 Cryopreservation of Tropical Plant Germplasm

Structural observations on potato shoot-tips after thawing from liquid nitrogen Ali M. Golmirzaie, Ana Panta and Cecilia Delgado CIP, Lima, Peru

Introduction Since 1995, the International Potato Center (CIP) has been applying a vitrification method for the cryopreservation of potato apical shoot-tips. Because the development of this technology and its application to all CIP's mandate crops are among CIP goals, research to improve the method and to achieve efficient long-term conservation of germplasm is continuous work at CIP. Currently, 75% of cryopreserved genotypes are successfully recovered. The average survival rate of recovered genotypes is 40%. In order to increase the recovery rate, the following assay was performed. A structural study of frozen-thawed shoot-tips was undertaken to define types of cellular damage caused by cryopreservation with the vitrification method. Determination of the type and cause of cell damage, modifications in freezing and thawing process would help in increasing the survival rate.

Procedures Four genotypes of Solanum stenotomum were taken from the long-term in vitro potato collection held at CIP. The CIP numbers of these genotypes are 703774, 703838, 702421 and 703709. Plants were micropropagated by single-node stem segments in magenta jars containing the potato propagation medium used at CIP (Golmirzaie and Panta 1997). The medium was poured into magenta jars in two layers. First, semi-solid medium (15 ml) was poured, replacing the gelling agent phytagel with agar. Then nine plant single nodes were planted. After 4–5 d, liquid MSA medium (10 ml) was added. Magenta jars were covered with a transparent polypropylene film with a filter (SIGMA, C6920). Apical shoot-tips (1.5 mm long) consisting of 4–5 leaf primordia and the apical dome, were isolated from in vitro-propagated plantlets. They were processed by vitrification, the cryopreservation method that is being adopted at CIP (Golmirzaie and Panta 1997). After thawing, shoot-tips were treated for electronic microscopy analysis following the protocol used by the Electronic Microscopy Unit at CIP (CIP 1997). Control treatments using shoot-tips from non–vitrified samples and vitrified but not frozen were included. Tissue slides 60–90 nm thick were fixed on 200-mesh grids covered with formvar. Samples were evaluated by observing at least 10 microscopy fields per treatment. Cells with and without damage symptoms were counted.

Results and discussion The ultrastructural analysis revealed that all assayed potato genotypes suffered damage during cryopreservation by the vitrification method. The symptoms observed with the highest percentage (Table 1) were abnormal cytoplasm aspect, cell plasmolysis at different stages and a large number of small vesicles. Posters 397

Symptoms of cell wall rupture and protoplast outflow were observed infrequently, not more than 5.2 and 6.8% in vitrified samples without and with freezing, respectively. Anomalous nucleus shape was observed mainly in frozen samples. No such damages were observed in non-treated material (controls). Damages related to cytoplasm aspect and cell plasmolysis seem to affect more the survival rate. Genotypes that had the higher percentage of these symptoms showed the lower survival rate. The highest percentage of cytoplasm with anomalous aspect, cell wall rupture and protoplast outflow was observed with genotype 703838. These damages would be responsible for the low survival rate of vitrified and frozen material. More studies are needed to know if nucleus shape alteration is overcome during post-thaw culture and if the cell function is not modified. Results stressed that the response to vitrification method is genotype dependent. A slight increase in the duration of the rehydration period (after thawing) and extremely careful manipulation of samples during thawing may allow reduction of damages. Assays to test this hypothesis are in progress.

Table 1. Percentage of cellular damage symptoms observed in vitrified shoot-tips of four genotypes of Solanum stenotomum by ultrastructure analyses under electronic microscopic in relation to survival rate Abnormal Cell plasmolysis cytoplasm Excessive Survival aspect V VF vesiculation rate (%) † ‡ § ¶ Genotype V V F L M L M V VF V VF CIP703774 22.5 53.4 13.5 3.7 32.5 8.9 37.1 60.3 62.5 18.0 CIP703838 47.0 75.1 27.4 6.7 47.5 7.0 37.6 44.6 50.0 6.1 CIP702421 16.1 69.9 12.0 1.7 10.8 5.1 34.0 60.4 47.5 26.9 CIP703709 0.0 31.1 24.7 1.0 10.1 8.7 41.6 36.1 45.0 35.4 † Control treatments, samples processed by vitrification without going through freezing. ‡ Samples frozen after treatment in vitrification solution. § Light cell plasmolysis symptom. ¶ Moderate cell plasmolysis symptom.

References Golmirzaie, A.M. and A. Panta. 1997. Advances in potato cryopreservation by vitrification. CIP Program Report. International Potato Center (CIP). Lima, Peru. Pp. 71–76. Centro Internacional de la Papa (CIP). 1997. Técnicas de Virología de Plantas. L.F. Salazar, U. Jayashinge (eds.). Manual de Capacitación. Lima, Peru. 398 Cryopreservation of Tropical Plant Germplasm

Cryopreservation of pineapple (Ananas comosus) apices by vitrification María Teresa González–Arnao¹, Manfred Márquez Ravelo², Caridad Urra 4 Villavicencio¹, Marcos E. Martínez Montero³ and Florent Engelmann ¹ Centro Nacional de Investigaciones Científicas, Cubanacán, Playa, La Habana, Cuba ² Universidad de la Habana, Fac. de Biología, La Habana, Cuba ³ Centro de Bioplantas-UNICA, CP 69450, Ciego de Avila, Cuba 4 IPGRI, 00145 Rome, Italy

Introduction Pineapple is a fruit crop of major importance in many tropical countries. Pineapple is vegetatively propagated and crosses between varieties produce botanical seeds. However, these seeds are highly heterozygous and therefore of limited interest for the conservation of specific gene combinations. Cryopreservation of apices is the most relevant strategy for long-term conservation of vegetatively propagated crops. The freezing methods employed are principally encapsulation-dehydration and vitrification, which do not require sophisticated equipment for freezing and produce high recovery rates with a wide range of materials (Engelmann 1997). As reported in this paper, vitrification and encapsulation-dehydration were used to freeze apices of in vitro plantlets of pineapple. The most efficient protocol was applied to apices of three different varieties.

Materials and methods The plant material consisted of apices sampled on in vitro plantlets of three pineapple [Ananas comosus (Stickm.) Merr.] varieties (Puerto Rico, Perolera and Smooth Cayenne). Plantlets were subcultured every 30 d and apices (up to 3 mm in length) dissected 15 d after the last subculture. For cryopreservation experiments, the encapsulation-dehydration technique and the vitrification procedure were applied. The experimental approach has been described in detail by González–Arnao et al. (1998).

Results and discussion Encapsulation-dehydration did not allow successful cryopreservation of pineapple apices under the conditions tested. These negative results can be related to the high sensitivity of pineapple apices to sucrose and dehydration. Indeed, pregrowth in media with sucrose concentrations higher than 0.5M was detrimental to survival and a prolonged treatment in 0.5M sucrose was required to improve survival after desiccation. The viability loss observed after freezing may be due to the crystallization of remaining intracellular freezable water upon freezing. This detrimental effect might be avoided by slowly cooling the encapsulated apices to allow freeze-induced dehydration to take place. Several plant species such as potato, grape and citrus cryopreserved by the encapsulation-dehydration technique have required a slow freezing regime Posters 399 to achieve optimal survival (Fabre and Dereuddre 1990; Plessis et al. 1993; González–Arnao et al. 1998). In contrast, survival of pineapple apices after cryopreservation was achieved using the vitrification technique, which has been successfully employed for freezing apices of a large number of different crops (Niino et al. 1992a, 1992b; Matsumoto et al. 1994; Kuranuki and Sakai 1995; Tagaki et al. 1997). Optimal conditions for vitrification of pineapple apices included a 2-d preculture on semi-solid MS medium supplemented with 0.3M sucrose, loading treatment for 25 min in medium with 0.75M sucrose + 1M glycerol and dehydration at 0°C for 7 h with the PVS2 vitrification soultion before rapid immersion in liquid nitrogen. The originality of the protocol developed with pineapple apices was the extended duration (7 h) of treatment with PVS2 required to achieve optimal survival of apices after freezing, in comparison with the much shorter optimal durations (around 1–1.5 h) suggested for most materials (Sakai et al. 1990; Matsumoto et al. 1994). This result is certainly due to the large size and compact structure of the pineapple apices employed in our experiments: the apices were around 3 mm long, and the apical dome was tightly covered by 2– 3 leaf primordia with a very thick cuticle. Extended treatment durations were therefore needed for the vitrification solution to sufficiently dehydrate these very compact structures. The vitrification protocol developed was sucessfully employed for cryopreserving apices of a total of eight pineapple varieties (Table 1).

Table 1. Effect of vitrification protocol on survival of apices from eight different pineapple varieties before (–LN) and after (+LN) cryopreservation. After loading with 1M glycerol and 0.75M sucrose for 25 min, apices were treated with PVS2 solution at 0°C for 7 h before freezing. (Reprinted from Gonzalez–Arnao et al. 1998, with permission). Survival (%) Variety –LN +LN Puerto Rico 80 65 Perolera 50 35 Smooth Cayenne 50 25 Cabezona 63 30 Piña Blanca 60 27 P3R5 53 20 Bromelia 33 10 Española Roja 47 13

Acknowledgement The authors gratefully thank IPGRI for the technical and financial support provided.

References Engelmann, F. 1997. In vitro conservation methods. Pp. 119–162 in Biotechnology and Plant Genetic Resources: Conservation and Use, B.V. Ford–Lloyd, J.H. Newburry, J.A. Callow, Eds., CABI, Wallingford, UK. 400 Cryopreservation of Tropical Plant Germplasm

Fabre, J. and J. Dereuddre. 1990. Encapsulation-dehydration: a new approach to cryopreservation of Solanum shoot-tips. Cryo–Letters 11: 413–426. González–Arnao, MT., M. Marquez Ravelo, C. Urra Villavicencio, M. Martinez Montero and F. Engelmann. 1998. Cryopreservation of pineapple (Ananas comosus) apices. Cryo– Letters 19: 375–382. Kuranuki, Y. and A. Sakai. 1995. Cryopreservation of in vitro grown shoot-tips of tea (Camellia sinensis) by vitrification. Cryo–Letters 16: 345–352. Matsumoto, T., A. Sakai and K. Yamada. 1994. Cryopreservation of in vitro grown apical meristems of wasabi (Wasabia japonica) by encapsulation-vitrification method. Plant Cell Reports 13: 442–446. Niino, T., A. Sakai, S. Enomoto, J. Magoshi and S. Kato. 1992a. Cryopreservation of in vitro grown shoot-tips of mulberry by vitrification. Cryo–Letters 13: 303–312. Niino, T., A. Sakai, H. Yakuwa and K. Nojiri. 1992b. Cryopreservation of in vitro grown shoot-tips of apple and pear by vitrification. Plant Cell Tissue and Organ Culture 28: 261– 266. Plessis, P., C. Leddet, A. Collas and J. Dereuddre. 1993. Cryopreservation of Vitis vinifera L. cv. Chardonnay shoot tips by encapsulation-dehydration: effect of pretreatment, cooling and postcultured conditions. Cryo–Letters 14: 309–320. Sakai, A., S. Kobayashi and I. Oiyama.1990. Cryopreservation of nucellar cells of navel orange (Citrus sinensis Osb. var. brasiliensis Tanaka) by vitrification. Plant Cell Reports 9: 30–33. Tagaki, H., N.T. Thinh, O.M. Islam and T. Senboku. 1997. Cryopreservation of in vitro grown shoot-tips of taro (Colocasia esculenta (L.) Schott) by vitrification. 1. Investigation of basic conditions of the vitrification procedure. Plant Cell Reports 16: 594–597. Posters 401

Research on cryopreservation of mango shoot-tips Xue–Lin Huang, Jie–Ning Xiao, Xiao–Ju Li, Yun–Feng Cheng and Jia–Rui Fu Department of Biology, Zhongshan University, Guangzhou, China

Introduction During the last 10 years, new cryopreservation techniques have been developed which allow the use of shoot apices, zygotic and somatic embryos for long-term germplasm conservation. Up to now, apices of more than 40 species have been successfully employed for cryopreservation (Bajaj 1995). Mango is a typical recalcitrant species whose seeds are extremely sensitive to desiccation. Experiments have shown that mango embryos could withstand desiccation to 12% moisture content, but could not survive after freezing in liquid nitrogen (Fu and Liang 1997). Cryopreservation of mango germplasm using embryonic axes is therefore impossible to envisage. Thus, alternative methods using explants other than embryonic axes should be explored. In this study, we investigated the possibility of using shoot-tips sampled from mature trees or young seedlings as the initial explants for cryopreservation.

Materials and methods

Selection of explants and sterilization methods for in vitro culture To determine the optimal sterilization conditions, shoot-tips sampled from mature trees or from 1-year-old seedlings were treated with several combinations of sterilizing treatments. The sterilized shoot-tips were rinsed at least five times with sterile water, cut to a size of about 0.5 cm and used as initial explants. Explants were cultured on MS (Murashige and Skoog 1962) basal medium containing 0.3% active charcoal. Culture conditions were a temperature of 27°C and a photoperiod of 12-h light /12-h dark.

Preliminary cryopreservation experiments Shoot-tips from 1-year-old or 18-d-old seedlings of mango cultivar Zi–Hua were sterilized with 75% ethanol for 30 s, then with 0.1% HgCl2 for 8 min, and rinsed in sterile water at least five times. The shoot-tips (1.5 to 2 mm long) with two to four leaf primordia were used as explants. Before cryopreservation, the explants were pretreated on solid (0.8% agar) MS medium with different sucrose concentrations at 4°C or at room temperature (30°C) for 1 d, and on semi-solid (0.4% agar) MS medium containing 13% sucrose or 0.2 mM putrescine and 0.2 mM spermidine or 50 µM abscisic acid (ABA) at 4°C for 1 or 7 d. The TTC (triphenyltetrazolium chloride) test was used for rapid estimation of shoot-tip viability after the different treatments. Fresh shoot-tips were used as viable controls; boiled ones as dead controls. The pretreated shoot-tips were then treated with the PVS2 vitrification solution and cryopreserved as described by Niino et al. (1992). After cryopreservation, shoot- tips were transferred to MS medium for assessment of survival. 402 Cryopreservation of Tropical Plant Germplasm

Results Shoot-tips sampled from mature trees were heavily contaminated (up to 100% contamination after 7–10 d in culture) even though various disinfectants and sterilization combinations were employed. Contamination of shoot-tips sampled from young seedlings could be controlled by spraying the mother- plants with the fungicide sporgon 7 d before samples were collected. The shoot-tips survived well on MS medium without addition of plant growth regulators. However, they grew very slowly and were difficult to multiply. Glucose at the concentration of 40% could improve growth of explants. This improvement was the same, with or without the addition of growth regulators in the medium. The TTC test allowed a good and rapid estimation of viability after the various treatments. The optical density value of viable shoot-tips was about 0.450 and that of non-viable shoot-tip lower than 0.200 under our experimental conditions. During preliminary experiments, it was found that the pretreatment conditions, and especially the temperature, strongly influenced viability of the shoot-tips. Shoot-tips were viable when they were precultured on MS medium containing successively 0.1, 0.3 and 0.5M sucrose for 5 h and 14 h at 4°C, but they lost viability when pretreated at room temperature (30°C). Shoot-tips pretreated with spermidine and putrescine or ABA were more resistant to chilling than those without such pretreatments. If shoot-tips remained viable after sucrose pretreatment, both treatment with PVS2 and immersion in liquid nitrogen resulted in rapid browning and death of all shoot-tips.

Discussion and conclusion Shoot-tips sampled from mature trees were very difficult to use for in vitro culture and cryopreservation because they were seriously contaminated. The contaminating fungus was a white mold identified as a Fusarium. Shoot-tips sampled from seedlings survived well when cultured on MS medium without the addition of plant growth regulators but they grew very slowly. Adding glucose at the concentration of 40% in the medium improved growth of explants. It has been shown that carbohydrates play an important role in the organogenesis of plant in vitro cultures (Welander and Pawlicki 1994). TTC test proved to be a sensitive method for rapid estimation of viability of shoot-tips at each step of the cryopreservation protocol. All compounds employed for pretreatment – including sucrose, glycerol, ABA, spermidine and putrescine – were not very effective in ensuring survival of shoot-tips after cryopreservation. PVS2 treatment also caused serious damage to shoot-tips. Additional experiments, including modifications in the pretreatment conditions and testing other cryopreservation techniques, are under way to achieve successful cryopreservation of mango shoot-tips. Posters 403

References Bajaj, Y.P.S. 1995. Cryopreservation of plant cell, tissue and organ culture for the conservation of germplasm and biodiversity. Pp. 3–28 in Biotechnology in Agriculture and Forestry Vol. 32. Cryopreservation of Plant Germplasm I (Bajaj, Y.P.S. ed.), Springer–Verlag, Berlin, Heidelberg. Fu, J.R. and Y.H. Liang. 1997. Cryopreservation of mango excised axes and regeneration of plantlets from buds and stem segments. Activity Report 1997. International Plant Genetic Resources Institute, Rome, Italy. Murashige, T. and F. Skoog. 1962. A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiologia Plantarum 15: 473–497. Niino, T., A. Sakai, S. Enomoto, J. Magoshi and S. Kato. 1992. Cryopreservation of in vitro- grown shoot tips of mulberry by vitrification. Cryo–Letters 13:303–312. Welander, W. and N. Pawlicki. 1994. Carbon compounds and their influence on in vitro growth and organogenesis. Pp. 83–93 in Physiology, Growth and Development of Plants in culture (P.J. Lumsden, J.R. Nicholas and W.J. Davies eds.). Kluwer Academic Publishers, Dordrecht. 404 Cryopreservation of Tropical Plant Germplasm

Cryopreservation of melon shoot primordia cultures using a slow prefreezing procedure Rie Ito–Ogawa¹, Masaya Ishikawa², Eiko Niwata³ and Katsuji Oosawa² ¹ Aichi Agricultural Research Center, Sagamine, Yazako, Aichi 480-1103, Japan ² National Institute of Agrobiological Resources, Kan’nondai, Tsukuba, Ibaraki 305- 8602, Japan ³ Aomori Green BioCenter, Yamaguchi, Nogi, Aomori 030-0142, Japan

Introduction In the case of melon, shoot primordia cultures which consist of numerous apical meristems and callus-like tissue can regenerate a large number of shoots. It is thus an effective method for mass propagation. Cryopreservation of shoot primordia cultures would provide a stand-by supply of inocula for mass propagation and conserve the genetic integrity of the germplasm stored. In the present study, we attempted to cryopreserve cultured shoot primordia of melon using a slow prefreezing procedure.

Materials and methods

Induction of shoot primordia Shoot primordia cultures were initiated by inoculating shoot apices of melon (Cucumis melo L. cv. prince melon) in Murashige and Skoog liquid medium containing sucrose (3%), benzylaminopurine (1 mg/L) and naphtalene acetic acid (0.01 mg/L). They were subcultured in standard medium (MS medium containing 3% sucrose and 1 mg/L BA at 3-week intervals). Cultures were incubated at 25°C under continuous light with cool-white fluorescent illumination (3000 lux) on a slanted rotary incubator revolving at 2 rpm.

Preculture and cryopreservation procedures Three-week-old cultured shoot primordia clumps were dissected into pieces of 1–2 mm or 2–3 mm of diameter and precultured for 3 d in standard medium. The precultured shoot primordia were then placed in 10-ml glass centrifuge tubes. One ml of cryoprotective solution was added directly to specimens at one time and they were incubated at room temperature for 0.5–3 h. Samples were then cooled at 0.5°C/min down to –8°C, at which temperature crystallization was induced in the cryoprotective medium and held for 30 min at –8°C. Subsequently, samples were cooled at 0.3 to 1.0°C/min down to prefreezing temperatures ranging from –25 to -40°C, held for 20 min at the prefreezing temperature, then immersed in liquid nitrogen (LN).

Thawing and reculture After 1 h storage in LN, samples were thawed rapidly in 40°C water. The cryoprotective medium was diluted 5-fold at room temperature by adding 3% (w/v) or 10% (w/v) sucrose, either slowly (drop by drop addition over 20 to 30 min) or rapidly (direct addition in one time). Posters 405

Cryopreserved shoot primordia were transferred on regeneration medium (MS gellan gum medium with 3% sucrose and 0.2 mg/L BA and 0.2–1.0 mg/L gibberellic acid, GA3). Viability was represented by the percentage of individual shoot primordia clumps which were green after 1 month of reculture. Shoot regeneration was also scored after 1 month of reculture. Viability was also checked by staining 50–100 mm sections of shoot primordia clumps with FDA after 2 weeks of reculture.

Results The cryoprotective solution termed CSP1 (Ishikawa et al. 1991, 1996) comprising 10% (w/v) sucrose, 10% (w/v) DMSO and 5% (w/v) glycerol, gave the highest viability (80%) and shoot regeneration rates (30%). The absence (0%) or high (10%) concentrations of glycerol in the cryoprotective medium resulted in decreased viability (36%). The different cooling rates tested (0.3, 0.5 and 1.0°C/min) from –8°C to – 30°C gave comparably high survival rates. To make the protocol less time- consuming, a cooling rate of 1.0°C/min can be used for cryopreservation of melon shoot primordia. The prefreezing temperatures tested (–25 to 40°C) gave fairly high survival, but the optimum was at –30°C, which gave 80–90% viability and 70–80% shoot regeneration rates. Preculture of shoot primordia clumps in standard medium for 3 d following dissection increased survival after cryopreservation. Samples incubated for 30 min to 1 h showed highest viability and regeneration rates after cryopreservation. Soaking for 3 h was toxic to the shoot primordia and reduced survival. Large clumps (2–3 mm) incubated in CSP1 for 30 min gave the best survival. The highest viability and regeneration of cryopreserved shoot primordia were obtained when CSP1 was diluted slowly with 3% sucrose at room temperature. When dilution was performed rapidly with 3% or 10% sucrose at room temperature, viability was nil and 22%, respectively. As a diluent for the dropwise slow dilution, 3% sucrose gave higher viability and shoot regeneration than 10% sucrose. When cryopreserved shoot primordia clumps were recultured on semi-solid medium, they regenerated shoots as vigorously as unfrozen controls within 1 month. FDA staining of sections of cryopreserved shoot primordia after 2 weeks of reculture revealed that clumps of shoot primordia had both stained apices (radiating fluorescence) and totally unstained apices. When cryopreserved shoot primordia clumps were placed in standard medium and cultured at 25°C under light for 1 month, they regrew and regained the original characters of melon shoot primordia cultures.

Discussion and conclusion We developed a cryopreservation protocol for cultured shoot primordia of melon (Cucumis melo L. cv. prince melon) using a slow prefreezing (two-step) method. Under the optimal conditions, more than 80% of the primordia clumps remained viable and shoot regeneration rates reached 60–80%. The 406 Cryopreservation of Tropical Plant Germplasm choice of an appropriate cryoprotective solution (CSP1), the dilution method (slow dilution with 3% sucrose at room temperature) and the use of glass containers were critical to obtain high survival rates. Preculture of dissected shoot primordia pieces for 3 d prior to cryopreservation improved survival. A relatively wide range of incubation durations in CSP1 (30 min to 2 h), of cooling rates (0.3 to 1.0°C/min) and prefreezing temperatures (–30 to –40°C) resulted in high survival.

References Ishikawa M., P. Tandon, A. Yamaguishi and S. Miyazaki. 1991. Cryopreservation of bromegrass cells using a rapid prefreezing and a slow prefreezing method. Proceedings of the Annual Meeting of Japanese Plant Physiology, 96. Ishikawa M., P. Tandon, M. Suzuki and A. Yamaguishi–Ciampi. 1996. Cryopreservation of bromegrass (Bromus inermis Leyss) suspension cultured cells using slow prefreezing and vitrification procedures. Plant Science 120: 81–88. Posters 407

Survival of dried axillary buds of micropropagated redgrass Manabu Katano and Yoshihiro Masuya Department of Agronomy, School of Agriculture, Kyushu Tokai University, Choyo- son, Aso-gun, Kumamoto 869-1404, Japan

Introduction We formerly succeeded in cryopreserving field-grown dormant shoot-tips suspended in distilled water or in aqueous solution with cryoprotectants of Malus domestica cv. Fuji (Katano et al. 1983), Prunus jamasakura (Katano 1990), Prunus yedoensis (Katano and Iryie 1991) and Enkianthus perulatus (Sakane and Katano 1997) and micropropagated apple cv. Fuji (Katano et al. 1989), which are popular deciduous fruit or ornamental trees, as well as field-grown Hordeum vulgare cv. Nishino–Gold (Katano 1990) and Trifolium repens (Katano 1990) after prefreezing at 0.5°C/min by addition of powdered dry ice to –30 or –40°C in an ethanol bath. Uragami et al. (1990) succeeded with the direct immersion in LN of axillary buds of Asparagus officinalis grown in vitro after culture on solid medium with 0.7M sucrose followed by dehydration with silica gel to replace freeze-induced dehydration. Yang and Katano (unpubl.) demonstrated that 100% of carrot embryogenic calluses induced from hypocotyls (Jeon et al. 1986), subcultured every 3 weeks for 3 years and precultured at 4°C for 3 weeks could survive after direct immersion in LN after desiccation to 9% water content, achieved with silica gel. However, the above- mentioned species were winter crops or deciduous trees originating from temperate zones. The objective of this study was to assess the possibility of freezing axillary buds of micropropagated redgrass (Alternanthera sessilis var. orforma), an aquarium Liliacean plant with origin in tropical Asia, using the protocol developed by Uragami et al. (1990).

Materials and methods Stem segments, 5 mm long, with one axillary bud of redgrass micropropagated through repeated subculture in vitro on 0.2% Gelrite solidified basic medium (BM) with LS inorganic and organic nutrients, 1 mg/L benzylaminopurine (BAP) and 3% (0.09M) sucrose were precultured on medium containing LS nutrients with 3% sucrose (Control) or with 25% sucrose (0.73M, high osmotic pressure; HOP) during 48 h, then transferred on 500-mm nylon mesh to 60x15 mm Petri dishes containing 15 g of silica gel for up to 24 h (Uragami et al. 1990) followed by immersion in LN. After rapid rewarming, stem segments were cultured on basal medium and survival was assessed after 10 d. The water content of stem segments was also measured in each treatment.

Results and discussion The water content of control stem segments drastically decreased and reached 87, 31, 16, and 6% after desiccation with silica gel for 0, 4, 5 and 6 h, respectively, while the water content of HOP stem segments was 70, 40, 18, 13, 408 Cryopreservation of Tropical Plant Germplasm

11 and 9% after 0, 4, 8, 12, 16 and 20 h desiccation, respectively (Table 1). The survival of axillary Posters 409

Table 1. Effects of preculture with 3% or 25% sucrose on the water content of single node segments and the survival of axillary buds of micropropagated redgrass Time in silica Water content Tested Survival † Preculture gel (h) (% FW) (no.) (no. /%) None (Control) 0 87 20 20 / 100 4 31 20 14 / 70 3% (0.09M) 5 16 20 4 / 20 Sucrose for 2 d at 25°C 6 6 20 0 / 0 8 8 20 0 / 0 12 8 20 0 / 0 16 6 20 0 / 0 20 6 20 0 / 0 24 5 20 0 / 0

Preculture 0 70 20 20 / 100 4 40 20 18 / 90 25% (0.73M) 8 18 20 13 / 65 Sucrose for 2 d at 25°C 12 13 20 0 / 0 16 11 20 0 / 0 20 9 20 0 / 0 24 7 20 0 / 0 † Survival was checked 10 d after the experiment. buds was closely related to the water content of stem segments. The water content at LD50 (representing half of axillary buds being alive) was estimated at 30% for Control and 18% for HOP. The critical water content of Asparagus stem segments was around 20% (Uragami et al. 1990); however, no survival of axillary buds of redgrass after immersion in LN could be achieved with either Control or HOP. Factors other than water content are also involved in the survival of tropical species like redgrass.

References Katano, M. 1990. In vitro culture of shoot-tips and their cold and freezing tolerance and the possibility of cryopreservation in liquid nitrogen. A project report for Grant-in-aid for Scientific Research (C) (No. 63560018) supported by the Ministry of Education in 1990. Katano, M. and R. Irie. 1991. Shoot-tip culture of Japanese flowering cherry (Prunus yedoensis Matsum.) and possible cryopreservation of shoot-tip in liquid nitrogen. Proceedings of the Faculty of Agriculture of Kyushu Tokai University 10:17–27. Katano, M., A. Ishihara and A. Sakai. 1983. Survival of dormant apple shoot-tips after immersion in liquid nitrogen. HortScience 18:707–708. Katano, M., A. Ishihara and A. Sakai. 1989. Survival of apple shoot tips cultured in vitro after immersion in liquid nitrogen. Japanese Journal of Breeding: 34 (Suppl. Issue 1): 212– 213. Sakane, I. and M. Katano. 1997. Shoot-tip culture of Enkianthus perulatus Schneid. and cryopreservation of shoot-tip in liquid nitrogen. Proceedings of the Faculty of Agriculture of Kyushu Tokai University 16:55–67. Uragami, A., A. Sakai and N. Nagai. 1990. Cryopreservation of dried axillary buds from plantlets of Asparagus officinalis L. grown in vitro. Plant Cell Reports 9:328–331. 410 Cryopreservation of Tropical Plant Germplasm

Cryopreservation of in vitro-grown shoot-tips of cassava cooled to –196°C by vitrification R. Charoensub¹, S. Phansiri¹, Akira Sakai² and W. Yongmanitchai¹ ¹ Scientific Equipment Center, Kasetsart University Research and Development Institute, Bangkok 10900, Thailand ² Asabu 1-5-23, Sapporo 001, Japan

Introduction Cassava is an important food crop in the tropics as it provides edible starch. According to 1995 statistics, Thailand is the largest cassava exporter in the world. Thus, much research has been done on cassava, especially breeding programmes, with the intention of minimizing costs and increasing efficiency of production. At present, more than 200 cassava cultivars are conserved in Thailand. An in vitro genebank has been developed at Kasetsart University, in which nearly 130 cultivars are maintained under slow-growth conditions. Problems of in vitro conservation include high costs of maintaining culture stocks, space problems and risks of contamination and somaclonal variation with increasing storage time. Cryopreservation appears to be a logical choice for long-term storage of cassava germplasm. Recently, cryopreservation of shoot-tips of cassava using a conventional slow-freezing method was reported (Escobar et al. 1997). However, this procedure is very complicated, time- consuming and gives low rates of recovery. The vitrification technique has been employed to simplify handling of explants and has secured a high level of recovery with numerous species. To our knowledge, there are no reports of successful cryopreservation of cassava by vitrification. This research was carried out to develop a suitable method for cryopreservation of cassava.

Materials and methods In vitro plantlets of cassava (Manihot esculenta Crantz) cv. CM3281–4 were used in this study. Stock cultures were maintained on Murashige and Skoog (1962) medium supplemented with 100 mg/L myo–inositol, 1 mg/L thiamine

HCl, 0.02 mg/L benzyladenine (BA), 0.1 mg/L gibberellic acid (GA3), 0.01 mg/L naphtalene acetic acid (NAA), 3% sucrose and 7.5 g/L agar–agar at pH 5.6. They were subcultured every 2 months and were grown at 25°C under a light intensity of 1500 lux with a 12-h light/12-h dark photoperiod. Shoot-tips of about 1 mm in length were dissected from 2-week-old plantlets and precultured on solidified MS medium containing 0.3M sucrose for 16 h, 2 d or 3 d. Precultured shoot-tips were treated with MS medium containing 2M glycerol + 0.4M sucrose (LS solution) for 20 min at 25°C. After removal of the LS solution, the shoot-tips were treated with PVS2 solution (Sakai et al. 1990) at 25°C for various durations. The shoot-tips were finally suspended in 0.5 ml of PVS2 in cryotubes and plunged into LN where they were kept for at least 2 h. After rapid rewarming in water at 45°C for about 1 min, the PVS2 solution was drained and replaced with MS medium containing 1.2M sucrose for 20 min. Shoot-tips were then transferred on sterilized filter paper discs placed Posters 411 over the culture medium in Petri dishes. After 1 d, they were again transferred to fresh medium. Shoot formation was recorded as the percentage of shoot-tips forming normal shoots 5 weeks after plating. Ten shoot-tips were tested for each of three replications.

Results and discussion It has been shown that increasing the dehydration tolerance to PVS2 (osmotolerance) of excised shoot-tips is the key for successful cryopreservation by vitrification. The preculture step considerably increased the rate of shoot formation of vitrified shoot-tips. However, shoot-tips precultured with 0.3M sucrose for 2–3 d showed a significant decrease in their rate of shoot formation compared with those precultured with 0.3M sucrose for 16 h. Treatment with the LS solution significantly improved the rate of shoot formation of vitrified shoot-tips (Table 1). During the treatment with LS solution, cells of shoot-tips are osmotically dehydrated and plasmolyzed. These cells are successively further dehydrated with PVS2 to be capable of vitrifying upon rapid cooling in LN. The osmoprotection produced by the LS solution may be explained by the mitigation of injurious effects during the dehydration process by decreasing osmostress and stabilizing membranes (Jitsuyama et al. 1997). The shoot formation of vitrified shoot-tips increased gradually with increasing time of exposure to PVS2 and reached a maximum (75%) for 45-min exposure, followed by a gradual decrease. The shoot-tips treated with PVS2 for up to 45 min without cooling in LN (treated control) retained high rates of shoot formation (about 81.8%). However, longer exposures decreased the shoot formation to nearly the same level as that of vitrified shoot-tips. Successfully vitrified shoot-tips resumed growth within 1 week and developed normal shoots without intermediary callus formation. Almost all the shoot-tips formed roots and were successfully transplanted. The vitrification protocol is easy to handle, not time-consuming and also gives a high rate of recovery growth. Further studies are necessary to apply this technique for a wide range of cassava germplasm. It is very interesting to note that in vitro shoot-tips of cassava, a tropical dicotyledon plant, were successfully cryopreserved using nearly the same vitrification protocol used for shoot-tips of both wasabi (Matsumoto et al. 1994) and several tropical monocotyledon plants (Thinh 1997). In view of the wide range of efficient and simple vitrification protocols available, many tropical plants could be amenable to cryopreservation, provided that the tissue culture protocols for apical meristems are sufficiently developed. 412 Cryopreservation of Tropical Plant Germplasm

Table 1. Effect of preculture and osmoprotection on shoot formation of cassava shoot-tips cooled to –196°C by vitrification Preculture Osmoprotection Shoot formation (% ±SD) None None 3.3 ± 4.7 0.3M sucrose 16 h None 56.7 ± 12.5 None LS solution 46.7 ± 17.0 0.3M sucrose 16 h LS solution 86.7 ± 4.7 Excised shoot-tips were precultured with 0.3M sucrose + MS medium for 16 h, then treated with LS solution (0.4M sucrose + 2M glycerol). They were dehydrated with PVS2 solution for 45 min at 25°C prior to direct immersion in LN. About 10 shoot-tips were tested for each of three replications.

Acknowledgements This study was supported by Kasetsart University Research and Development Institute, Thailand, under the Genetic Engineering and Biotechnology Research Program.

References Escobar, R.H., G. Mafla and W.M. Roca. 1997. A methodology for recovering cassava plants from shoot tips maintained in liquid nitrogen. Plant Cell Reports 16:474–478. Jitsuyama, Y., T. Suzuki, T. Harada and S. Fujikawa. 1997. Ultrastructural study of mechanism of increased freezing tolerance to extracellular glucose in cabbage leaf cells. Cryo–Letters 18:33–44. Matsumoto, T., A. Sakai and K. Yamada. 1994. Cryopreservation of in vitro-grown apical meristems of wasabi (Wasabia japonica) by vitrification and subsequent high plant regeneration. Plant Cell Reports 13:442–446. Murashige, T. and F. Skoog. 1962. A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiologia Plantarum 15: 473–497. Sakai, A., S. Kovayashi and I. Oiyama. 1990. Cryopreservation of nucellar cells of navel orange (Citrus sinensis Osb. var. brasiliensis Tanaka) by vitrification. Plant Cell Reports 9: 30–33. Thinh, N.T. 1997. Cryopreservation of germplasm of vegetatively propagated tropical monocots by vitrification. Doctoral Papers of Kobe University, Department of Agronomy, Japan. Posters 413

Cassava cryopreservation – I Roosevelt H. Escobar¹, Graciela Mafla² and William M. Roca¹ ¹ Biotechnology Research Unit, CIAT, Cali, Colombia ² Genetic Resources Unit, CIAT, Cali, Colombia

Introduction Vegetative propagation, bulkiness of planting material and risks of genetic erosion make cassava an ideal candidate for the application of innovative germplasm conservation methods. In vitro-grown shoot-tips of cassava were successfully cryopreserved by programmed slow freezing (Escobar et al. 1997). Several factors contributed to successful cryopreservation of cassava apices, including tissue desiccation, shoot size and pretreatment with sorbitol and DMSO.

Objective To develop a cryopreservation protocol that enables establishment of an in vitro-based genebank (IVBG) at CIAT for the long-term conservation of cassava genetic resources.

Methodology (Adapted from Escobar et al. 1997)

1. Small shoot-tips (2–3 mm long) were dissected from 3 to 4-month-old in vitro cultures. 2. Pretreatment of shoot-tips on CIAT–4E medium (Roca 1984) for 3 d. 3. Preculture on solid C4 medium for 3 d, in the dark, at 26–28°C. 4. Cryoprotection in liquid medium for 2 h, on ice. 5. Surface drying for 1 h on filter paper at room temperature.

6. Programmed freezing using a Cryomed 1010 to –40 °C. 7. Immersion and storage in liquid nitrogen for at least for 3 h. 8. Thawing at 37°C, for 45 s. 9. Re-culture steps: A. Equilibrium media R1 and R2 for 2 d each; B. transfer to semi-solid CIAT–4E medium, 10. Evaluation: tissue viability (tissue survival) and shoot formation (plant recovery) after 1 month.

Media composition -7 · CIAT 4E: Full MS salts (Murashige and Skoog 1962), 1.7x10 M BAP, -7 -7 -6 -4 1.44x10 M GA3, 1.07x10 M NAA, 2.96x10 M thiamine–HCl, 5.55x10 m–inositol, 0.058M sucrose, agar 0.7%, pH 5.7–5.8. -8 -8 -6 · CIAT 17N: 1/3 MS salts, 2.88x10 M GA3, 5.37x10 M NAA, 2.96x10 M -4 thiamine–HCl, 5.55x10 myo–inositol, 0.058M sucrose, 25 mg/L ®Plantex (10:52:10) agar 0.7%, pH 5.7–5.8. · Pretreatment C4: 4E medium, 1M sorbitol, 0.1M DMSO, 0.11M sucrose. · Cryoprotection solution: 1M sorbitol, 1.28M DMSO, 0.11M sucrose pH 5.7–5.8. 414 Cryopreservation of Tropical Plant Germplasm

· Recovery R1: 0.75M sucrose, 0.2% activated charcoal, agar 0.7%, pH 5.7– 5.8. -3 · Recovery R2: ½ MS salts, 0.35M sucrose, 5.56x10 M m–inositol, agar 0.7% pH 5.7–5.8. · Recovery 4E ss: 4E with ½ agar concentration.

Results and discussion Using the above protocol we were able to recover viable cassava plants (50– 60%) from shoot-tips frozen in liquid nitrogen using cultivar MCol 22. Thereafter, the technique was reproduced with several cultivars, representing a wide geographic distribution (Escobar et al. 1997) (Table 1).

Table 1. Survival rate of control (–LN) and cryopreserved (+LN) shoot-tips of different cassava cultivars Shoot recovery (%) Cassava cultivar –LN +LN MCol 22 100 52.2 CM 922-2 100 48.6 MArg 2 100 37.4 MEcu 48 100 21 MPar 193 100 14 MPan 125 100 13 MCub 27 100 11 MCol 1468 100 10 MDom 2 100 3 MCR 113 100 3 MGua 14 100 2 MBra 161 100 0 MPer 303 100 0 MVen 232 100 0 MMex 71 100 0

Cassava shoot-tips proved sensitive to high osmotic pressure, with DMSO and sucrose being detrimental when used at high concentration. When sorbitol was used at high concentration, the morphogenic response was modified, callus formation becoming the most common response after freezing. Some other cvs (MVen 232 and MMex 71) improved their response when the osmotic concentration was adjusted in the preculture media (Escobar et al. 1992) (Table 2). It seems that the cryopreservation response can be related to the edaphoclimatic origin of cassava. The best-responding cultivars (MCol 22 and CM 922-2) are drought-tolerant, and cultivar MArg 2 is well adapted to subtropical conditions. Cultivars MDom 2, MVen 232 and MMex 71 showed high sensitivity to osmotic concentration, manifested through bleaching before freezing (Escobar et al. 1997). Posters 415

Table 2. Effect of preculture medium on recalcitrant cassava cultivars after freezing in LN MCol 22 MMex 71 MVen 232 † Medium % viability % shoots % viability % shoots % viability % shoots 1 88 72 40 8 32 18 2 84 56 32 4 40 20 3 92 52 4 0 5 0 † 1: 0.5M sorbitol / 0.01M DMSO / 0.1M sucrose; 2: 0.5M sorbitol / 0.001M DMSO / 0.25M sucrose; 3: 1M sorbitol / 0.1M DMSO / 0.1M sucrose.

Our results show that the use for long periods of preculture media containing high osmotic concentration reduces shoot recovery even prior to freezing. The variation of cassava cultivars in response to cryopreservation can be linked to stress factors involved in the process. Homogenizing the age and size of explants under normal growth conditions prior to freezing can help identify genotypic responses. Preculturing shoot-tips on 4E medium increased survival and plant recovery after freezing (Escobar et al. 1994). The use of cytokinins in the recovery medium influenced the response after freezing, with kinetin being (Fig. 1) more effective than 2iP, BAP, TDZ and adenine. When the BAP concentration was increased, a drastic effect on shoot recovery was noted (Escobar et al. 1995). Recovered plants were rooted on 17N medium (Roca 1984) and placed in soil for monitoring performance and stability.

Fig. 1. Effect of cytokinin on plant recovery from cassava shoot-tips cryopreserved in LN (cv. MCol 22). 416 Cryopreservation of Tropical Plant Germplasm

Conclusion We have been able to consistently recover cassava plants from cryopreserved shoot-tips. We have reproduced the methodology with cultivars representing a wide geographic distribution. Several factors contributed to successful cryopreservation, including tissue desiccation, shoot-tip size and pretreatment with sorbitol and DMSO. The response of recalcitrant cultivars to cryopreservation could be improved by employing lower concentrations of osmoticums in the preculture medium.

References Escobar, R.H., G. Mafla and W.M. Roca. 1994. Cryopreservation for long term conservation of cassava genetic resources. Pp. 190–193 in First International Scientific Meeting of the Cassava Biotechnology Network, Bogor, Indonesia, 22–26 August 1994, CBN II. Vol 1. W.M. Roca and A.M. Thro (eds.). CIAT, Cali, Colombia. Escobar, R.H., G. Mafla and W.M. Roca. 1997. A methodology for recovering cassava plants from shoot tips maintained in liquid nitrogen. Plant Cell Reports 16:474–478. Escobar, R.H., W.M. Roca and C. Guevara. 1995. Cryopreservation of cassava shoot tips in liquid nitrogen. Pp. 82–84 in Biotechnology Research Unit Annual Report. CIAT, Cali- Colombia. Escobar, R.H., W.M. Roca and G. Mafla. 1992. Cryopreservation of cassava shoot tips. Pp. 116–121 in First International Scientific Meeting of the Cassava Biotechnology Network. Cartagena, Colombia 25–28 August 1992 CBN I. W.M. Roca and A.M. Thro (eds.). CIAT, Cali, Colombia. Murashige, T. and F. Skoog. 1962. A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiologia Plantarum 15: 473–497. Roca, W.M. 1984. Cassava. Pp. 269–301 in Handbook of Plant Cell Culture: Crop Species. Vol 2. (W.R. Sharp, D.A. Evans, R.V. Ammirato and Y. Yamada (eds.). MacMillan Publ. New York. Posters 417

Cassava cryopreservation – II Roosevelt H. Escobar¹, Juan D. Palacio¹, Maria P. Rangel¹ and William M. Roca¹ ¹ Biotechnology Research Unit, CIAT, Cali, Colombia

Introduction The development of a simple and reliable method for cryopreservation would allow more widespread use of this technique with different cassava genotypes. Variability in survival due to genotypic differences can be overcome by choosing an appropiate method (Reed and Yu 1995). New cryopreservation techniques are based on vitrification, i.e. the transition of water directly from liquid phase into an amorphous phase or glass, whilst avoiding the formation of crystalline ice (Towill 1996). Four cryopreservation procedures are based on vitrification: encapsulation-dehydration, desiccation, pregrowth-desiccation and vitrification.

Objectives · To develop a practical protocol using rapid freezing of cassava shoot-tips. · To develop the encapsulation-dehydration technique as an alternative to the current cassava cryopreservation technique.

Results and discussion

Rapid freezing Considering the large number of accessions in the world cassava germplasm collection maintained at CIAT, the rapid freezing technique would not only expedite the process, but make it more operational in terms of cost per accession (Escobar and Roca 1997). Using direct immersion in liquid nitrogen, no differences were found in comparison with the programmed freezing protocol previously developed for cassava (Escobar et al. 1997) (Table 1).

Encapsulation-dehydration Cassava shoot-tips were encapsulated in 3% sodium alginate and dropped into

0.1M CaCl2. The beads were then pretreated in sucrose medium for 3 d, slowly dehydrated to 15–20% water content and plugged directly into liquid nitrogen (LN). Dehydration with silica gel was more reproducible than in a flow chamber and is not temperature-dependent. The methodology was tested with five cassava cultivars. Some of these (MVen 232 and MMex 71) have been considered recalcitrant to the programmed freezing protocol (Table 2). Their response after freezing using the encapsulation-dehydration was more consistent and shoot growth was rapid and direct (fewer callus formations per explant). The sucrose concentration in the liquid medium and the duration of the dehydration period with silica gel affected the recovery rate after freezing. Cassava shoot-tips showed low response when they were treated with direct exposure to high sucrose level. To avoid detrimental effects, the use of 418 Cryopreservation of Tropical Plant Germplasm sequential media, with increased sucrose concentration and treatment duration, improved shoot regeneration (Escobar et al. 1995). Suboptimal water content of beads after dehydration on silica gel can affect shoot recovery. With 15–20% water content, the maximum shoot recovery after freezing was obtained. Benson et al. (1992) report 30–35% water content as optimal for cassava shoot-tips. We were not able to obtain any response with this level of water content. Currently, we are modifying the regrowth conditions and preliminary observations show an increase in shoot growth recovery. We will test this methodology with a subcore cassava collection comprising 64 cultivars.

Table 1. Effect of the freezing protocol on cryopreservation of cassava shoot-tips Genotype Freezing protocol† % Viability % Shoot recovery MCol 304 Programmed 63.6 18.1 Rapid 100 54.5 MCol 1389 Programmed 90.9 9.1 Rapid 90.0 10.0 MCol 1468 Programmed 53.8 0 Rapid 84.6 0 MPar 71 Programmed 84.6 76.9 Rapid 92.8 71.4 MCol 22 Programmed 76.5 49.7 Rapid 80.2 55.5 † Programmed protocol according to Escobar et al. (1997); Rapid freezing (direct immersion) according to Escobar and Roca (1997).

Table 2. Response of cassava genotypes to the encapsulation- dehydration technique† of cryopreservation Genotype % Viability % Shoot recovery MCol 22 100 82 MVen 232 94 12 MBra 507 92 43 MCol 1468 79 17 MMex 71 53 17 † Encapsulation-dehydration technique according to Palacio (1998) and Escobar et al. (1998).

Conclusions · Using a rapid freezing technique we have obtained similar rates of shoot recovery after freezing in LN in comparison with the slow programmed freezing protocol. · Both techniques (rapid freezing and encapsulation-dehydration) allowed the direct immersion of cassava shoot-tips in liquid nitrogen, avoiding the use of expensive programmable freezing equipment. · Dehydration with silica gel was more effective than in the flow chamber, and was not dependent on the room temperature. · For cassava cultivars susceptible to high sucrose concentration, sequential exposure to increasing levels of sucrose in the medium improved recovery. Posters 419

References Benson, E.E., N. Chabrillange and F. Engelmann. 1992. The development of cryopreservation methods for the long-term in vitro storage of cassava (Manihot spp.). Rapport de fin d'étude, ORSTOM, Montpellier, France. Escobar, R.H., G. Mafla and W.M. Roca. 1997. A methodology for recovering cassava plants from shoot tips maintained in liquid nitrogen. Plant Cell Reports 16:474–478. Escobar, R.H., W.M. Roca and C. Guevara. 1995. Cryopreservation of cassava shoot tips in liquid nitrogen. Pp. 82–84 in Biotechnology Research Unit Annual Report. CIAT, Cali, Colombia. Escobar, R.H. and W.M. Roca. 1997. Cryopreservation of cassava shoot tips through rapid freezing. Andean Journal of Root and Tuber Crops 2: 214–215 Escobar, R.H., J.D. Palacio, M.P. Rangel and W.M. Roca. 1998. Crioconservación de ápices de yuca mediante encapsulación-deshidratación. in Proceeding of the III Latin-american Meeting on Plant Biotechnology REDBIO’98, Habana, Cuba, 1–5 June 1998. Palacio, J.D. 1998. Crioconservación de ápices de yuca (Manihot esculenta Crantz) utilizando la técnica de encapsulación-deshidratación. Tesis CIAT, Cali, Colombia. Reed, B. and X. Yu. 1995. Cryopreservation of in vitro-grown gooseberry and currant meristems. Cryo–Letters 16:131–136 Towill, L. 1996. Germplasm preservation. Pp. 291–296 in Plant Tissue Culture Concepts and Laboratory Exercises, R. Trigiano and D.J. Gray (eds.). CRC Press, Boca Raton, Florida. 420 Cryopreservation of Tropical Plant Germplasm

Cryopreservation of shoot apices of yams (Dioscorea species) by vitrification P.M. Kyesmu¹ and Hiroko Takagi² JIRCAS, Ishigaki-City, Okinawa 907, Japan ¹ Present address: Department of Botany and Microbiology, P.M.B. 2084, University of Jos, Jos, Nigeria ² Present address: Research Information Division, 1-2 Ohwashi, Tsukuba, Ibaraki 305, Japan

Introduction Yams are vegetatively propagated tuber crops that belong to the family Dioscoreaceae within the order Dioscorales and genus Dioscorea. Dioscorea alata has the widest global distribution of all the yams. Its cultivation is mostly in southeast Asia, the Caribbean (where it is the most important species) and in West Africa (where it is second to D. rotundata) (Onwueme and Zanga 1996). Dioscorea rotundata, being native to West Africa, remains the principal yam cultivated, though its cultivation has now spread to other parts of the world, the West Indies and some parts of East Africa. Dioscorea bulbifera, D. esculenta and D. opposita are mostly cultivated in southeast Asia, Oceania, the Caribbean, China and Japan (Onwueme 1996). At present the conventional method of yam germplasm conservation is ex situ (as field or ‘tuber seeds’ collections). Field collections present special problems because of the requirement for extensive labour and large field areas, and the crops are exposed to pests, diseases and climatic uncertainties. These difficulties have led to the development of safe and cost-effective techniques for the long-term conservation of germplasm, i.e. cryopreservation. In this work, we present the first report on the successful cryopreservation of shoot apices of D. rotundata by vitrification. To the best of our knowledge, there has never been any published report on the cryopreservation of shoot apices of D. rotundata by vitrification except that reported by us (Kyesmu et al. 1997). Other limited available reports on Dioscorea species are those on the cryopreservation of shoot apices of D. wallchi, D. alata, D. bulbifera and D. floribunda by the encapsulation-dehydration method (Mandal et al. 1996).

Materials and methods Eight clones of in vitro shoot cultures of D. rotundata (TDr131, TDr179, TDr205, TDr608, TDr742, TDr743, TDr747 and NDR/BD10) obtained from the International Institute for Tropical Agriculture (IITA), Nigeria were used as study materials. Other Dioscorea species were obtained from JIRCAS, ICRS and Ishigaki collections. Apices of 5 to 7-d-old in vitro shoot cultures (0.5–1.0 mm in length) were precultured on Murashige and Skoog (MS) medium supplemented with 0.3M sucrose at 25°C under a 16-h light/8-h dark photoperiod. They were then dehydrated with the PVS2 vitrification solution (Sakai et al. 1990) and plunged into liquid nitrogen (LN). Exposure time of apices to PVS2 and preculture duration were evaluated. After cryopreservation, apices were Posters 421 thawed at 38°C for 60 s and unloaded in 1.2M sucrose for 20 min. Unloaded apices were then cultured on recovery medium (MS medium supplemented with 0.1M sucrose and 0.1M BAP).

Results The results of preculture duration on shoot apices of D. rotundata var. TDr743 indicated that post-thaw shoot recovery was highest for apices precultured for 72 h compared with those precultured for 24 or 48 h. Apices exposed to PVS2 for either 20 or 30 min before cryopreservation showed higher post-thaw shoot recovery than those exposed for 10, 40 or 50 min (Table 1). The method was applied to other clones with relative success (Kyesmu and Takagi, unpubl.): TDr131 (43%), TDr179 (73.75%), TDr608 (75.25%), TDr743 (67.50%), TDr747 (53.50%) and NDR/BD10 (64.25%); Dioscorea species such as D. alata var. UM680 (21.20%), D. bulbifera var. bulbifera (14.25%) and D. opposita var. Nishiki (10.80%).

Discussion/conclusion Preculture duration of plant cells on high sucrose levels has been reported to increase freezing tolerance. As a result, it is widely used for the cryopreservation of plant materials (Lee and Chen 1993). Durations of 1 to a few days have been reported to give successful results (Engelmann 1991; Towill 1995). Variation observed in clones as well as species could be attributed to physiological status or size of shoot apices.

Table 1. Effects of preculture duration (h) and PVS2 exposure time (min) on post-thaw shoot recovery of apices of D. rotundata var. TDr743 † ‡ Post-thaw shoot recovery (% ± SE ) Preculture duration (h) Main effects§ PVS2 (min) 24 48 72 Lsd = 10.7 10 12.50 ± 4.33 14.24 ± 5.90 46.50 ± 8.88 27.42b 20 21.75 ± 7.25 43.00 ± 0.00 60.50 ± 6.70 41.75a 30 57.00 ± 5.71 53.50 ± 3.50 67.50 ± 3.50 59.33a 40 10.50 ± 3.50 17 75 ± 3.75 32.50 ± 3.50 20.25bc 50 7.00 ± 4.04 7.00 ± 4.04 14.25 ± 5.90 9.42c Main effects 23.55b 27b 44.25a Lsd 13.9 NB: Apices precultured on MS + 0.3M sucrose at 25°C/16-h photoperiod, exposed to PVS2 at 25°C, LN for 60 min, thawed at 38°C for 60 s and unloaded in 1.2M sucrose for 20 min then transferred onto recovery medium (MS supplemented with 0.1M sucrose and 0.1M BAP). Data represent means of seven apices per treatment replicated four times. Means followed by the same letter are significantly different at probability level P<0.05. † =mean percentage shoot regeneration. ‡ =standard error of the mean. § Lsd=least significant difference.

From the study above, a protocol for the cryopreservation of D. rotundata was developed, which consisted of the following procedures: · dissecting apices from 5 to 7-d-old in vitro shoot cultures · dissected apices precultured on MS + 0.3M sucrose for 72 h at 25°C · precultured apices dehydrated in PVS2 at 25°C for 20–30 min, then 422 Cryopreservation of Tropical Plant Germplasm

cryopreserved · cryopreserved apices thawed at 38–50°C for 60 s; thereafter, unloaded in 1.2M sucrose for 20 min followed by immediate transfer onto recovery medium.

The study demonstrated for the first time that shoot apices of Dioscorea species could be cryopreserved by vitrification.

Acknowledgements The authors wish to thank S.Y.C. Ng of IITA, Nigeria for providing the in vitro shoot cultures of D. rotundata and Prof. A. Sakai for his positive criticisms and continuous encouragements. This work was supported by a Fellowship offered to P.M. Kyesmu under the JIRCAS Visiting Research Fellowship Program, Ministry of Agriculture, Fisheries and Forestry, JAPAN.

References Engelmann, F. 1991. In vitro conservation of tropical plant germplasm—a review. Euphytica 57: 227–243 Lee, S.P. and T.H.H. Chen. 1993. Molecular cloning of abscisic acid-responsive mRNAs expressed during freezing tolerance in bromegrass (Bromus inermis Leyss) suspension culture. Plant Physiology 101: 1089–1096 Kyesmu, P.M., H. Takagi and S.Yashima. 1997. Cryopreservation of shoot apices of white th yams (Dioscorea rotundata Poir) by vitrification. Abstract of the 15 Meeting of the Japanese Society for Plant Cell Molecular Biology, 2D–13: 162 Mandal, B.B., K.P.S. Chandel and S. Dwivedi. 1996. Cryopreservation of yam (Dioscorea spp) shoot apices by encapsulation-dehydration. Cryo–Letters 17: 165–174 Onwueme, I.C. 1996. Dioscorea. Pp. 85–90 in Plant Resources of Southeast Asia (9)-Plants Yielding Non-seed Carbohydrates, M. Flach and F. Rumawas (eds.). Backhuys Publishers, Leiden. Onwueme, I.C. and M. Zanga. 1996. Dioscorea alata L. Pp. 90–92 in Plant Resources of Southeast Asia (9)-Plants Yielding Non-seed Carbohydrates, M. Flach and F. Rumawas (eds.). Backhuys Publishers, Leiden. Sakai, A., S. Kobayashi and I. Oiyama. 1990. Cryopreservation of nucellar cells of navel orange (Citrus sinensis Osb. var. brasiliensis Tanaka) by vitrification. Plant Cell Reports 12: 525–529 Towill, L.E. 1995. Cryopreservation by vitrification. Pp. 99–111 in Genetic Preservation of Plant Cells In Vitro, B. Grout (ed.). Springer–Verlag, New York. Posters 423

Survival and recovery of yam (Dioscorea spp.) apices after cryopreservation using encapsulation-dehydration Bernard Malaurie¹, Marie–France Trouslot¹, Nathalie Chabrillange¹ and

Florent Engelmann ² ¹ GeneTrop, Unité de Génétique et d’Amélioration des Plantes (GAP), Centre ORSTOM, Montpellier, France ² IPGRI, 00145 Rome, Italy

Introduction Yams, which are edible or medicinal tuber crops, are very important crops in many developing countries. Efficient conservation methods are needed for these crops. Medium- and long-term conservation have been explored with Dioscorea (Malaurie et al. 1998a, 1998b, 1998c, 1998d). In addition to previous works by Mandal et al. (1996), Malaurie et al. (1998b) have investigated the effect of three factors on the survival of encapsulated apical shoot-tips of in vitro plantlets of two yam (Dioscorea bulbifera L. and D. alata L.) genotypes after cryopreservation. The factors studied were: (i) the pretreatment duration in sucrose liquid medium, (ii) the sucrose concentration, and (iii) the duration of desiccation with silica gel. Techniques and the most important points of the note are presented here.

Material and methods Two clones – Brazo fuerte for D. alata, and Nouméa Imboro for D. bulbifera – from the in vitro collection of yam germplasm maintained at ORSTOM Montpellier (Malaurie et al. 1993) were used. Nodal cuttings were subcultured on MS standard medium. Mother microplants, 5–8 months old, were used for massive shoot production; after 18 ± 4 d in culture, apices were excised from the young growing shoots. All cultures were maintained under the following standard environmental conditions: at 27 ± 1°C, under a light intensity of 36 µE m-² s-¹ (PAR), with a 12-h light/12-h dark photoperiod. Excised apical shoot-tips (2–5 mm long) were placed overnight in Petri dishes on solidified medium containing 5% (w/v) sucrose. Apices were encapsulated in 3% (w/v) calcium alginate. Encapsulated apices were pretreated for 3–10 d (D. alata) or 13 d (D. bulbifera) in liquid MS standard medium with various sucrose concentrations (0.75 to 1.1M), in 125-ml Erlenmeyer flasks, on a rotary shaker (91 rpm) under standard environmental conditions. After sucrose pretreatment, beads were dehydrated for 4–23 h in 125-ml airtight boxes filled with 40 g silica gel. Twenty beads were used per treatment: 10 dried beads were placed in a 2-ml polypropylene sterile cryotube and frozen directly in liquid nitrogen where they were kept for at least 2 h; the 10 remaining beads were placed in Petri dishes onto 2GG medium (Malaurie et al. 1993) without activated charcoal and maintained on 30 g/L sucrose for dehydration treatments up to 12 h, and supplemented with 50 g/L sucrose, 1 mg/L benzylaminopurine, 0.01 mg/L naphtalene acetic acid for longer desiccation periods. The residual water content of the alginate beads was expressed in g 424 Cryopreservation of Tropical Plant Germplasm water per g dry weight. Dry matter was determined after desiccating 20–50 beads in 125-ml airtight boxes containing 40 g of dry silica gel, for up to 30 d

(DW30) (Table 1). Posters 425

Table 1. Dry mass and water content of sucrose-pretreated alginate beads, determined after 30 d of drying with silica gel in airtight boxes at room temperature DW30 (% FW) estimated by Water content before Sucrose concentration linear regression† dehydration (g/g DW) 0.75M 28.8 2.47 0.9M 33.3 2.00 1.0M 36.3 1.76 1.1M 39.3 1.54 † From mean values over 13–15 replicates for each of the four sucrose concentrations (y= 6.4319 + 29.872x; N= 4; r= 0.999). Similar results were obtained from replicate data (y= 6.4177 + 29.883x; N= 55; r= 0.960); data not shown. (Adapted from Malaurie et al. 1998b, with permission).

Results The duration of sucrose pretreatment had a significant effect on the survival of cryopreserved D. alata apices only, and optimal pretreatment durations were between 3 and 7 d. The sucrose concentration employed during pretreatment had a strong effect on both genotypes. With D. alata, the highest survival was noted with 0.9M sucrose, and with 0.9 up to 1.1M sucrose in the case of D. bulbifera. With both genotypes, high survival rates after cryopreservation were obtained for bead water contents lower than 0.15 g H2O/g DW only. With D. alata, the highest survival rate (67%) was achieved after pretreatment with 0.9M sucrose followed by 11–12 h of dehydration, and survival rates were not significantly different after 9–23 h of dehydration (Fig. 1). With D. bulbifera, the highest survival rate (65%) was achieved after pretreatment with 1M sucrose followed by 14–16 h of dehydration. Survival was not significantly different for desiccation periods between 11 and 23 h. After 3 months of culture on medium without growth regulators, 60% of cryopreserved D. bulbifera shoot-tips had developed into plantlets, whereas this was the case with 19% of frozen D. alata apices only, owing to browning and/or callusing of numerous shoot-tips.

Discussion and conclusion This study demonstrated that apices of D. alata cv. Brazo fuerte and D. bulbifera cv. Nouméa Imboro could be successfully cryopreserved using the encapsulation-dehydration technique. For the two species, the water content of encapsulated apices had to be decreased down to 0.15 g H2O/g DW in order to obtain high survival after freezing. The percentage of water loss was of 67, 62, 58 and 55% FW (± 1%) for 0.75, 0.9, 1 and 1.1M sucrose pretreatments, respectively. Our results demonstrated that, in most cases, survival increased when dehydration was extended to a defined threshold, around 0.13–0.15 g H2O/g DW, which was obtained after desiccation periods from 10 to 18 h. It seemed that, with this soft desiccation process, we could rub out differences in residual water, which certainly exist between apices from a same plot. 426 Cryopreservation of Tropical Plant Germplasm

100 1.0 Da (0.9M+1M) -LN 90 A +LN 0.9 80 WatCont 0.8 70 0.7 60 0.6 50 .47 0.5 40 0.4 Survival (%) 30 .26 0.3

20 .17 Water Content (g/gDW) .15 .15 .12 0.2 10 0.1 0 0 0 2 4 6 8 10 12 14 16 18 20 22 24 Dehydration duration (hours) 100 1.0 90 B Db (1M+1.1M) -LN +LN 0.9 WatCont 80 0.8 70 0.7 60 0.6 50 .41 0.5 40 0.4 30 .22 0.3 Water Content (g/gDW) Survival (%) 20 .18 .13 .13 .11 0.2 10 0.1 0 0 0 2 4 6 8 10 12 14 16 18 20 22 24 Dehydration duration (hours)

Fig. 1. Effect of dehydration duration on the water content of beads and survival rate of control (–LN) and cryopreserved (+LN) encapsulated apices of D. alata Brazo Fuerte after pretreatment with sucrose (0.9M+1M) (A), and D. bulbifera Nouméa Imboro after pretreatment with sucrose (1M+1.1M) (B). Each point corresponds to a mean over two sucrose concentrations and all the 6 or 8 pretreatment durations used depending on the clone. (Reprinted from Malaurie et al. 1998b, with permission). Posters 427

References Malaurie, B., O. Pungu, R. Dumont and M.F. Trouslot. 1993. The creation of an in vitro germplasm collection of yam (Dioscorea spp.) for genetic resources preservation. Euphytica 65: 113–122. Malaurie, B., M.F. Trouslot and J. Berthaud. 1998a. Conservation et échange de germoplasme chez les ignames (Dioscorea spp.). Pp. 135–161 in L’igname : Plante Séculaire et Culture d’Avenir. Actes du Séminaire International Cirad–Inra–Orstom-Coraf, J. Berthaud, N. Bricas and J.-L. Marchand eds. Montpellier, France, 3–6 June 1997. Malaurie, B., M.F. Trouslot, F. Engelmann and N. Chabrillange 1998b. Effect of pretreatment conditions on the cryopreservation of in vitro-cultured yam (Dioscorea alata 'Brazo Fuerte' and D. bulbifera 'Nouméa Imboro') shoot apices by encapsulation-dehydration. Cryo– Letters 19 : 15–26 Malaurie, B., M.F. Trouslot, J. Berthaud, N. Chabrillange, C. Récalt and S. Dussert. 1998c. The use of slow growth condition culture and cryopreservation in liquid nitrogen for medium and long term conservation and utilisation of in vitro yam (Dioscorea spp.) germplasm. In: Proceedings of the Workshop on Conservation & Utilisation of Cassava, Sweetpotato and Yam Germplasm in Sub-Saharan Africa. Nairobi, 11–13 November, 1997 (in press). Malaurie, B., M.F. Trouslot, J. Berthaud, M. Bousalem, A. Pinel and J. Dubern. 1998d. Medium-term and long-term in vitro conservation and safe international exchange of rd yam (Dioscorea spp.) germplasm. P. 190 in Abstracts of the 3 Latin-American Meeting on Plant Biotechnology, REDBIO’98, June 1–5 1998, Habana, Cuba. Mandal, B.B., K.P.S. Chandel and S. Dwivedi. 1996. Cryopreservation of yam (Dioscorea spp.) shoot apices by encapsulation-dehydration. Cryo–Letters 17, 165–174. 428 Cryopreservation of Tropical Plant Germplasm

Cryopreservation of cassava and yam shoot-tips by fast freezing Shou Yong Choy Ng and Nyat Quat Ng IITA, Oyo Road, Ibadan, Nigeria

Introduction With the recent focus on collecting activities for germplasm of cassava and yam, IITA now has a collection of about 2500 accessions of cassava (Manihot esculenta) and over 3000 accessions of yam (Dioscorea spp.). The collections are maintained in the field and about 25% of cassava and 50% of the yam are duplicated in vitro under reduced growth conditions. Both field and in vitro maintenance require periodic planting/subculturing of the conserved germplasm. Cryopreservation allows long-term conservation, but the high cost of the equipment required for achieving controlled slow freezing has limited the research and its use. Recently, with the development of simple and reliable cryopreservation protocols, the number of plant species that could be cryopreserved has increased (Sakai 1997). The objectives of this research were to develop simple and less expensive cryopreservation protocols by fast freezing for cassava and yam, to evaluate the reproducibility of the protocol on a wide range of germplasm, and to transfer the technology to collaborators in the region.

Materials and methods Cassava genotypes (TME2, TME3, I60142, I70775, I63397 and M86/00106) and yam genotypes [TDr179 and TDr608 (D. rotundata), TDb3058 (D. bulbifera) and TDa1170 (D. alata)] were used in these studies. Cassava and yam shoot-tips were excised from in vitro plantlets. Cassava shoot-tips were precultured for 3 d on cassava shoot-tip culture medium as described by Ng and Hahn (1985) and yam on yam shoot-tip culture medium described by Ng (1992) with elevated sucrose concentration (0.7M). They were then transferred to the vitrification solution for 20 min in all experiments except for the study on the effects of vitrification time. The vitrification solution was composed of 0.7M sucrose, 15% ethylene glycol, 15% dimethyl sulfoxide (DMSO) and 30% glycerol. Shoot-tips after treatment with the vitrification solution were transferred to cryovials and immersed directly in liquid nitrogen (LN). After at least 1 h storage in LN (–196°C) samples were retrieved and thawed either by slow thawing at room temperature (28°C) or fast thawing in a water-bath set at 40°C. The shoot-tips after rinsing with sterile washing solution were cultured on the appropriate shoot-tip culture medium with 0.7M sucrose, and incubated under dark conditions for 3 d. They were then transferred to normal shoot-tip culture medium, and incubated under light to resume growth. Also, the effects of further dehydration by air-drying shoot-tips in the flow hood after treatment with the vitrification solution were investigated. Responses of cassava shoot-tips and nodal cuttings to cryopreservation were Posters 429 also compared. 430 Cryopreservation of Tropical Plant Germplasm

Results and discussion Studies on the tolerance of shoot-tips to vitrification indicated that, depending on genotype, the survival rate of cassava shoot-tips ranged from 60 to 85% and those of yam from 25 to 75%. Preliminary results obtained from the study on the effects of thawing showed that fast thawing gave a higher survival rate than slow thawing with some genotypes. Among the genotypes studied, M86/00106 and I63397 had higher survival rates. With slow thawing, none of the shoot-tips survived except in genotype I70775. TME 2 and TME 3, two local cassava cultivars, did not survive after freezing. This indicated that there are genotypic differences in response to the cryopreservation protocol. A positive correlation was obtained between the survival rate of cassava shoot-tips and nodal cuttings and time of exposure to the vitrification solution before freezing (Table 1). A survival rate as high as 70% was recorded in shoot-tips treated with vitrification solution for 50 min. In general, shoot-tips had a higher survival than nodal cuttings. The recovery (percentage bud/shoot formation) also increased with the increase in time exposure to the vitrification solution with a maximum of 30% being obtained. However, none of the nodal cuttings formed buds. This indicated that shoot-tips are better sources of explants for use in cassava cryopreservation. Using shoot-tips of cassava genotype I70775 and 40-min vitrification, 40% recovery was obtained by slow thawing and 35% recovery by fast thawing (Table 2). Air-drying of the shoot-tips after treatment with the vitrification solution also increased the percentage recovery after freezing. The survival rate of yam shoot-tips after freezing was lower than that of cassava. Preliminary results showed that D. bulbifera had a higher survival rate than D. rotundata and D. alata. Using D. rotundata genotype TDr179, a survival rate of 40% and a recovery rate of 20% was obtained from shoot-tips exposed to vitrification solution for 30 and 40 min followed by freezing in LN (Table 3). Air-drying of the shoot-tips for 5–20 min after vitrification, then freezing in LN gave a slight increase in the survival rate.

Conclusions Results obtained so far clearly showed that the cryopreservation protocol by vitrification and fast freezing has immense potential for the cryopreservation of cassava and yam germplasm. This provides an opportunity to develop a simple and less expensive cryopreservation protocol for routine use with cassava and yam which will reduce the cost of the process and make it more accessible to researchers working on these two important crop species. Further research is under way to test a wide range of genotypes and improve the recovery rate. Posters 431

Table 1. Effects of duration of exposure to the vitrification solution on the survival and recovery of cassava shoot-tips and nodal cuttings after freezing Exposure to PVS2 Survival (%) Recovery (%) (min) Shoot-tip Nodal cutting Shoot-tip Nodal cutting 0 10 0 0 0 10 30 0 0 0 20 30 50 0 0 30 40 50 20 0 40 50 30 20 0 50 70 50 30 0 Control 100 90 90 80

Table 2. Effects of fast and slow thawing on survival and recovery of cassava (I70775) shoot-tips (vitrification time, 40 min) Treatment Survival (%) Recovery (%) Control 100 75 Fast thawing 75 35 Slow thawing 75 40

Table 3. Effects of duration of exposure to the vitrification solution on the survival and recovery of yam shoot-tips after freezing (TDr 179) Exposure duration (min) Survival (%) Recovery (%) 0 0 0 10 0 0 20 20 0 30 40 20 40 40 20

References Ng, S.Y.C. 1992. Micropropagation of white yam (Dioscorea rotundata Poir). Pp. 135–159 in Biotechnology in Agriculture and Forestry, Vol. 19. Y.P.S. Bajaj (ed.). Springer–Verlag, Berlin. Ng, S.Y. and S.K. Hahn. 1985. Application of tissue culture to tuber crops at IITA. Pp. 29–40 in Proceedings of the Inter-centre Seminar on International Agricultural Research Centres (IARCs) and Biotechnology. IRRI, the Philippines. Sakai, A. 1997. Potentially valuable cryogenic procedures for cryopreservation of cultured plant meristems. Pp. 53–66 in Conservation of Plant Genetic Resources In Vitro, Vol. 1: General Aspects. M.K. Razdan and E.C. Cocking (eds.). Science Publishers, Inc., USA. 432 Cryopreservation of Tropical Plant Germplasm

Cryopreservation of tissue-cultured tropical forest trees Emilio Maruyama and Katsuaki Ishii Bio-Resources Technology Division, Forestry and Forest Products Research Institute, Tsukuba Norinkenkyu Danchi-Nai, Ibaraki, 305-8687 Japan

Introduction Several important tropical forest trees have become seriously threatened in terms of amount and genetic resources due to extensive and selective cutting, and burning for shifting cultivation or raising livestock. Cedrela odorata L. (cedro) belonging to the Meliaceae family (mahogany family) and Guazuma crinita Mart. (bolaina blanca) belonging to the Sterculiaceae family (cacao family), are two important forest tree species in tropical America. Cedro wood is greatly valued for its good quality and is used for making fine furniture, cigar boxes, interior construction, general carpentry and for decorative veneer production. Owing to the continuous selective felling of good-quality cedro for several decades, the resources of this tree are on a steady decline. In addition, cedro and other Meliaceae plantations have been damaged severely by the Meliaceae shoot borer, Hypsipyla grandella Zeller (Lepidoptera, Pyralidae). Bolaina blanca has a soft, light wood with good working properties which has been used for light construction, panelling, interior joinery, mouldings, boxes, crates and matches, and because of its fast growth and excellent adaptability to a wide range of sites, is considered one of the potential species for reforestation in lowlands of the Peru–Amazon region. Conservation of germplasm of cultivated species and their wild relatives is necessary for sustainable exploitation systems and as a means of maintaining species diversity and genetic diversity to prevent genetic erosion. Cryopreservation of tissue-cultured tropical forest trees may be a reliable method for long-term preservation of plant genetic resources without apparent risk of genetic instability using a minimum space and with less maintenance labour. We report a cryopreservation approach for the useful tropical forest trees C. odorata and G. crinita, using tissue-cultured plant materials.

Materials and methods

Plant materials for cryopreservation Shoot-tips and root-tips (about 2 mm long) and adventitious bud clusters (about 1.0–1.5 cubic segment) from 2 to 3-month-old in vitro plantlets regenerated by methods described by Maruyama et al. (1989, 1996) were used as plant material for cryopreservation experiments.

Cryoprotectant solutions The following cryoprotectant solutions, modified from Sakai et al. (1990, 1991) and Towill (1990), containing (w/v), A: 20% glycerin+15% sucrose; B: 30% glycerin +15% sucrose+15% ethylene glycol+15% dimethyl sulfoxide (DMSO); C: 25% Posters 433 glycerin+15% sucrose+15% ethylene glycol+13% DMSO+2% polyethylene glycol 4000 (PEG); D: 35% ethylene glycol+10% DMSO+5% PEG, were tested.

Cryopreservation methods

Simple freezing Explants were treated with cryoprotectant solution A at 25°C for 5–60 min and then cooled in a freezer at –30°C for 1 h prior to immersion in LN and kept there for at least 1 h.

Rapid freezing Explants were treated with cryoprotectant solutions A, B, C, D, alone or in succession at 25°C for different periods of time (0–90 min) and then directly immersed in LN and kept there for at least 1 h.

Slow prefreezing Explants, were treated with cryoprotectant solutions A, B, C, alone or in succession at 25°C for different periods of time (0–45 min) and then cooled to -40°C at a rate of 0.5°C/min prior to immersion in LN and kept there for at least 1 h.

Survival and plant recovery Explants were thawed by rapidly transferring cryotubes to a water-bath at 37°C. After thawing, cryoprotectant solutions were drained from the cryotubes and replaced with medium containing 40% (w/v) sucrose and kept for 20 min. Then, shoot-tip explants were transferred to sterilized filter paper discs over solidified medium. Root-tips and adventitious bud cluster explants were transferred into liquid medium and cultured on a bio-shaker at 70–75 rpm.

Results and discussion The results of the cryopreservation methods on the survival and plant conversion are shown in Table 1. Slow prefreezing was the optimum method for cryopreservation of shoot-tip explants. When shoot-tips were treated with cryoprotectants A and B for 15–20 and 10–15 min, respectively, and then cooled to –40°C at a rate of 0.5°C/min prior to storage in LN, survival and plant recovery rates of 50–20% and 50–15% were obtained in cryopreserved shoot-tips of C. odorata and G. crinita, respectively. For root tip explants, no differences between the rapid freezing and slow prefreezing methods were found. The rapid freezing method was effective for the cryopreservation of adventitious bud clusters of G. crinita. Survival-plant recovery rates of 85–25% were achieved in explants treated with cryoprotectants B or C for 15–60 min. Survival of explants after immersion in LN was not achieved in any of the treatments tested with the simple freezing method. No morphological abnormalities were observed in the plants regenerated from cryopreserved explants. Although the plant recovery rates should be improved in the near future for a more effective cryopreservation system, in our opinion these results can be 434 Cryopreservation of Tropical Plant Germplasm used at present because, considering that more than 170 000 and 800 000 shoots can be obtained in a year from only one shoot-tip explant of C. odorata and G. crinita, respectively (Maruyama et al. 1989, 1996), further propagation from a few cryopreserved surviving explants is really possible. Thus, cryopreservation by using both the slow prefreezing and rapid freezing methods can be considered to be a suitable alternative for the long-term storage of C. odorata and G. crinita germplasm. Storage of selected germplasm by cryogenic procedures is an attractive possibility for the conservation of genetically superior tropical forest trees for many decades.

Table 1. Effects of different cryopreservation methods on the survival (S) and plant conversion (PC) rates of C. odorata and G. crinita explants after cooling in LN Method Slow Simple freezing Rapid freezing prefreezing Species Explant S (%) PC (%) S (%) PC (%) S (%) PC (%) C. odorata Shoot-tip 0 0 10 10 50 20 G. crinita Shoot-tip 0 0 0 0 50 15 Root tip 0 0 30 5 30 5 Adv. bud cluster 85 25

References Maruyama, E., K. Ishii, A. Saito and K. Migita. 1989. Micropropagation of Cedro (Cedrela odorata L.) by shoot-tip culture. Journal of the Japanese Forestry Society 71: 329–331. Maruyama, E., K. Ishii, I. Kinoshita, K. Ohba and A. Saito. 1996. Micropropagation of bolaina blanca (Guazuma crinita Mart.), a fast-growing tree in the Amazon region. Journal of Forestry Research 1: 211–217. Sakai, A., S. Kobayashi and I. Oiyama. 1990. Cryopreservation of nucellar cells of navel orange (Citrus sinensis Osb. var. brasiliensis Tanaka) by vitrification. Plant Cell Reports 9: 30–33. Sakai, A., S. Kobayashi and I. Oiyama. 1991. Cryopreservation of nucellar cells of navel orange (Citrus sinensis Osb.) by simple freezing method. Plant Science 74: 243–248. Towill, L.E. 1990. Cryopreservation of isolated mint shoot tips by vitrification. Plant Cell Reports 9: 178–180. Posters 435

Cryopreservation of grape in vitro-cultured axillary shoot- tips by three-step vitrification Toshikazu Matsumoto¹ and Akira Sakai² ¹ Shimane Agricultural Experiment Station, Izumo, Shimane, 693-0035, Japan ² Hokkaido University, Kitaku, Sapporo, 001-0045, Japan

Introduction Economically, grape is one of the most important fruit crops for table food. It is also important as a processing agent for the production of wines and dry fruits. To date, grape germplasm has been conserved mainly in the field. However, field genebanks are faced with serious problems, including natural disasters, attacks by pests and pathogens, and high maintenance costs. Cryopreservation in LN appears to be a logical choice by the long-term storage of germplasm with minimum space and maintenance requirements. At present, potentially valuable cryogenic procedures would be the vitrification and encapsulation- dehydration techniques for cryopreservation of in vitro-grown plants. The vitrification technique especially offers various advantages over the encapsulation-dehydration in terms of both its high recovery growth and its simple procedure. To our knowledge, there are few or no reports of successful cryopreservation of grape by vitrification. Thus, this study was performed to develop a simple and reliable procedure for the cryopreservation of grape germplasm.

Materials and methods Cabernet Sauvignon, one of the most popular cultivars for wine, was mainly used in this study. Axillary shoot-tips were dissected from 4 to 5-month old plants cultured on modified Murashige and Skoog medium (1962) (half strength of KNO3 and NH4NO3, termed ½ MS medium) containing 1 mg/L benzyladenine (BA), 3% (w/v) sucrose and 0.2% (w/v) gellan gum with a 16-h light/8-h dark photoperiod at 25°C. Excised shoot-tips were precultured on solidified ½ MS medium containing 0.3M sucrose for 3 d at 25°C, then treated with a mixture of 2M glycerol + 0.4M sucrose (LS solution) for 20 min at 25°C before dehydration with a vitrification solution (PVS2 solution, Sakai et al. 1990). Following this procedure, excised shoot-tips were directly dehydrated with the PVS2 solution for 80 min at 0°C (two-step procedure), or first dehydrated with 50% PVS2 solution (half strength of glycerol, ethylene glycol and DMSO), followed by PVS2 solution for different lengths of time at 0°C (three-step procedure) prior to immersion in LN. After the shoot-tips were kept for more than 1 h in LN, they were rewarmed rapidly in a water-bath set at 40°C. Vitrified shoot-tips were expelled into a 1.2M sucrose solution for 20 min at 25°C, and then placed on filter papers over solidified ½ MS medium containing 1 mg/L BA and 3% sucrose for regrowth. 436 Cryopreservation of Tropical Plant Germplasm

Results and discussion In our previous experiments, a high recovery (approx. 60%) after cooling to -196°C by vitrification was obtained from shoot-tips precultured with 0.3M sucrose for 3 d and subsequently treated with LS solution for 20 min at 25°C, and dehydrated with the PVS2 solution for 80 min at 0°C (two-step procedure, Matsumoto et al. 1998). These experiments suggest that preculture and LS treatment are two necessary steps to produce a high recovery rate for vitrified shoot-tips of grape. However, the buds regenerated tended to form clusters and did not elongate into normal shoots. Thus, to improve the recovery of vitrified shoot-tips, shoot-tips precultured and treated with LS solution were dehydrated in two steps, first with a 50% PVS2 solution for 30 min, followed by PVS2 solution for 50 min at 0°C before immersion in LN. The recovery of vitrified shoot-tips cryopreserved by the three-step procedure amounted to approximately 80% and normal shoots were produced within 7–10 d after plating. Recovery rates by the two-step and three-step procedures were compared with three other Vitis vinifera cultivars: Muscat of Alexandria, Rizamat and Thompson Seedless. The recovery rates of all cultivars cryopreserved by the three-step procedure were higher than that of the two- step procedure (Table 1). Thus, this three-step vitrification procedure appears promising for the cryopreservation of grape germplasm.

Table 1. Recovery growth of shoot-tips† from four cultivars of grape cooled to –196°C by two methods of vitrification Recovery rate (%) Cultivar two-step three-step Cabernet Sauvignon 66.7±3.3 85.0±5.0 Muscat of Alexandria 65.0±5.0 76.7±6.7 Rizamat 23.3±3.3 46.7±8.8 Thompson Seedless 25.0±5.0 56.7±3.3 † Excised shoot-tips were precultured with 0.3M sucrose for 3 d and then treated with a mixture of 2M glycerol and 0.4M sucrose for 20 min at 25°C. After preculture and loading, the shoot-tips were dehydrated with PVS2 for 80 min at 0°C (two-step) or with 1/2 PVS2 for 30 min, then with PVS2 for 50 min at 0°C (three-step) before being immersed in LN. Recovery rate: 30 d after reculture.

References Matsumoto, T., A. Sakai and Y. Nako. 1998. Cryopreservation of in vitro cultured axillary shoot tips of grape (Vitis vinifera) by vitrification. Journal of the Japanese Society of Horticultural Science 67 (Supplement 1): 78. Murashige, T. and F. Skoog. 1962. A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiologia Plantarum 15: 473–497. Sakai, A., S. Kobayashi and I. Oiyama. 1990. Cryopreservation of nucellar cells of navel orange (Citrus sinensis Osb. var. brasiliensis Tanaka) by vitrification. Plant Cell Reports 9: 30–33. Posters 437

Cryopreservation of shoot apices of lawngrass by encapsulation-dehydration Tomohiro Minami and Akira Sawai Kagoshima Agricultural Experiment Station Osumi Branch, Laboratory of Forage Grass Breeding, Kushira, Kagoshima, 893-1601, Japan

Introduction A number of lawngrass germplasm have been conserved as ex situ collections of plants in several locations in Japan. These collections are exposed to pests and pathogens as well as to natural disasters. In addition, it is costly to maintain these collections in the field. Cryopreservation has become a very important tool for long-term storage of germplasm. In recent years, simplified cryogenic procedures such as vitrification (Sakai et al. 1990), encapsulation-dehydration (Fabre and Dereuddre 1990) and encapsulation-vitrification (Matsumoto et al. 1995) have been developed, and the number of species to be cryopreserved has increased. These three procedures enable cryopreservation of cells, meristems, somatic embryos and hairy root cultures by direct immersion in liquid nitrogen without the need for special equipment. However, in most cases, the cryopreserved species are limited to horticultural crops such as vegetable crops, flowers and ornamental plants and fruit trees, and there have been only a few reports on forage crops such as white clover (Yamada et al. 1991) and gramineous crops such as rice (Huang et al. 1995) and sugarcane (Paulet et al. 1993). The objective of this study was to determine optimal conditions for cryopreservation of Japanese lawngrass (Zoysia japonica Steud.) by the encapsulation-dehydration method.

Materials and methods Shoot apices were excised from 50 to 70-mm-long stolon segments of field- grown Japanese lawngrass. Shoot apices precultured for 3, 6 and 9 d on MS medium containing 30 g/L sucrose and 8 g/L agar at 26°C were suspended in

MS liquid medium devoid of CaCl2, supplemented with 2% sodium alginate. Each shoot apex from this medium was dropped into MS liquid medium with

100 mM CaCl2 for encapsulation. Encapsulated shoot apices (beads) were pretreated on MS medium with 0.2–0.8M sucrose for 2 d to induce desiccation tolerance. Sixteen pretreated beads and control beads without apices were placed on an aluminium dish (5 cm in diameter), and three of the dishes were placed on a larger Petri dish (16 cm in diameter) with 90 g silica gel. During desiccation, the water content of the control beads was determined by measuring the weight of beads at 2-h intervals during desiccation and drying them at 105°C for 16 h at the end of the desiccation period. The beads were put into 1.8-ml cryotubes after being dried to a water content of about 20%, then plunged into LN and stored for at least 1 h at this temperature. The cryotubes were rapidly thawed in a 40°C water-bath for 30 s, and the beads were directly placed on MS solid medium containing 0.1M sucrose. Viability was determined 438 Cryopreservation of Tropical Plant Germplasm based on regrowth or shoot regeneration after 3 weeks of culture. Values were indicated as the means with standard deviation (SD) of three replicates of 16 shoot apices.

Results and discussion In many reports about encapsulation-dehydration, optimal survival has been achieved when beads were desiccated to a water content of 15–25%. Thus, the dehydration of beads pretreated with different sucrose levels was investigated to determine the approximate length of dehydration. The water content of beads pretreated with 0.8M sucrose decreased to approximately 20% after 8–10 h desiccation, while treatments with lower sucrose concentrations required longer dehydration periods. In order to determine the optimal pretreatment conditions, encapsulated shoot apices were pretreated on MS medium with 0.2, 0.4 and 0.8M sucrose for 2 d before dehydration for 12 or 18 h. The highest dehydration tolerance, leading to a survival rate of over 90%, was induced by placing encapsulated apices on medium with 0.8M sucrose for 12 h. Although beads treated with 0.8M sucrose for 18 h had a lower water content, their survival rate was 45% only. Therefore, all the beads conserved in liquid nitrogen were pretreated with 0.8M sucrose for 2 d. The duration of the preculture period also affected the survival rate. The highest survival after immersion in LN was observed when beads were precultured for 3 or 6 d. Although the survival rate of desiccated apices decreased gradually in line with bead water content, the survival rate of cryopreserved apices reached 75% after 10–12 h desiccation. However, in spite of their high viability (over 75–90%), most of the surviving shoot apices showed poor regrowth and low regeneration after a few months in culture. Japanese lawngrass shoot apices were successfully cryopreserved by encapsulation-dehydration, but further studies are required to improve plant recovery from cryopreserved shoot apices.

Acknowledgements We thank Dr S. Fukai (Kagawa University) for critical reading of the manuscript and helpful advice. This work was supported in part by the Kagoshima Biotechnology Institute.

References Fabre, J. and J. Dereuddre. 1990. Encapsulation-dehydration: A new approach to cryopreservation of Solanum shoot-tips. Cryo–Letters 11:413–426. Huang, C.N., J.H. Wang, Q.S. Yan, X.Q. Zhang and Q.F. Yan. 1995. Plant regeneration from rice (Oryza sativa L.) embryogenic suspension cells cryopreserved by vitrification. Plant Cell Reports 14:730–734. Matsumoto, T., A. Sakai, C. Takahashi and K. Yamada. 1995. Cryopreservation of in vitro- grown apical meristems of wasabi (Wasabia japonica) by encapsulation-vitrification method. Cryo–Letters 16:189–196. Paulet, F., F. Engelmann and J.C. Glaszmann. 1993. Cryopreservation of apices of in vitro plantlets of sugarcane (Saccharum sp. hybrids) using encapsulation/dehydration. Plant Cell Reports 12:525–529. Posters 439

Sakai, A., S. Kobayashi and I. Oiyama. 1990. Cryopreservation of nucellar cells of navel orange (Citrus sinensis Osb. var. brasiliensis Tanaka) by vitrification. Plant Cell Reports 9:30–33. Yamada, T., A. Sakai, T. Matsumura and S. Higuchi. 1991. Cryopreservation of apical meristems of white clover (Trifolium repens L.) by vitrification. Plant Science 78:81–87. 440 Cryopreservation of Tropical Plant Germplasm

Cryopreservation of garlic apical meristems by vitrification Eiko Niwata¹, Masahiko Suzuki¹ and Masaya Ishikawa² ¹ Aomori Pref. Green Biocenter, Aomori 030-0142, Japan ² NIAR, Ibaraki 305-0856, Japan

Introduction Garlic (Allium sativum L.) is one of most important and widely cultivated crops in the world for food and medicine, and the conservation of garlic genetic resources is a priority objective for developing breeding programmes. Since most garlic cultivars are sterile, their conservation is performed through field collections. The maintenance of field collections is a major consumer of time, labour, space and materials, and the plants remain exposed to diseases and environmental stresses. Thus, the safe conservation of garlic germplasm is impossible to consider without a long-term storage technique. In this study, apical meristems excised from post-dormant bulbs of garlic were successfully cryopreserved by vitrification with subsequent high plant regeneration rates, thus making the long-term conservation of garlic possible.

Materials and methods Shoot-tips excised from garlic (Allium sativum L. cv. Fukuchi-howaito) were mainly used in the present study. Bulbs harvested in July were stored first at 30–35°C to break summer dormancy, then at room temperature until use from September to November. After removal of a few outer older leaves and protective leaves, scales were surface-sterilized by dipping them in 70% ethanol, then in a sodium hypochlorite solution (effective chlorine concentration: 0.5%) for 15 min. Apical meristems, about 2 mm in length and width including a few leaf primoldia and a 0.5-mm basal plate were dissected. They were then precultured on solidified MS medium containing 3% sucrose and 0.2% gellan gum at 20°C for several days under supplementary lighting to provide a 12-h light/12-h dark photoperiod with an intensity of about 40 µmol -2 -1 m s . The vitrification procedure comprised the following steps (Niwata 1995): 1. Exposure of meristems to PVS2 vitrification solution consisting of 30% (w/v) glycerol, 15% (w/v) ethylene glycol, 15% (w/v) DMSO and 0.4M sucrose within MS medium (Sakai et al. 1991). 2. Direct immersion of meristems in LN in 0.5 ml PVS2 solution. 3. Rapid rewarming in a water-bath at 30°C. 4. Dilution of PVS2 by slow addition of a 3% sucrose solution over 15 min or a 1.2M sucrose solution over 20 min. 5. Plating meristems on MS medium containing 0.5 mg/L gibberellic acid.

Results Apical meristems excised from post-dormant bulbs of garlic were successfully cryopreserved by vitrification with subsequent high plant regeneration rate. Posters 441

Apical meristems were precultured on solidified Murashige and Skoog (1962) medium at 20°C for at least 2 d. The precultured meristems were treated with highly concentrated cryoprotective solution (PVS2; Sakai et al. 1991) at 25°C for 10–15 min before being directly immersed into liquid nitrogen. After dilution of PVS2, cryopreserved meristems on regrowth medium resumed growth within 5-7 d and directly developed normal shoots without intermediary callus formation. The average rate of shoot formation determined 30 d after plating was nearly 100%. The regenerated plants successfully formed garlic bulbs in the field. This protocol was most successful with post-summer dormant bulbs and produced lower survival and plant regeneration rates when applied to bulbs vernalized by cold storage. Both cryopreserved and untreated, excised from post-summer dormant bulbs did not initiate bulbing during regrowth (further in vitro culture) at 20°C while meristems dissected from cold-stored bulbs formed minibulbs, irrespective of the cryopreservation treatment. This means that cryopreservation and dissection did not affect the physiological phase (vegetative or vernalized) of the meristems of garlic bulbs used. This procedure was successfully applied to 11 other garlic cultivars with high subsequent plant regeneration. This vitrification procedure is very simple and appears promising for cryopreserving post-dormant garlic meristems.

Conclusions The critical points for successful cryopreservation of garlic meristems are: 1. Apical meristems used were in the vegetative phase, i.e. they were post- summer dormant, before being vernalized by cold storage. 2. Apical meristems were dissected to about 2 mm height and diameter, and comprised 3–4 leaf primordia. They were precultured on solidified MS medium at 20°C for at least 2 d under a 12-h light/12-h dark photoperiod. 3. The optimal period of exposure to PVS2 was 10–15 min. 4. Cooling and rewarming of apices were performed rapidly. After rewarming and diluting the cryoprotectant, the meristems were placed on medium containing 0.5 mg/L gibberellic acid. 5. The percentage of shoot formation of cryopreserved garlic meristems was increased to 100% by using a glass tube. The vitrification protocol described here will be a useful tool for the conservation of garlic genetic resources. These results may be applied for the cryopreservation of meristems of similar summer-dormant types (Mediterranean type) of plants which require cold storage for vernalization: with such plants, cold treatment does not necessarily increase the suitability for cryopreservation, unlike with cold-resistant plant materials.

References Murashige, T. and F. Skoog. 1962. A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiologia Plantarum 15:473–497. Niwata, E. 1995. Cryopreservation of apical meristems of garlic and high subsequent plant regeneration. Cryo–Letters 16:102–107. 442 Cryopreservation of Tropical Plant Germplasm

Sakai, A., S. Kobayashi and I. Oiyama. 1991. Survival by vitrification of nucellar cells orange (Citrus sinensis L. var. brasiliensis Tanaka) cooled to –196°C. Journal of Plant Physiology 137:465–470. Posters 443

Cryoexposure of in vitro shoot-tips of mangosteen – effects of sucrose and desiccation M.N. Normah and B.S. Tan Department of Botany, Faculty of Life Sciences, Universiti Kebangsaan Malaysia, 43600 UKM, Bangi, Selangor, Malaysia

Introduction Mangosteen (Garcinia mangostana L.) is a tropical fruit species with recalcitrant seeds. The seeds are considered relatively large (13.0 ± 0.2 mm width, 18.0 ± 0.6 mm length) (Normah et al. 1997) and do not have differentiated embryos. In fact, the whole seed structure is all axis with no defined meristems. On size alone, their highly hydrated seeds could neither be rapidly dehydrated nor cryopreserved. The use of in vitro shoot-tips/buds for cryopreservation is thus suggested. Cryopreservation using vitrification and encapsulation-dehydration techniques of shoot-tips has been shown to be successful for some plant species such as gooseberry and currant (Reed and Yu 1995), sugarbeet (Vandenbussche and de Proft 1998) and white clover (Yamada et al. 1991). Both techniques were tested for mangosteen shoot-tips but from preliminary results no success was obtained (Aliudin 1997). Preculturing on medium with high concentration of sucrose followed by desiccation and direct immersion in liquid nitrogen has proved to be successful for Asparagus officinalis (Uragami et al. 1990). The present study was undertaken to investigate the effects of sucrose and desiccation on the survival of mangosteen shoot-tips exposed to liquid nitrogen. Regeneration systems for shoot-tips and buds of mangosteen have been established (Aliudin 1997) and were used in the present study.

Materials and methods Freshly harvested mangosteen fruits were obtained from Ulu Langat, Selangor. Seeds were sterilized in 20% Clorox with a few drops of Tween 20 for 30 min, followed by rinsing three times in sterile distilled water. The seeds were then cut into six segments before culturing on in vitro shoot proliferation medium reported by Normah et al. (1995). In vitro shoots obtained were excised and precultured on MS (Murashige and Skoog 1962) medium supplemented with 0.1M and 0.3M sucrose for 48 h followed by desiccation under laminar airflow for 0, 0.5, 1 and 2 h. The shoot-tips were then wrapped in aluminium foil envelopes and plunged directly into liquid nitrogen for 24 h. Thawing was carried out in a water-bath (40°C). The shoot-tips were cultured on post- treatment media which consist of MS basal medium, MS medium supplemented with 2.5 mM NAA (a–naphthaleneacetic acid) and 20 mM BAP (6–benzylaminopurine), or MS medium with 2.5 mM NAA, 20 mM BAP, 0.2 mg/L GA3 (gibberellic acid). Activated charcoal at the concentration of 2 g/L was added to all media. A triphenyltetrazolium chloride (TTC) test was carried out in addition to observations of growth of shoots in culture. The cultures were incubated at 25°C under an 8-h light/16-h dark photoperiod -2 -1 with a light intensity of 25 mmol m s . Each treatment consisted of 10 explants 444 Cryopreservation of Tropical Plant Germplasm and was repeated three times.

Results and discussion Table 1 shows the viability of the shoot-tips after cryopreservation based on TTC test. The results show that desiccation for 2 h gave low percentages of viability for the control (16.67–56.67%) and no survival was obtained with cryopreserved shoot-tips. However, viable tissues (68–93%) were observed for cryopreserved shoot-tips desiccated for 0, 0.5 and 1 h following preculture treatment with 0.1M and 0.3M sucrose for 48 h. On post-treatment medium including 2.5 mM NAA and 20 mM BAP, the explants remained green for a longer period of time (4–5 d) compared with explants on the other two media where they turned yellow and died after 2–3 d in culture. The cryopreserved shoot-tips, however, did not respond further in growth after about 7 d in culture. Two hours of desiccation gave low viability of the shoot-tips even before cryopreservation. This is probably due to an injury that must have occurred during desiccation. As for the other periods of desiccation, with 0.1M sucrose preculture, viability was high even without desiccation under the laminar flow. Nevertheless, the viability results here need to be confirmed with regeneration of the shoot-tips. When observed under a light microscope, most of the cells that showed positive results were at the base of the explant and others were scattered throughout the shoot-tip. The number of cells that survived is probably not enough to cause recovery and allow further development of the shoot. The post-treatment medium may need to be improved to stimulate recovery of the shoot-tips after desiccation and cryopreservation. It was also observed that explants of 2–4 mm in size with the meristem dome exposed responded better to cryoexposure. The large surface area of the dome probably causes fast freezing to occur, hence allowing better survival. The concentration of sucrose in the preculture medium did not produce obvious differences in shoot-tip viability. For further studies, both the effects of concentration and desiccation on viability after cryopreservation probably still need to be investigated as it is important to reach the optimum moisture level for desiccation and freezing tolerance. Mycock et al. (1995) used a similar procedure for successful cryopreservation of somatic embryos of Coffea arabica, Manihot esculenta, Phoenix dactylifera and Pisum sativum. The potential for the procedure to work with mangosteen shoot-tips is shown in the present study. More work, however, needs to be done in order to obtain a successful technique for full regeneration and development of the cryopreserved shoot- tips. Posters 445

Table 1. Percentage viability (TTC test) of mangosteen shoot-tips after cryopreservation (5 d after culture) Sucrose concentration (M) in preculture medium Desiccation (h) Viability (%) Control 0.1 0 96.7 0.5 76.6 1 83.3 2 56.7 0.3 0 89.7 0.5 90.0 1 46.6 2 16.7 Cryopreserved 0.1 0 93.3 0.5 85.7 1 72.7 2 0 0.3 0 68.2 0.5 76.5 1 77.8

Acknowledgements The authors express their thanks to the International Foundation for Science (IFS) for funding the project. This work was also supported by the Malaysian National IRPA grant.

References Aliudin, R. 1997. Tissue culture and cryopreservation of mangosteen buds. MSc. Thesis, Universiti Kebangsaan Malaysia, Bangi, Selangor. Murashige, T. and F. Skoog. 1962. A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiologia Plantarum 15: 473–497. Mycock, D.J., J. Wesley–Smith and P. Berjak. 1995. Cryopreservation of somatic embryos of four species with and without cryoprotectant treatment. Annals of Botany 75: 331–336. Normah, M.N., A.B. Nor–Azza and R. Aliudin. 1995. Factors affecting in vitro shoot proliferation and ex vitro establishment of mangosteen. Plant Cell Tissue and Organ Culture 43: 291–294. Normah, M.N., D.R. Saraswathy and G. Mainah. 1997. Desiccation sensitivity of recalcitrant seeds – a study on tropical fruit species. Seed Science Research 7:179–183. Reed, B.M. and X. Yu. 1995. Cryopreservation of in vitro-grown gooseberry and currant meristems. Cryo–Letters 16: 131–136. Uragami, A., A. Sakai and M. Nagai. 1990. Cryopreservation of dried axillary buds from plantlets of Asparagus officinalis L. grown in vitro. Plant Cell Reports 9: 328–331. Vanderbussche, B. and M.P. de Proft. 1998. Cryopreservation of in vitro sugar beet shoot tips using encapsulation-dehydration technique: influence of abscisic acid and cold acclimation. Plant Cell Reports 17: 791–793. Yamada, T., A. Sakai, T. Matsumura and S. Higuchi. 1991. Cryopreservation of apical meristems of white clover (Trifolium repens L.) by vitrification. Plant Science 78: 81–87. 446 Cryopreservation of Tropical Plant Germplasm

Cryopreservation of almond apices using encapsulation- dehydration and vitrification Mohamad A. Shatnawi¹,², Florent Engelmann³, Andrea Frattarelli² and Carmine Damiano² ¹ Plant Tissue Culture Laboratory, Biotechnology Section, Center for Agriculture Research and Production, Jordan University of Science and Technology, Irbid, Jordan ² Istituto Sperimentale per la Frutticoltura, 00040 Ciampino Aeroporto, Rome, Italy ³ IPGRI, 00145 Rome, Italy

Introduction Prunus is a genus of the Rosaceae family which comprises 77 species including a large number of fruit trees, ornamentals and rootstocks (e.g. plum, peach, apricot, cherry and almond). Many of these species are of great economic importance in temperate regions. These species are usually propagated vegetatively to maintain clonal genotypes and genetic resources of these species are traditionally maintained as whole plants in field genebanks. Field genebank conservation presents certain drawbacks that limit its efficiency and threaten its security (Withers and Engels 1990). Cryopreservation of apices represents the only current option for the safe long-term conservation of vegetatively propagated crops. Only a limited number of publications report cryopreservation experiments with Prunus using apices (Brison et al. 1995; Helliot and de Boucaud 1997) and zygotic embryos (de Boucaud et al. 1996). In the case of almond, one report only has been published on the cryopreservation of embryonic axes after partial desiccation (Chaudhury and Chandel 1995). In this paper, we present some of the results obtained with the cryopreservation of apices of almond in vitro plantlets using the vitrification and encapsulation-dehydration techniques (Shatnawi et al. 1999).

Materials and methods

Plant material The in vitro cultures of rootstock M51 and cultivar Ferragnes used in this study originated from apices which had been sampled on 1-year-old plants and introduced and propagated in vitro.

In vitro culture M51 in vitro plantlets were cultured on modified MS medium (Murashige and Skoog 1962). Plants were grown at 24±1°C, under a photoperiod of 16-h -2 -1 light/8-h dark, with a light intensity of 37 µmol m s . Subcultures were performed every 15 d.

Cryopreservation Mother-plants were cold-hardened at 5°C for 0–5 weeks. For vitrification, Posters 447 apices were cultured for 24 h on solid medium containing 0.3M sucrose. They were loaded 10 min with a solution containing 2M glycerol + 0.4M sucrose in liquid medium, then treated with PVS2 vitrification solution (Sakai et al. 1990) at 0°C for 30–180 min. Cryotubes were then immersed directly in liquid nitrogen. Rewarming was performed in a water-bath thermostated at 40°C. Apices were rinsed in a 1.2M sucrose solution for 10 min, then placed on solid medium with 0.4M sucrose for 24 h and transferred to standard culture medium for growth recovery. For encapsulation-dehydration, apices were cultured for 24 h on solid medium containing 0.3M sucrose. They were then encapsulated and pregrown for various durations (1–7 d) in liquid medium with sucrose concentrations ranging between 0.3 and 1.25M. Desiccation was performed by placing the beads in airtight containers with silica gel for 0–30 h. After desiccation, beads were immersed rapidly in liquid nitrogen. Thawing was performed by placing the cryotubes in the air current of the laminar flow cabinet for 3–4 min. Beads were then transferred to Petri dishes containing standard medium. Apices were placed in the dark for 1 week, then cultured under standard conditions. Apices showing any sign of regrowth after 2 weeks were extracted from the beads. The necrotic basal part was removed and apices were placed directly on the medium for further regrowth.

Results Increasing the duration of cold-hardening treatment allowed control apices to withstand longer exposures to the PVS2 vitrification solution (Table 1). Very low survival of apices was achieved after cryopreservation, and only after the longest cold-hardening treatment. Preliminary experiments (data not shown) had indicated that optimal pregrowth conditions for almond apices included 3 d of culture in liquid medium with 0.75M sucrose and that high survival rates were obtained after desiccation of beads to moisture contents between 20.3 and 18% (fresh weight basis). Survival of control apices of both genotypes tested decreased in line with decreasing bead moisture contents (Table 2). Survival of cryopreserved apices was higher with accession M51 than with cultivar Ferragnes, and the highest survival was achieved for slightly higher moisture content (20.3–20.1% for M51; 19% for Ferragnes). Apices which were alive after cryopreservation remained green and started producing new shoots within 1 week. However, they could grow and develop into whole plantlets only if they were extracted from the bead and placed directly on culture medium.

Discussion/conclusion This study demonstrated that almond apices could be cryopreserved using both the vitrification and encapsulation-dehydration techniques. However, survival of apices after vitrification was very low under the conditions tested. The effect of increased durations of cold-hardening treatment of mother-plants should be tested and other vitrification solutions (e.g. PVS3; Nishawaza et al. 1992) or cryopreservation techniques (e.g. encapsulation-vitrification) could be tested to try improving the survival rates. 448 Cryopreservation of Tropical Plant Germplasm

Table 1. Effect of cold-hardening and duration of treatment with PVS2 vitrification solution on the survival (%) of control (–LN) and cryopreserved (+LN) apices of almond selection M51 (Adapted from Shatnawi et al. 1999, with permission) Duration of cold-hardening (weeks) Duration of PVS2 0 3 5 treatment (min) –LN +LN –LN +LN –LN +LN 0 100 0 100 0 100 0 60 50 0 81 0 90 0 120 20 0 33 0 80 10 150 10 0 33 0 70 9 180 5 0 20 0 40 0

Table 2. Effect of desiccation on the survival of control (–LN) and cryopreserved (+LN) apices of almond selection M51 and cultivar Ferragnes. Apices were pretreated for 3 d in medium containing 0.75M sucrose before dehydration and cryopreservation (Adapted from Shatnawi et al. 1999, with permission) Desiccation M51 Ferragnes duration (h) Bead MC (% FW) –LN +LN –LN +LN 8 20.3 90 60 80 33 9 20.1 80 62 66 40 14 19.0 62 45 56 50 20 18.0 54 44 40 20

Acknowledgements M.A. Shatnawi gratefully acknowledges the support provided by a Special Skills Training Award within the Italian Government funded training scheme of IPGRI. The authors thank the Istituto Sperimentale per la Frutticoltura for providing the research facilities.

References Brison, M., M.T. de Boucaud and F. Dosba. 1995. Cryopreservation of in vitro grown shoot tips of two interspecific Prunus rootstocks. Plant Science 105: 235–242. de Boucaud, M.T., B. Helliot and M. Brison. 1996. Desiccation and cryopreservation of embryonic axes of peach. Cryo–Letters 17: 379–390. Chaudhury, R. and K.P.S. Chandel. 1995. Cryopreservation of embryonic axes of almond (Prunus amygdalus Batsch.) seeds. Cryo–Letters 16: 51–56. Helliot, B. and M.T. de Boucaud. 1997. Effect of various paramaters on the survival of cryopreserved Prunus Ferlenain in vitro plantlet shoot tips. Cryo–Letters, 18: 133–142. Murashige, T. and F. Skoog. 1962. A revised medium for rapid growth and bioassays with tobacco tissue cultures. Plant Physiology 15, 473–497. Nishizawa, S., A. Sakai, Y. Amano and T. Matsuzawa. 1992. Cryopreservation of asparagus (Asparagus officinalis L.) embryogenic suspension cells and subsequent plant regeneration by a simple freezing method. Cryo–Letters 13: 379–388. Sakai, A., S. Kobayashi and I. Oiyama. 1990. Cryopreservation of nucellar cells of navel orange (Citrus sinensis Osb. var. brasiliensis Tanaka) by vitrification. Plant Cell Reports 9: 30–33. Shatnawi, M., F. Engelmann, A. Frattarelli, C. Damiano and L.A. Withers. 1999. Cryopreservation of apices from in vitro plantlets of almond (Prunus dulcis Mill.). Cryo– Letters 20: 13–20. Withers, L.A. and J.M.M. Engels. 1990. The test tube genebank – a safe alternative to field conservation. IBPGR Newsletter for Asia and the Pacific 3:1–2. Posters 449

Cryopreservation of shoot-tips of two leguminous trees (Acacia mangium and Paraserianthes falcataria) using encapsulation-dehydration and vitrification Enny Sudarmonowati¹, Rosmithayani¹, E.S. Mulyaningsih¹, D. Priadi¹ and Akira Sakai² ¹ R&D Centre for Biotechnology-Indonesian Institute of Sciences, Indonesia ² Asabucho 1-5-23, Kitaku, Sapporo 001, Japan

Introduction Cryopreservation of plant organs has been successfully applied to numerous plant species. However, this technique has been mainly applied to annual crops, especially those grown in temperate regions. Research on cryopreservation of tropical forest tree species has been very limited and the success achieved has been very low. Attempts to preserve Acacia mangium and Paraserianthes falcataria, as model species of tropical forest trees, using simple cryopreservation techniques were therefore carried out. Acacia mangium and P. falcataria are both leguminous multipurpose trees which mainly provide raw material for the pulp and paper industry. Their wood could be used for furniture, artificial boards and handicraft, as it has reasonable physical and mechanical properties. These two species have been recommended by the Government of Indonesia for the development of industrial timber estates as they could be planted in marginal lands and are considered fast-growing trees (harvesting age is around 7–8 years). The aim of this research was to determine the most appropriate cryopreservation method for A. mangium and P. falcataria by investigating some factors affecting their survival rate after immersion in liquid nitrogen (–196°C).

Materials and methods

Source of explants Shoot-tips of A. mangium and P. falcataria were excised from multiple shoots derived from seeds germinated in vitro on MS hormone-free medium.

Culture medium and prefreezing conditions Multiple shoots of A. mangium and P. falcataria were maintained on MS medium containing 2 mg/L BAP. This medium was also used for regenerating shoot-tips after immersion in liquid nitrogen. Shoot-tips of both species were precultured for 10 d on MS medium containing 0.4M sucrose. All cultures were incubated at 25±1°C under a 16-h light/8-h dark photoperiod.

Vitrification procedure After incubation on high sucrose-containing medium, shoot-tips of A. mangium and P. falcataria were subjected to various vitrification solutions for 20, 25 or 30 min. Eight and two compositions of vitrification solution were tested for 450 Cryopreservation of Tropical Plant Germplasm

P. falcataria and A. mangium, respectively.

Encapsulation-dehydration procedure Shoot-tips were encapsulated in 3% sodium alginate prior to dehydration on MS medium containing 0.4M sucrose. After pregrowth on this medium for 10 d, the encapsulated shoot-tips were air-dried under the laminar airflow or in Petri dishes containing silica gel for 3 or 5 h.

Regeneration after storage in liquid nitrogen Frozen shoot-tips were quickly thawed in a water-bath set at 40°C for 2 min. The thawed, encapsulated shoot-tips were cultured on sterile filter paper placed on MS medium of the same composition as the preculture medium. The alginate beads were removed after 3 d on this medium.

Results The highest percentage of surviving shoot-tips (70%) of A. mangium was obtained using encapsulation-dehydration, while that of P. falcataria (50%) was obtained using vitrification (Tables 1 and 2). It appears that different species require different cryopreservation methods. These results are in accordance with those of Benson et al. (1996) who also compared these two cryopreservation techniques for Ribes nigrum. For this latter species, encapsulation-dehydration was superior to vitrification in terms of both plant recovery and genotypic coverage. Encapsulation-dehydration has ensured high survival rates after storage in liquid nitrogen of many plant species. Desiccation with silica gel for 5 h gave the best result in terms of survival rate, with both A. mangium and P. falcataria. Silica gel has been used for dehydrating shoot-tips of several species such as sugarbeet (Vandenbussche and de Proft 1996) and horseradish (Punchindawan et al. 1997). Vitrification has been the most appropriate technique for certain species such as navel orange (Sakai et al. 1990) and taro (Takagi et al. 1997). Of eight different compositions of the vitrification solution, six contained the same compounds as PVS2 but their concentrations were different; the other two solutions contained a combination of sorbitol and sucrose or a mixture of glycerol and sucrose. The results obtained indicated that PVS2 (Sakai et al. 1990) seemed to be the best for cryopreservation of shoot-tips of P. falcataria. PVS2 has led to the highest survival rates for many plant species such as Ribes nigrum (Benson et al. 1996), horseradish (Punchindawan et al. 1997) and taro (Takagi et al. 1997). A 20-min exposure to the vitrification solution seemed to be optimal as this period gave the highest survival rate of shoot-tips of P. falcataria after immersion in liquid nitrogen. This result supports the results obtained by Takagi et al. (1997) with taro apices. As the regeneration of plants from shoot-tips was fast and easy, these organs were used for cryopreservation research with both A. mangium and P. falcatria. Shoot-tips have been used as materials to be cryopreserved with many plant species. Posters 451

Table 1. Cryopreservation of shoot-tips of A. mangium and P. falcataria using the encapsulation-dehydration procedure Desiccation Species Method Duration (h) Survival rate (%) A. mangium Control – 100 –, LN 0 Air-drying 3 100 3, LN 10 5 100 5, LN 40 Silica gel 3 100 3, LN 0 5 100 5, LN 70 P. falcataria Control – 100 –, LN 0 Air-drying 3 100 3, LN 0 5 100 5, LN 0 Silica gel 3 100 3, LN 0 5 80 5, LN 40

Table 2. Cryopreservation of shoot-tips of A. mangium and P. falcataria using the vitrification method Species Exposure to vitrification solution (min) Survival rate (%) A. mangium – 100 –, LN 0 10 80 10, LN 0 20 30 20, LN 0 P. falcataria – 100 –, LN 0 10 90 10, LN 0 20 90 20, LN 50

Conclusion The success of cryopreservation was affected by numerous factors including: the species (genotype-dependent), the type of tissues, the composition of the vitrification solution, the period of exposure to the solution, the desiccation methods, and the regeneration procedure. 452 Cryopreservation of Tropical Plant Germplasm

Encapsulation-dehydration as well as vitrification could give high survival rates with A. mangium and P. falcataria, respectively. The protocols developed to conserve shoot-tips of A. mangium and P. falcataria might be used as a model for other tropical forest tree species with recalcitrant seeds. Further research to obtain higher survival rates and normal plantlet regeneration needs to be carried out.

References Benson, E.E., B.M. Reed, R.M. Brennan, K.A. Clacher and D.A. Ross. 1996. Use of thermal analysis in the evaluation of cryopreservation protocols for Ribes nigrum L. germplasm. Cryo–Letters 17: 347–362. Punchindawan, M., K. Hirata, A. Sakai and K. Miyamoto. 1997. Cryopreservation of encapsulated shoot primordia induced in horseradish (Armoracia rusticana) hairy root cultures. Plant Cell Reports 16: 469–473. Sakai, A., S. Kobayashi and I. Oiyama. 1990. Cryopreservation of nucellar cells of navel orange (Citrus sinensis Osb. var. brasiliensis Tanaka) by vitrification. Plant Cell Reports 9: 30–33. Takagi, H., N.T. Thinh, O.M. Islam, T. Senboku and A. Sakai. 1997. Cryopreservation of in vitro-grown shoot tips of taro (Colocasia esculenta (L.) Schott) by vitrification. 1. Investigation of basic conditions of the vitrification procedure. Plant Cell Reports 16: 594–599. Vandenbussche, B. and M.P. de Proft. 1996. Cryopreservation of in vitro sugarbeet shoot tips using encapsulation-dehydration technique: development of a basic protocol. Cryo– Letters 17: 137–140. Posters 453

Cryopreservation of in vitro-grown apical meristems of terrestrial orchids (Cymbidium spp.) by vitrification Nguyen Tien Thinh and Hiroko Takagi JIRCAS, Okinawa Sub-Tropical Research Center, Ishigaki, Okinawa 907, Japan

Introduction In various humid, tropical countries, orchid germplasm is threatened due to rapid deforestation and excessive exploitation of rare and/or beautiful species. The orchid micropropagation industry also has to deal with the costly and laborious in vitro maintenance of large numbers of commercial hybrid clones. Therefore, cryopreservation appears as a promisingly safe and economical tool for the conservation of the orchid genepool. Currently, information on the cryopreservation of orchid germplasm is very limited. Na and Kondo (1996) reported the conservation of cultured shoot primordia of Vanda pumila in LN by abscisic acid preculture and dessication. Ishikawa et al. (1997) cryopreserved cultured zygotic embryos of a Japanese terrestrial orchid (Bletilla striata) by the vitrification technique. In this report, we present a simple and efficient vitrification technique for the cryopreservation of apical meristems of several commercial clones of Cymbidium orchids.

Materials and method

Materials Three-month-old clonally propagated in vitro plantlets of cv. Suva Royal Velvet (cv. Suva) were used as donors providing three kinds of meristems for the cryopreservation experiments. These dissected meristems were: (i) meristems with apical dome fully covered (FC type), (ii) meristems with apical dome partly covered (PC type), and (iii) meristems with apical dome uncovered (O type) by the outer leaf primordia. The vitrification protocol used with meristems of cv. Suva was later tested on the meristems of four additional cultivars: Nandy Green Mist, Sensation Night, Sayonara and Miretta.

Vitrification method All dissected meristems were precultured overnight in the dark on MS basic medium supplemented with 0.3M sucrose. Precultured meristems (5–7 meristems) of the same type were wrapped together in a piece of tissue paper (referred to as TP) and subjected to loading treatment for 20 min at 25°C in a solution of 2M glycerol + 0.4M sucrose. Next, the meristems were dehydrated for 5–60 min at 25°C in PVS2 solution (Sakai et al. 1990), then inserted in a 0.7- ml cryotube, filled with fresh PVS2 and rapidly plunged into LN. After 1 h of storage in LN, the cryotubes were rapidly thawed (90 s in a water-bath at 40°C). The meristems were then unloaded for 15 min in liquid MS basic medium supplemented with 1.2M sucrose. Next, the meristems were released out of the TP and blotted for 1 night in the dark on a filter paper laid over the 454 Cryopreservation of Tropical Plant Germplasm gel surface of MS basic medium supplemented with 0.3M sucrose. For post- thaw regrowth, the blotted meristems were subcultured on three different media composed by either Knudson (1943), Morel (1960) or Wimber (1963) (these media were reviewed by Arditti and Ernst 1992). Cultures were incubated for 10 d in dim light at 25°C, then transferred to a light intensity of -2 -1 about 80–90 mE m s and a photoperiod of 16-h light/8-h dark. After 1 month of culture, meristems that could produce regenerative protocorms were considered living.

Results and discussion Loaded meristems of Cymbidium cv. Suva tolerated quite well the dehydration by PVS2 at 25°C. For example, more than 90, 70 and 50% of PC meristems could survive 30, 40 and 60 min of treatment with PVS2, respectively. This enabled a thorough dehydration of meristems by PVS2 with less damage; in other words, the well-dehydrated meristems could be vitrified at LN temperature (–196°C). After cooling in LN, the results showed that PC meristems showed the highest rate of post-thaw survival (93.3%) within dehydration periods of 10–20 min, followed by the O (86.6%) and the FC ones (40%). Thus, PC meristems and a 10-min PVS2 treatment were selected as the most suitable explants and dehydration time for further applications to other cultivars. Seventy-six to 90% of meristems from the four other Cymbidium cultivars were successfully cryopreserved by the vitrification procedure established with cv. Suva. With all cultivars tested, surviving meristems produced protocorms and healthy plants similar to the controls (which were precultured, loaded and dehydrated, but not frozen in LN). The regenerated plants were successfully acclimatized in pots in the greenhouse, where no differences between growth of plants surviving cryopreservation and control plants were observed. The successful recovery of cryopreserved meristems reported above was obtained by using the culture medium described by Wimber (1963). In descending order, the media used for Cymbidium meristem culture reported by Morel (1960) and Knudson (1943) resulted in significantly lower rates of regrowth of cryopreserved meristems [see Arditti and Ernst (1992) for the composition of these media]. This vitrification procedure appears to be a simple, fast and efficient cryotechnique suitable for the conservation of commercial Cymbidium clones. Its applicability to meristems of other hybrid clones as well as of wild species should be tested.

Acknowledgements Thanks are due to Prof. Dr A. Sakai (Hokkaido, Japan) for his enthusiastic encouragement and for the documents provided on orchid propagation, and to Dr S.D. Hamill (Queensland Horticulture Institute, Australia) for correction of English. Nguyen Tien Thinh is grateful to JIRCAS for providing a Visiting Research Fellowship. Posters 455

References Arditti, J. and R. Ernst. (eds.). 1992. Micropropagation of orchids. John Wiley & Son, Inc. New York, USA. Ishikawa, K., K. Harata, M. Mii, A. Sakai, K. Yoshimatsu and K. Shimomura. 1997. Cryopreservation of cultured zygotic embryos of a Japanese terrestrial orchid (Bletilla striata) by vitrification. Plant Cell Reports 16: 754–757. Knudson, L. 1943. Nutrient solutions for orchid seed germination. American Orchid Society Bulletin 12: 77–79. Morel, G. 1960. Producing virus-free Cymbidium. American Orchid Society Bulletin 29: 495– 497. Na, H.Y. and K. Kondo. 1996. Cryopreservation of tissue-cultured shoot primordia from shoot apices of cultured protocorms in Vanda pumila following ABA preculture and desiccation. Plant Science 118: 195–201. Sakai, A., S. Kobayashi and I. Oiyama. 1990. Cryopreservation of nucellar cells of navel orange (Citrus sinensis Osb. var brasiliensis Tanaka) by vitrification. Plant Cell Reports 9: 30–33. Wimber, D.D. 1963. Clonal multiplication of Cymbidium through tissue culture of the shoot meristem. American Orchid Society Bulletin 32: 105–107. 456 Cryopreservation of Tropical Plant Germplasm

Cryopreservation of hairy root cultures of useful plants by encapsulation-dehydration Kazumasa Hirata and Kazuhisa Miyamoto Environmental Bioengineering Laboratory, Graduate School of Pharmaceutical Sciences, Osaka University, 1-6 Yamada-oka, Suita, Osaka 565-0871, Japan

Introduction Cryopreservation has become important as a means of ensuring the long-term conservation of plant germplasm. In the case of higher plants, roots are considered to be an appropriate material for cryopreservation since many uniform root samples can be taken from plants growing in the field without inflicting lethal damage. In vitro-transformed hairy root cultures constitute a very useful model material for investigating cryopreservation of roots because of the high levels of uniformity of their morphology and their high regeneration ability. However, there are only a few reports in the literature on the cryopreservation of root cultures by the conventional slow-prefreezing technique. Recently, cryopreservation by vitrification and encapsulation- dehydration techniques has been applied to various plant materials. In contrast to the vitrification technique, the encapsulation-dehydration technique does not require toxic cryoprotectants like dimethylsulfoxide and ethylene glycol, and the encapsulation of the thin hairy roots in gel beads makes their handling much easier. In this study, horseradish hairy root cultures which showed high levels of productivity of the clinically important enzyme peroxidase were employed to investigate the applicability of the encapsulation-dehydration technique to the cryopreservation of plant roots.

Materials and methods Hairy root cultures of horseradish were subcultured in the dark every 4 weeks in Murashige and Skoog (MS) liquid medium at 25°C. The cultures easily produced shoot primordia in the presence of light. Root tips excised from 2- week-old cultures were suspended in a 2% (W/V) sodium alginate solution, and then immediately dropped into a 50 mM calcium chloride solution. After 5 min to allow the formation of Ca–alginate beads (5 mm in diameter), the beads were precultured for 1 d on MS medium in Petri dishes. The beads were then slowly dehydrated by placing the Petri dishes in a desiccator with silica gel for about 60 h to reduce the water content to 33%. Dehydrated beads were placed in cryotubes which were then immersed in LN. After storage for 3 d, the beads were rewarmed rapidly at 40°C and then directly transferred onto MS medium. The survival rate was recorded as the percentage of beads showing definite elongation of root tips after cultivation for 4 weeks.

Results and discussion With the encapsulation-dehydration technique, sucrose is generally added to both the preculture and encapsulation media to allow the acquisition of desiccation tolerance. Recently, Matsumoto and Sakai (1995) reported that the Posters 457 addition of glycerol together with sucrose was more effective than sucrose alone for obtaining a high survival rate of the encapsulated adventitious buds of lily. We also found that the desiccation tolerance of horseradish shoot primordia increased following the addition of glycerol and sucrose, the survival rate after cooling in LN being more than 90% (Phunchindawan et al. 1997). Therefore, this procedure was applied to the root tips but the survival rate of the root tips dropped to zero after cooling in LN under optimal concentrations of glycerol (1M) and sucrose (0.5M) for the shoot primordia. Hence, combinatorial optimization of the concentrations of glycerol and sucrose to increase survival rate was done. Addition of 0.5M glycerol and 0.3M sucrose gave the highest survival rate (33%) among the various conditions tested. To obtain a higher survival rate, the effect of ABA on the survival rate was investigated. ABA is a well-known phytohormone that plays an important role in the acquisition of desiccation tolerance. The hormone has also been found to be effective in conferring tolerance against freezing and for obtaining high survival rates in cryopreservation (Kendall et al. 1993). When 2 mM ABA was added to the preculture medium and beads, the survival rate declined more slowly as the water content was reduced by desiccation in comparison with that in the non-treated control. Compared with 33% water content, a survival rate of about 60% was obtained in ABA-treated cryopreserved root tips, higher than that in non-treated root tips. A significant increase in the proline content of ABA-treated root tips was observed after preculture for 1 d. These results suggest that endogenous synthesis of proline was stimulated by preculture with ABA and that the ABA added and/or proline synthesized play important roles in attaining a high survival rate after cooling in LN. Determining the optimal conditions for ABA treatment is necessary both to increase the survival rate and to establish a generally applicable cryopreservation technique for cultured and normal roots.

References Kendall, E. J., K. K. Kartha, J. A. Qureshi and P. Chermak. 1993. Cryopreservation of immature spring wheat zygotic embryos using an abscisic acid pretreatment. Plant Cell Reports 12:89–94. Matsumoto, T. and A. Sakai. 1995. An approach to enhance dehydration tolerance of alginate-coated dried meristems cooled to –196°C. Cryo–Letters 16:299–306. Phunchindawan, M., K. Hirata, A. Sakai and K. Miyamoto. 1997. Cryopreservation of encapsulated shoot primordia induced from horseradish (Armoracia rusticana) hairy root cultures. Plant Cell Reports 16:469–473. 458 Cryopreservation of Tropical Plant Germplasm

Differential response and freezing tolerance of horticultural plant tissues by sugar-incubation in aid of cryopreservation Yutaka Jitsuyama¹, T. Suzuki¹, T. Harada¹ and Seizo Fujikawa² ¹ Department of Horticulture, Faculty of Agriculture, Hokkaido University, Sapporo 060-8589, Japan ² Institute of Low Temperature Sciences, Hokkaido University, Sapporo 060-8589, Japan

Introduction Sugar-incubation before freezing is widely used in procedures developed for cryopreservation of plant tissues by both conventional (two-step freezing) and vitrification methods (Kartha 1985). Preculture in media with a high concentration of sugars is clearly effective for increasing freezing tolerance in some plant tissues such as cabbage leaves (Sakai and Yoshida 1968). In other plant tissues such as onion bulbs, however, sugar-incubation is not effective for increasing freezing tolerance. In order to elucidate the different effects of sugar-incubation on the freezing tolerance of these two plant tissues, we investigated: (i) the changes in survival during sugar-incubation as well as the osmotic sensitivity, (ii) the changes in freezing tolerance after sugar-incubation, and (iii) the processes of sugar loading into the tissue cells.

Materials and methods Onion bulbs (Allium cepa L. cv. Sapporo–Ki) and cabbage leaves (Brassica oleracea L. cv. Early Ball) were used as experimental materials. The pieces of material were incubated with 0.8M sorbitol solutions, for specific times. For analysis of survival during sugar-incubation, the samples were incubated with 0.8M sorbitol solution, for specific times. For analysis of the osmotic sensitivity, samples incubated with 0.8M sorbitol solution were directly transferred into sorbitol solution diluted to 0.4M. Survival was evaluated by measurement of electrolyte leakage and observation of vital staining with fluorescein diacetate (FDA), using confocal laser scanning microscopy (CLSM). For equilibrium freezing, the samples were frozen at 0.05°C/min (onion) or 0.1°C/min (cabbage) to desired temperatures. For analysis of survival, samples were thawed rapidly to room temperature. LT50 (freezing temperature for 50% survival) was calculated from the survival curve measured by electrolyte leakage. The samples, which were incubated with 0.8M sorbitol solution containing 1 mg/ml Lucifer Yellow carbohydrazide (LYCH) for specific times, were washed with 0.8M sorbitol solutions and the process of sugar-loading (by incorporation of LYCH as a marker for incorporation of sugar into cells) was observed using CLSM. The effect of sugar transport for loading of sucrose into cells was evaluated using incubation with 1 mM p–chloromercuribenzene sulfonic acid (PCMBS), Posters 459 as an inhibitor of sucrose transporter, for 1 h in the presence of 0.8M sucrose, as described previously (Thierry et al. 1997).

Results and discussion The temperatures of constitutive freezing tolerance of onion bulbs and cabbage leaves before sugar-incubation, as measured by electrolyte leakage, were –2.7°C and –3.3°C, respectively. Samples of both tissues were incubated with 0.8M sorbitol solution for specific times and the change in viability during incubation was examined by electrolyte leakage. A reduction in viability occurred abruptly soon after immersion into sorbitol solution in the case of onion bulbs, while cabbage showed gradual reduction of survival in line with increasing immersion periods. The osmotic response was also different in two species. When the onion bulbs incubated with a 0.8M sorbitol solution were transferred into a 0.4M sorbitol solution, survival was distinctly reduced. Such a reduction did not occur in cabbage leaves. The same tendency was shown by observation with FDA vital staining. These results may indicate that onion bulb tissues are more sensitive to osmotic stress than cabbage leaves. In the case of cabbage leaves, incubation with a 0.8M sorbitol solution resulted in an increase in freezing tolerance to –9 to –13°C, depending on the duration of the incubation period. In the case of onion bulbs, however, sugar- incubation had little effect on freezing tolerance. Because the increase in freezing tolerance by sugar-incubation may be attributed to incorporation of sugars during incubation, the process of sugar incorporation and subsequent distribution of sugars within cells were examined by CLSM. With both species, incubation with 0.8M sorbitol solution resulted in plasmolysis. Plasmolysis began to occur after 10 min of incubation and reached a maximum within 1 h, without further distinct changes. In onion, sorbitol was incorporated into cells by endocytotic vesicles ranging from 0.1 to 1 µm at 10-min incubation, although the concentration of LYCH in vesicles was very low, such that the same laser intensity as for cabbage hardly detected the incorporated vesicles. At higher laser intensity, incorporation of LYCH was still barely observable in onion. Incorporation of vesicles was restricted to cytoplasm but not in vacuoles. The appearance of incorporated vesicles, in respect to the intensity and distribution, did not change even after incubation for 1–3 h. In the case of onion, incubation for 1 h brought about vesiculation of tonoplasts as observed with FDA staining, while such additional changes were not observed in cabbage even after more prolonged incubation. In cabbage leaves, incorporation of vesicles ranging from 0.1 to 5 µm was also observed at 10-min incubation. The incorporated vesicles increased gradually in size (1–10 µm) and number within 1 h, while remaining restricted to the cytoplasm in their distribution within the cell. The incorporation of vesicles was essentially unchanged from 1 to 48 h. Some of the incorporated vesicles showed movement around cytoplasmic organelles. Furthermore, even after freezing (to non-lethal temperatures) and thawing of the cells, the incorporated vesicles remained intact and without disruption. On the other 460 Cryopreservation of Tropical Plant Germplasm hand, the possibility that some sugars might be incorporated during incubation independently of vesicles is suggested by careful observations of cells after prolonged incubation which shows the dispersion of LYCH, though faintly, throughout the entire background of the cytoplasm. To test more precisely the possibility of sugar incorporation other than by endocytotic vesiculation, cabbage cells were incubated with PCMBS, an inhibitor of sucrose transporter, in the presence of 0.8M sucrose. The presence of PCMBS did not change incorporation of vesicles, but freezing tolerance was significantly reduced (P<0.05). It is suggested that freezing injury may be prevented by sugars incorporated through carriers, rather than those incorporated by endocytotic vesicles.

Conclusion In onion bulbs, sugar-incubation itself brought about injury due to the inherent osmotic sensitivity. In cabbage leaves, sugar-incubation resulted in increased freezing tolerance by incorporation of sugars by transporter, but not by endocytotic vesiculation.

References Kartha, K.K. (ed.). 1985. Cryopreservation of Plant Cells and Organs. CRC Press, Boca Raton, Florida. Sakai, A. and S. Yoshida. 1978. The role of sugar and related compounds in variations of freezing resistance. Cryobiology 5: 160–174. Thierry, C., H. Tessereau, B. Florin, M.C. Meschine and V. Pétiard. 1997. Role of sucrose for the acquisition of tolerance to cryopreservation of carrot somatic embryos. Cryo–Letters 18: 283–292. Posters 461

Cryopreservation at CATIE: an additional tool for the conservation of tropical agricultural crops and forest species María Elena Aguilar¹, Nelly Vásquez¹, Emelda Yah¹, Florent Engelmann² and François Côte¹ ¹ CATIE (Tropical Agricultural Research and Higher Education Center), Biotechnology Laboratory, Turrialba 7170, Costa Rica ² IPGRI, 00415 Rome, Italy

Introduction CATIE is a regional centre of research and post-graduate education in agriculture and natural resources. One of CATIE’s missions is to improve and conserve genetic resources of Central America and the Caribbean region. Cryo- preservation is a useful tool to support these objectives. This paper gives a brief overview of cryopreservation research activities currently ongoing in CATIE. The collection of Coffea arabica at CATIE is one of the largest wordwide, with a total of 1820 accessions. To ensure long-term conservation of this collection, CATIE has initiated a collaborative research programme with ORSTOM, France and IPGRI on cryopreservation of coffee seeds. Since Coffea arabica is self-fertile, many cultivated accessions can be considered homozygous. For these accessions, seeds could be cryopreserved after self-pollination. A core collection to be cryopreserved will be defined using passport genetic diversity structure and characterization data. CATIE is deploying important efforts toward the rescue, characterization and conservation of endangered tropical forest species. Many of these species have recalcitrant seeds. A project aiming at developing conservation methods for forest tree species has been initiated at CATIE. Different cryopreservation methods including encapsulation-dehydration, vitrification and slow freezing are tested with shoot-tips and embryonic axes. Bananas and plantains are seriously threatened by pests and numerous viral and fungal diseases. Non-conventional improvement methods are being used for parthenocarpic edible bananas and plantains. A cryopreservation technique has been developed to conserve embryogenic cell suspensions of different Musa cultivars which are produced in CATIE with these programmes. The objectives of CATIE’s cryopreservation programme are: · To rationalize CATIE’s coffee collection by adopting ORSTOM’s cryopreservation protocol for long-term storage of coffee seeds · To develop cryopreservation protocols for shoot-tips, seeds and embryonic axes that could be applied to recalcitrant seeds and vegetative material from forest tree species · To optimize a cryopreservation technique for embryogenic cell suspensions of Musa cultivars. 462 Cryopreservation of Tropical Plant Germplasm

Cryopreservation of coffee germplasm The first step of the seed cryopreservation coffee project is to transfer the cryopreservation protocol (Dussert et al. 1998) to CATIE. Experiments will be first performed with 2–3 genotypes and will focus on the optimization of the seed osmoconditioning procedure after thawing. Depending on the results, trials will be carried out under nursery/greenhouse conditions, either to achieve direct germination of osmoconditioned seeds or to improve the transfer of germinated embryos to nursery/greenhouse. Optimized protocols will then be applied to 13 C. arabica accessions chosen from wild and cultivated accessions, based on their variability in biochemical composition and size of their seeds. The first results will provide preliminary information on the feasibility of cryopreserving a broad genetic diversity using a single protocol.

Cryopreservation of tropical tree germplasm In tropical forest tree species, several methods will be tested including encapsulation-dehydration, vitrification and slow freezing. Experiments will be carried out with 2–3 species to establish technical procedures. The first experiments are being performed for encapsulation-dehydration with mahogany (Swietenia macrophylla) shoot-tips. Trials are under way to determine the optimal conditions for pregrowth (sucrose concentration (0.3–0.75M) and pretreatment duration); bead desiccation under the laminar airflow (0, 2, 4, 6 h); direct immersion in liquid nitrogen; rapid thawing and recovery. The culture medium utilized for these experiments was developed for microcutting culture (Orellana 1997). Limited survival (7%) of cryopreserved mahogany shoot-tips was achieved after pregrowth for 3 d in liquid medium with 0.5M sucrose, desiccation of beads for 6 h (27% water content), followed by direct immersion in liquid nitrogen and rapid thawing. Studies on in vitro conservation have not been reported for this species. However, Maruyama et al. (1997) reported shoot-tip encapsulation and storage at 12–25ºC in Cedrela odorata. The best storage temperature was 12ºC with a plant conversion rate of 80% after 12 months. Our objective for the short term is to improve the results obtained by performing additional experiments including modifications of the pregrowth and desiccation periods. Regrowth of mahogany shoot-tips after cryopreservation opens new possibilities that could be applied to other species.

Cryopreservation of Musa germplasm Embryogenic cell suspensions of Musa cv. Dominico (AAB) were obtained according to Grapin et al. (1996) and Côte et al. (1996). Cryopreservation was performed using a method derived from Panis et al. (1990). For cryoprotective treatment, DMSO was added to the final concentration of 7.5% (v/v). For slow freezing the samples were cooled at 1°C/min to –40°C prior to transfer to liquid nitrogen (–196°C). Crystallization of the cryoprotective medium at –10°C was performed automatically by the apparatus (CRYOMED). With Musa cv. Dominico, crystallization induction during freezing was indispensable to obtain regrowth after thawing (Fig. 1). Pregrowth of the cell Posters 463 suspensions for 7 d with sucrose (0.39M) was the most efficient. Optimal sucrose concentrations during pretreatment were 0.39 and 0.53M. Cell regrowth was more efficient after high-density plating on solid medium. The optimized protocol was applied to various embryogenic cell suspensions of Musa and four out of five cultivars withstood cryopreservation (Dominico 1, SF 265, Curraré 3, Col 49 2.8). The efficiency of the protocol was calculated by the ratio of the number of embryos produced after plating a cryopreserved cell suspension against the number of embryos produced by a non-cryopreserved cell suspension (Fig. 2). The data ranged from 0 to 26% depending on the cultivar. This efficient system of quantification should be optimized for Musa and could also be applied to other species.

Fig. 1. Effect of crystallization induction in Fig 2. Germination of somatic embryos Musa cv. Dominico. Left: with induction, derived from cryopreserved cell suspension right: without induction. in Musa cv. Dominico. Top: Control, Right: with induction, Left: without induction.

Conclusion The level of cryopreservation research in CATIE as well as the range of species covered by the programmes is expanding progressively. Mastering cryopreservation techniques will allow CATIE to conserve genetic resources of problem crops in a safer and more cost-effective manner.

References Côte, F.X., R. Domergue, S. Monmarson, J. Schwendiman, C. Teisson and J.V. Escalant. 1996. Embryogenic cell suspension from the male flower of Musa AAA cv. Grande naine. Physiologia Plantarum 97: 285–290. Dussert, S., N. Chabrillange, F. Engelmann, F. Anthony, J. Louarn and S. Hamon. 1998. Cryopreservation of seeds of four coffee species (Coffea arabica, C. costatifructa, C. racemosa and C. sessiliflora): importance of water content and cooling rate. Seed Science Research 8:9–15. Grapin, A., J. Schwendiman and C. Teisson. 1996. Somatic embyogenesis in plantain and banana. In Vitro Cell Development Biology–plant 32: 666–671. 464 Cryopreservation of Tropical Plant Germplasm

Maruyama, E., Y. Kinoshita, K. Ishii, K. Ohba and A. Saito. 1997. Germplasm conservation of the tropical forest trees, Cedrela odorata L., Guazuma crinita Mart., and Jacaranda mimosaefolia D. Don., by shoot tip encapsulation in calcium-alginate and storage at 12 – 25ºC. Plant Cell Reports 16:393–396. Orellana, M. 1997. Desarrollo de un sistema de cultivo in vitro para los explantes nodales de caoba (Swietenia macrophylla. King). Tesis de Maestría, CATIE, Turrialba. 94p. Panis, B.J., L.A. Withers and E.A.L. De Langhe. 1990. Cryopreservation of Musa suspension cultures and subsequent regeneration of plants. Cryo–Letters 11: 337–350. Posters 465

In vitro conservation and cryopreservation of papaya Sarah Ashmore, Rodney Saunders and Roderick Drew School of Biomolecular and Biomedical Science, Griffith University, Nathan, Brisbane, Queensland 4111, Australia

Introduction Papaya (Carica papaya L.) is an important fruit species in many tropical and subtropical countries. It is vulnerable to a number of diseases, including papaya ringspot virus (PRSV), and has been the subject of recent attempts to introduce genes for resistance using either transformation techniques or interspecific hybridization (Manshardt and Drew 1998). The conservation of germplasm of both C. papaya and related species is important to ensure future access to valuable genes for the improvement of this crop. In vitro conservation techniques are important in papaya as stored seeds are only viable for 1–2 years, and clonal propagation is required for the conservation of elite genotypes. QDPI (Queensland Department of Primary Industries) holds a collection of C. papaya in slow growth (Drew 1992), including elite genotypes, breeding lines and wild species. Cryopreservation can offer an alternative or complementary approach for the conservation of species for which seed storage is not an option, and is now routinely used for a small number of crop species (Ashmore 1997). There are, however, no reports of the use of cryopreservation in papaya. This paper provides information on the in vitro collection of papaya held at QDPI and presents results of cryopreservation trials on shoot-tips of papaya. Some preliminary trials have also been carried out on embryogenic cultures of papaya.

In vitro collection of papaya The Queensland Horticulture Institute (QHI) of QDPI holds a small collection of papaya germplasm in tissue culture. Germplasm is maintained as single, apically dominant plants on DeFossard salts (DeFossard et al. 1974) containing fructose in place of sucrose, and without added plant growth substances (Drew 1992). The storage temperature is 25°C, and subculture interval is about every 9–12 months. This collection of Carica spp. includes: 32 elite genotypes of papaya which have superior cropping qualities in the field, 6 other Carica species and a related species (Jacaratia spinosa), and 11 interspecific hybrids. Limitations for the continued and sole use of slow-growth techniques for storage of this germplasm include the cost of maintenance (particularly if the collection continues to increase) and concerns over the potential for somaclonal variation. For these reasons, cryopreservation is under investigation.

Cryopreservation methods Cryopreservation methods chosen for these trials are based on the newer cryopreservation techniques which involve a rapid freezing process, and rely on the principle of vitrification of tissue (Fahy et al. 1984). These techniques are relatively simple, do not require the use of expensive equipment, and have proved successful for a range of species (Engelmann 1997). These factors are 466 Cryopreservation of Tropical Plant Germplasm important in considering the adoption of techniques at genebank facilities for large numbers of genotypes. Modifications in the techniques involve differences in the pretreatment steps of the procedure. Pretreatments which will have been trialed here are pregrowth with high sucrose followed by desiccation (Dumet et al. 1993) for embryogenic cultures, the droplet-freezing technique which involves exposure to 10% DMSO (Schäfer–Menuhr 1995) and exposure to vitrification solutions (Sakai et al. 1990) for shoot-tips. Embryogenic tissue, which was produced using the method previously described (Drew et al. 1995), was cultured on high sucrose (0.3M) for 4 d, then desiccated for 75 min prior to freezing. Axillary buds from apically dominant in vitro papaya plants were used as a source of shoot-tips for cryopreservation. These plants were propagated using the method developed by Drew (1992) and buds were removed from the plants using a sterile hypodermic needle. Shoot-tips, 1–2 mm in size and including 2– 3 leaf primordia, were incubated overnight in liquid medium. Pretreatment was either incubation in 10% DMSO for 2 h or treatment at 0 or 25°C in 20% PVS2 (30% glycerol, 15% ethylene glycol, 15% DMSO) for 1 h followed by 20 min in 100% PVS2 solution. All pretreated samples were placed on sterile foil strips and rapid freezing was achieved by plunging directly into liquid nitrogen. Rapid thawing after 0.5–2 h involved direct plunging into medium with elevated sucrose concentration (1.4M) at room temperature. For regeneration, shoot-tips were placed on Murashige and Skoog (MS) salts (Murashige and Skoog 1962) or DeFossard salts containing 3% sucrose, 0.2 mg/L GA3 (gibberellic acid), 0.5 mg/L IAA (indole–3–acetic acid) and 0.1 mg/L BA (6–benzyl adenine). Cultures were incubated at 25°C in the dark for the first 48 h, and were then placed in the light at the same temperature. Embryogenic cultures were regenerated according to the method previously described by Drew et al. (1995).

Results of cryopreservation Two cryopreservation trials on embryogenic cultures produced regeneration rates between 5 and 10% after preculture in sucrose followed by desiccation for 75 min prior to freezing. This compares with control regeneration rates of 50%. Where 10% DMSO was used as a pretreatment for shoot-tips in two trials, there was less than 5% regeneration after cryopreservation. For the trials in which vitrification was used as a pretreatment, 100% of shoot-tips had regenerated at 21 d for control samples (16 shoot-tips in two trials), for samples treated at 0°C with vitrification solution but not frozen (16 shoot-tips in two trials) and for samples treated at 25°C with vitrification solution but not frozen (9 shoot-tips in one trial). Samples treated at 0°C with vitrification solution followed by freezing in liquid nitrogen showed a mean regeneration of 65% (27 shoot-tips in three trials), whereas samples treated at 25°C with vitrification solution followed by freezing in liquid nitrogen showed a mean regeneration of 71% (42 shoot-tips in three trials). Some callus tissue was seen in the first of these trials, but more recent trials showed no callus. Initial greening and regeneration were delayed by about 7 d for frozen samples compared with controls, and samples treated with vitrification at 25°C generally appeared more vigorous than those treated at 0°C. These results are summarized in Table Posters 467

1. 468 Cryopreservation of Tropical Plant Germplasm

Table 1. Mean regeneration rates of papaya shoot-tips 21 d after a range of treatments in cryopreservation trials. +LN indicates that samples were plunged into liquid nitrogen for 0.5–2 h Treatment Mean % regeneration at 21 d Comments Control 100% (16 shoot-tips, 2 trials) No callus Vitrification (0°C) 100% (16 shoot-tips, 2 trials) No callus Vitrification (25°C) 100% (9 shoot-tips, 1 trial) No callus Vitrification (0°C) +LN 65% (27 shoot-tips, 3 trials) Some callus, reduced size compared with control Vitrification (25°C) +LN 71% (42 shoot-tips, 3 trials) Some callus, reduced size compared with control

Discussion This work illustrates significant potential for the use of simple cryopreservation protocols for the long-term storage of papaya germplasm. The cryopreservation of shoot-tips using vitrification pretreatments looks particularly promising. The plants, which have been regenerated after cryopreservation using the vitrification pretreatment, are continuing to grow in culture, and will be monitored for morphology and growth characteristics over the next few months. Further work will replicate these trials and refine a technique for routine application.

Acknowledgements We would like to acknowledge the work of Khyl Domrow in the generation of some of these results and Leigh Towill, who led a cryopreservation workshop held at the New Zealand Institute for Crop & Food Research, Christchurch, which was attended by one of us.

References Ashmore, S.E. 1997. Status Report on the Development and Application of In Vitro Conservation and Use of Plant Genetic Resources. IPGRI, Rome, Italy. DeFossard, R.A., A. Myint and E.C.M. Lee. 1974. A broad spectrum tissue culture experiment with tobacco (Nicotiana tabacum L.) with pith tissue cells. Physiologia Plantarum 31:125–130. Drew, R. 1992. Improved techniques for in vitro propagation and germplasm storage of papaya. HortScience 27:1122–1124. Drew, R.A., J.N. Vogler, P.M. Magdalita, R.E. Mahon and D.M. Persley. 1995. Application of biotechnology to Carica papaya and related species. Pp. 321–326 in Current Issues in Plant Molecular and Cellular Biology. M. Terzi, R. Cella and A. Falavigna (eds.). Kluwer Academic Publishers, Netherlands. Dumet, D., F. Engelmann, N. Chabrillange and Y. Duval. 1993. Cryopreservation of oil palm (Elaeis guineensis Jacq.) somatic embryos involving a desiccation step. Plant Cell Reports 12: 352–355. Engelmann, F. 1997. In vitro conservation methods. Pp. 119–162 in Biotechnology and Plant Genetic resources: Conservation and Use. B.V. Ford–Lloyd, J.H. Newburry and J.A. Callow, Eds., CABI, Wellingford. Posters 469

Fahy, G.M., D.R. MacFarlane, C.A. Angell and H.T. Meryman. 1984. Vitrification as an approach to cryopreservation. Cryobiology, 21: 407–426. Manshardt, R.M. and R.A. Drew. 1998. Biotechnology of Papaya. Acta Horticulturae 461:65– 73. Schäfer–Menuhr, A. 1995. Refinement of cryopreservation techniques for potato. Year-end IPGRI project report. IPGRI, Rome, Italy. Murashige, T. and F. Skoog. 1962. A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiologia Plantarum 15:473–497. Sakai, A., S. Kobayashi and I. Oiyama. 1990. Cryopreservation of nucellar cells of navel orange (Citrus sinensis Osb. var. brasiliensis Tanaka) by vitrification. Plant Cell Reports 9:30–33. 470 Cryopreservation of Tropical Plant Germplasm

Cryopreservation of seeds, embryos, embryonic axes and pollen at National Cryobank of NBPGR Rekha Chaudhury National Bureau of Plant Genetic Resources, Pusa Campus, New Delhi 110012, India

Introduction Cryopreservation technology for germplasm conservation has been used by several research groups during the past decade. The technology has been tested on various germplasm stored in the form of seeds, embryos, embryonic axes, meristems, shoot-tips, pollen, etc. and has been especially advocated for recalcitrant seed species (Engelmann et al. 1995). However, at the moment, the use of cryopreservation is limited to small laboratory collections and its use on a large scale is only exceptional. In this regard, a National Facility for cryopreservation of plant germplasm has been established in India at the National Bureau of Plant Genetic Resources, New Delhi. Studies have been conducted on desiccation and freezing sensitivity of several suborthodox and recalcitrant seed species to determine factors responsible for success, low recovery percentages or failures during cryopreservation (Chandel et al. 1995). New cryopreservation techniques like vitrification, desiccation, pregrowth- desiccation and encapsulation-dehydration have also been tested. The main objective has been to establish pilot cryopreserved collections for species for which cryopreservation protocols have been standardized. Efforts have been made to use simplified procedures which can be practised routinely and which enable handling of large collections for cryostorage.

Materials and methods

Orthodox and suborthodox seed species Diverse germplasm of orthodox seed species totaling more than 1240 seed accessions of millets, vegetables, oilseeds, horticultural fruits, medicinal and aromatic plants belonging to genera and species were received from different parts of the country. In suborthodox seed species like black pepper (Piper nigrum), cardamom (Elettaria cardamomum), neem (Azadirachta indica) and almond (Prunus amygdalus), fresh fruits were collected and seeds extracted in the laboratory. Moisture content (MC) of seed lots was determined by the low constant temperature oven method. Seed samples of orthodox seed species with more than 7% MC were desiccated to 5–7% MC in a desiccator over silica gel or seed dryer under forced air at 25°C and 20% relative humidity. Seeds of suborthodox species were desiccated over regularly charged silica gel to 12– 14% MC. Viability was tested by germination before and after regular periods of storage. Seed samples with >85% germinability were stored in polypropylene cryovials in the vapour phase of liquid nitrogen. Posters 471

Recalcitrant seed species Fruits of tea (Camellia sinensis), jackfruit (Artocarpus heterophyllus), neem (Azadirachta indica), litchi (Litchi sinensis) and oak (Quercus leucotrichophora) were collected at different stages of maturity and seeds extracted. Desiccation and freezing were done as described earlier (Chandel et al. 1995) and axes were cultured on modified MS (Chin et al. 1988) medium.

Pollen MC of fresh pollen was determined by heating to a constant weight at 80°C. Samples were desiccated to 8–15% MC for two-celled and 15–20% for three- celled pollen over charged silica gel and viability tested by in vitro germination. Pollen samples were then packed in cryovials and suspended in the vapour phase of LN.

Results and discussion Large-scale cryostorage of seed accessions of cereals and millets (210), pulses (117), vegetables (254), horticultural crops (28), spices and condiments (59), medicinal and aromatic plants (367), and oilseed crops (208) and 61 pollen samples of nine genera has been successful. The initial viability and vigour of seed samples and percentage germination of pollen has been maintained over different periods of storage varying from 2 to more than 11 years. Orthodox seeds and pollen can be stored routinely following simple protocols. Recalcitrant seed species along with suborthodox species were investigated for desiccation and freezing sensitivity of whole seeds and embryonic axes. Some critical factors like maturity status, duration of desiccation and initial (at the time of harvest) and final (after specified desiccation duration) moisture content have been found to be important. The most suitable method so far for cryopreservation of jackfruit, litchi, tea, Quercus, neem and almond is the desiccation of embryonic axes in the sterile air of a laminar flow cabinet followed by direct plunging into liquid nitrogen (Table 1). Success in cryopreservation was obtained in partially mature axes of jackfruit and litchi, whereas for the other species tested fully mature axes were most amenable to cryostorage. With all the species optimal survival (lowest of 25% in oak to maximum of 90% in neem and tea) after cryopreservation has been observed for axes desiccated to 12–18% moisture content before freezing and 45–100% survival for seeds desiccated to 12–14% moisture content. Regeneration of axes was direct with no intervening callus. The technology thus developed does not necessarily require the application of cryoprotectants or use of programmable freezers. 472 Cryopreservation of Tropical Plant Germplasm

Table 1. Successful cryopreservation of recalcitrant and suborthodox seed species at NBPGR Optimal MC (% fresh wt Results Plant Tissue basis) (% survival) Jackfruit † Embryonic axes 14.5–14.8 25–30 Litchi † Embryonic axes 12.1–18.3 22–35 Tea Embryonic axes 13.2–14.3 80–90 Oak Embryonic axes 13.0–14.0 15–25 Neem Embryonic axes 10.0–12.0 70–80 Seeds 4.0–13.5 50–70 Almond Embryonic axes 7.0–10.0 70–100 Black pepper Seeds 6.0–12.0 10–45 Cardamom Seeds 7.0–14.0 70–80 † Partially mature embryonic axes were used for studies.

Acknowledgements The author gratefully acknowledges IPGRI's support for the project funding obtained through the Department for International Development (DFID, UK). Sincere thanks are due to Dr Florent Engelmann, IPGRI, Rome for his guidance and cooperation for the author's participation in the Workshop.

References Chandel, K.P.S., R. Chaudhury, J. Radhamani and S.K. Malik. 1995. Desiccation and freezing sensitivity in recalcitrant seeds of tea, cocoa and jackfruit. Annals of Botany 76: 443–450. Chin, H.F., B. Krishnapillay and Z.C. Alang. 1988. Media for embryo culture of some tropical recalcitrant species. Pertanika 11: 357–363. Engelmann, F., K.P.S. Chandel, B. Krishnapillay and Y.L. Hor. 1995. Interest of cryopreservation for the long term storage of recalcitrant seed species. In Abstracts of the th 24 Congress of the International Seed Testing Association, Copenhagen, Denmark, 7–16 June 1995. Posters 473

Systematic determination of an adequate method for large-scale sweet potato cryopreservation at CIP Ali M. Golmirzaie, Ana Panta and Sandra Diaz CIP, Lima, Peru

Introduction Ex situ conservation of sweet potato biodiversity is another responsibility of the International Potato Center (CIP). CIP maintains the largest worldwide sweet potato collection under field and in vitro conditions. To ensure safeguarding of this material, continuous improvement of conservation methods is necessary. In recent years, CIP has made efforts to apply cryopreservation in the long-term germplasm conservation of its mandate crops. Currently, the application of potato cryopreservation on a large scale is being refined. The experience obtained on potato has paved the way to start research on sweet potato cryopreservation. This report describes assays that are being performed for the systematic determination of an efficient cryopreservation method applicable on a large scale.

Procedures Sweet potato genotypes were taken from the in vitro collection held at CIP. They were micropropagated by single-node stem segments in magenta jars containing MPB medium (Murashige–Skoog salts, 2 mg/L calcium panthotenate, 10 mg/L gibberellic acid, 100 mg/L arginine, 200 mg/L ascorbic acid, 20 mg/L putrescine, 3% sucrose, and 3.5 g/L SIGMA phytagel). Cryopreservation assays were performed as follows.

First assay Shoot-tips 0.5–0.7 mm long of cultivars, Maria Angola, Jonathan, Chugoku, Morada Inta, Tib 10 and Jewel were tested by a vitrification method based on Towill and Jarret's research (1992). Shoot-tips were exposed for 1, 5, 10 and 60 min to 100, 80, 60 and 40% PVS2, respectively. Thawing was done 1 d after freezing and survival evaluation was performed 2 months later.

Second assay Somatic embryo cultures of five cultivars (María Angola, Jonathan, Chugoku, Morada Inta and Tanzania) obtained using the protocol of Salinas et al. (1991), were cryopreserved using the dehydration of encapsulated and non- encapsulated embryo methods developed by Blakesley et al. (1995, 1996).

Third assay Shoot-tips (2.5 mm long) of cv. Jonathan were cultured in preculture medium 1 (Murashige and Skoog salts supplemented with 2 mg/L calcium panthotenate, 15 mg/L gibberellic acid, 100 mg/L ascorbic acid, 20 mg/L putrescine, 100 mg/L arginine and 0.09M sucrose) for 24 h. They were then exposed for 6 h to preculture medium 2, which is equal to preculture medium 1 except for 474 Cryopreservation of Tropical Plant Germplasm the sucrose content. Several sucrose concentrations in this medium were tested: 0.3M, 0.4M, 0.5M and 0.6M. After preculture, shoot-tips were transferred to post-thaw medium. Survival was evaluated 4 weeks after.

Fourth assay Shoot-tips (2.5 mm long) of cv. Jewel were processed as described in the third assay. Preculture medium 2 with 0.4M sucrose was used. Then, they were dehydrated with the vitrification solution developed by Steponkus et al. (1992) testing different time periods (20, 25 and 30 min). Half of the shoot-tips were rinsed by shaking in liquid post-thaw medium (Murashige and Skoog salts, with 2 mg/L calcium panthotenate, 20 mg/L gibberellic acid, 100 mg/L ascorbic acid, 100 mg/L calcium nitrate, 20 mg/L putrescine, 100 mg/L arginine, 5 ml/L coconut milk and 3% sucrose). After 24 h, shoot-tips were removed and transferred to semi-solid post-thaw medium (liquid post-thaw medium jellified with 2.2 g/L phytagel). The other half was transferred directly to semi-solid post-thaw medium without rinsing. Survival was evaluated after 4 weeks of culture in post-thaw medium.

Results and discussion In the first assay, regrowth was observed in four genotypes. María Angola had a 24% of survival, Jonathan 17%, Morada Inta 8%, and Jewel 26%. Because survival was low and not all genotypes tested presented survival, other alternative explants were looked for. A second assay using somatic embryos was carried out; unfortunately no survival was observed with the five cultivars tested. Blakeskey et al. (1995, 1996) were successful in testing other cultivars. The difference in results may be due to the different genotypes used, which means that genotype dependence is affecting the results. According to these results, the vitrification method was identified as the best alternative method. During the first assay, the short shoot-tip size and several vitrification steps made it difficult to manipulate the samples, which could affect the survival rate. For this reason, assays to look for other simpler vitrification methods using bigger shoot-tips are being performed. According to preliminary experiments (third and fourth assays) testing the method of Schnabel–Preikstas et al. (1992), we found that a protocol using 2.5-mm long shoot-tips, preculture medium 2 with 0.4M sucrose, dehydration time 25 min in Steponkus' vitrification solution (Steponkus et al. 1992), and rinsing with post-thaw liquid medium after dehydration is working well for sweet potato. A broad range of sweet potato genotypes will be tested following this protocol.

References Blakesley, D., S. Al–Mazrooei and G.G. Henshaw. 1995. Cryopreservation of embryogenic tissue of sweet potato (Ipomea batatas): use of sucrose and dehydration for cryoprotection. Plant Cell Reports 15: 259–263. Blakesley, D., S. Al–Mazrooei, M.H. Bhatti and G.G. Henshaw. 1996. Cryopreservation of non-encapsulated embryogenic tissue of sweet potato (Ipomea batatas). Plant Cell Reports 15: 873–876. Salinas, R., A. Gutierrez, R. Alvarez and J.H. Dodds. 1991. Regeneration of plants from Posters 475

somatic embryos of sweet potato (Ipomoea batatas (L.) Lam. ACEVIV Scientific Bulletin 3:5– 16 Schnabel–Preikstas, B., E.D. Earle and P.L. Steponkus. 1992. Cryopreservation of sweet potato shoot tips by vitrification. Cryobiology 29:738–739. Steponkus, P.L., R. Langis and S. Fujikawa. 1992. Cryopreservation of plant tissues by vitrification. Pp. 1–61 in Steponkus, P.L. (Ed.) Advances in low temperature biology Vol.1. JAI Press Ltd, London. Towill, L.E. and R.L. Jarret. 1992. Cryopreservation of sweet potato (Ipomoea batatas L. Lam.) shoot tips by vitrification. Plant Cell Reports 11:175–178. 476 Cryopreservation of Tropical Plant Germplasm

Pilot plant cell culture collections cryopreserved in liquid nitrogen Masaya Ishikawa, Tadashi Yokoyama and Kenichi Higo Department of Genetic Resources II, National Institute of Agrobiological Resources, Tsukuba, Ibaraki 305-8602, Japan

Introduction A number of plant cell cultures have been established by scientists as model systems and/or experimental systems such as the tobacco BY–2 cell line. In recent years, a number of transgenic cell cultures have been produced. Most of these cell cultures require frequent subculturing for their maintenance. The relevant original characters or the cultures themselves are sometimes lost during prolonged subcultures or due to contamination, personnel changes, etc. Cryopreservation will be a useful tool to store them safely and subeternally and recover them when necessary with the desired original characters. There have been requests for such stable preservation of plant cell cultures in our institute and we have recently started to cryopreserve plant cell culture lines that are important as experimental systems. At the moment, we have four suspension cultured cells successfully stored in the vapour phase of liquid nitrogen as working collections. Here we report the successful cryopreservation of tobacco BY–2 cells and soyabean suspension cultured cells.

Materials and methods Tobacco BY–2 developed by Nagata et al. (1992) and soyabean suspension cultured cells developed by Yokoyama (unpubl.) were used for cryopreservation studies. These cultures were maintained by subculturing in their respective liquid medium every 7 d as detailed elsewhere (Nagata et al. 1992). Six-day-old cells were used for cryopreservation as we found that the early stationary stage gives the highest survival following cryopreservation and various stress treatments in bromegrass cell cultures (Ishikawa et al. 1990, 1995). A slow prefreezing procedure using the cryoprotectant medium CSP1 (Ishikawa et al. 1996) and a rapid freezing method using the cryoprotectant medium RPF2 (Ishikawa 1994) developed in our laboratory were used for cryopreservation of both tobacco and soyabean cell cultures. For each method, about 0.1 g cells were allocated into 5–10 glass vials. The cells treated with 1 ml of CSP1 for 20 min were ice-inoculated at –8ºC before being cooled at 0.3ºC/min to –30ºC and plunged into liquid nitrogen. With the rapid prefreezing procedure, the cells treated with 0.3 ml of RPF2 for 5 min were directly placed in a –30ºC freezer for 30 min before immersion in liquid nitrogen. Following 7 d of cryostorage, cells were rapidly thawed in a water- bath. Following slow dilution of CSP1 with 3% sucrose or rapid dilution of RPF2 with 1.2M sucrose, cells were collected by blotting on filter paper and incubated on the respective semi-solid medium to check the survival by regrowing capability. We checked the growth and characters of the recovered Posters 477 cultures following transfer to liquid medium and 2–3 subculture cycles. 478 Cryopreservation of Tropical Plant Germplasm

Results and discussion We obtained good recovery of tobacco BY–2 cells cryopreserved using the slow prefreezing procedure with CSP1 but no recovery of cells cryostored using the rapid freezing procedure with RPF2. In the meantime, good recovery of soyabean suspension cultured cells was obtained using the rapid freezing method but no recovery with the slow prefreezing method. The reasons for the differential behaviour of soyabean and tobacco suspension cultured cells under different cryopreservation protocols are not clear. For sensitive cultured cells like soyabean and tobacco, employment of more than one method is recommended to obtain reasonable recovery. When cryopreserved tobacco cells were recovered on semi-solid medium and transferred to liquid cultures, they regained original cell characteristics after two subculture cycles, such as fast growth rates and the tendency to become single cells. These were comparable to non-cryopreserved cells. Besides tobacco and soyabean cultures, rice and bromegrass supension cultures have been successfully cryopreserved using both methods described earlier (Ishikawa et al. 1996). These cell cultures have been stored in the vapour phase of liquid nitrogen (–170ºC) for more than 6 months. We are planning to steadily increase the number of accessions to meet the requests of scientists. We are accepting requests only from nearby institutes as we need close collaboration to check the cell culture conditions and characters before and after cryopreservation. We are planning to register the cryopreserved cultures in our genebank systems with passport data in the near future. What is different from the existing genebanks at our institute is that delivery may be made upon request by the person who developed the culture.

Acknowledgements The authors would like to thank Ms Hatanaka, T. Kitashima and Suzuki for their technical assistance in maintaining the cultures.

References Ishikawa, M. 1994. Recent progress in cryopreservation of plant genetic resources. JIRCAS International Symposium Series No2: 155–167. Ishikawa, M., A.J. Robertson and L.V. Gusta. 1990. Effect of temperature, light, nutrients and dehardening on abscisic acid induced cold hardiness in Bromus inermis Leyss suspension cultured cells. Plant Cell Physiology, 31:51–59. Ishikawa, M., A.J. Robertson and L.V. Gusta. 1995. Comparison of viability tests for assessing cross adaptation to freezing, heat and salt stresses induced by abscisic acid in bromegrass (Bromus inermis Leyss) suspension cultured cells. Plant Science 107: 83–93. Ishikawa, M., P. Tandon, M. Suzuki and A. Yamaguishi–Ciampi. 1996. Cryopreservation of bromegrass (Bromus inermis Leyss) suspension cultured cells using slow prefreezing and vitrification procedures. Plant Science 120: 81–88. Nagata, T., Y. Nemoto and S. Hasegawa. 1992. Tobacco BY–2 cell line as the "He La" cell in the cell biology of higher plants. International Review of Cytology 132:1–30. Posters 479

Cryopreservation of some Malaysian tropical urban forestry species M. Marzalina and Z.N.A. Nashatul Forest Research Institute Malaysia, 52109 Kepong, Kuala Lumpur, Malaysia

Introduction Although Malaysia comprises only 0.2% of the world’s land area, it is estimated to harbor 6% of the world’s flowering plant species. Its tropical rainforests are priceless reservoirs of plant germplasm and play a vital role in maintaining global environment stability. Meanwhile the majority of the rainforest species – especially timber trees – produce recalcitrant seeds (Marzalina et al. 1994). The importance and the difficulty of conservation of these species are well recognized, especially with species endemic to narrow habitat zones. As many are not being preserved in genebanks (Normah and Marzalina 1996) their conservation is dependent upon wild stands or arboreta.

Constraints Most tropical timber tree species have very complex life cycles. This includes: a long juvenile phase, a sporadic phase of flowering and fruiting, a short production phase, seeds with no dormancy, seeds that deteriorate rapidly, and a high level of seed moisture content (36–90%).

Conservation activities Owing to the natural constraints which arise, ex situ conservation has been explored. This includes cryopreservation. To date at FRIM, the activity focuses on forestry species, including those of urban forestry.

Materials and methods

Whole seeds Mature seeds were used in all studies. The screening mechanism was done stepwise by determining the moisture content (MC,%), control viability test (GT,%) and whole seeds were desiccated under the laminar flow to a range of MCs between 5 and 25%. Whole seeds were subjected to either storage in a deep freezer (–20°C), direct plunge into liquid nitrogen (LN) at –196°C or slow cooling using a controlled freezing system set at –1°C/min to –40°C followed by rapid immersion into LN. If more than 60% of the seeds died, embryo axes were used instead.

Embryo axes Embryos from mature seeds were dissected aseptically under the laminar flow and their MC% and GT% determined. Then desiccation of embryos was carried out down to the range of less than 10% MC. Embryos were subjected to direct plunging in LN, controlled freezing and encapsulation-dehydration. 480 Cryopreservation of Tropical Plant Germplasm

All materials tested were stored in LN for 1 month to 3 years. They were thawed either slowly under the laminar flow or rapidly by immersion in a water-bath thermostated at 38–40°C. Viability of whole seeds was tested by germinating the seeds on paper in Petri dishes at 30°C while the embryos were placed on standard Murashige and Skoog medium.

Results Table 1 presents the results of cryopreservation studies on tropical forest tree germplasm which were carried out by the Seed Technology Section, FRIM from 1992 to the present.

Discussion and conclusion It was found that whole seeds of most orthodox and intermediate species could be cryopreserved without much problem as the lowest safe moisture content could be reduced to less than 10%. Viability could be maintained above 15% after cryopreservation. However, whole seeds of truly recalcitrant species could not be cryopreserved, but when embryos were used instead, there was some recovery. Further modifications to the cryopreservation protocols are needed including: · Recovery conditions for embryos · Suitable culture media · Application of encapsulation techniques · Application of vitrification techniques.

Acknowledgements The authors wish to thank IPGRI and Prof. Akira Sakai for the financial support to MM to attend this meeting.

References Marzalina, M., M.N. Normah and B. Krishnapillay. 1994. Artificial seeds of Swietenia nd macrophylla. Pp. 132–134 in Proceedings of the 2 National Seed Symposium (B.Krishnapillay et al., eds.). MAST, Kuala Lumpur. Normah, M.N. and M. Marzalina. 1996. Achievements and prospects of in vitro conservation for tree germplasm. Pp. 253–261 in In vitro conservation of plant genetic resources (M.N.Normah, M.K. Narimah and M.M. Clyde, eds.). UKM, Bangi, Malaysia. Posters 481

Table 1. Summary of cryopreservation experiments performed in FRIM with tropical tree species Optim. Part cryo- Storage temperature/ MC Via- ‡ Species preserved method (%) bility Year Acacia mangium Whole seed –196°C, direct plunge 7.6 76% 1997 A Adenanthera pavonina Whole seed –20°C, deep freeze R 9.7 97% 1996 † Albizia falcataria Whole seed –20°C, deep freeze A 6.6 100% 1996 Alstonia angustiloba Whole seed –20°C, deep freeze A 5.0 81% 1997 Bambusa arundinacea Whole seed –196°C, direct plunge 10.7 73% 1993 † A Calamus manan Embryonic –196°C, direct plunge 7.8 60% 1992 axis A † Cassia nodosa Whole seed –20°C, deep freeze R 9.5 17% 1996 † Cassia spectabilis Whole seed –196°C, direct plunge 9.5 20% 1996 A † Casuarina sumatrana Whole seed –196°C, direct plunge 11.2 23% 1996 R Dendrocalamus Whole seed –196°C, direct plunge 8.5 53% 1993 membranaceus A Dentrocalamus Whole seed –196°C, direct plunge 7.3 48% 1993 brandisii A Dialium platysepalum Whole seed –20°C, deep freeze A 10.6 37% 1997 Dipterocarpus alatus Whole seed –196°C, direct plunge 10.0 68% 1992 A Dipterocarpus Whole seed –196°C, direct plunge 6 – 8 70% 1992 intricartus A † Dyera costulata Whole seed –196°C, direct plunge 6.7 37% 1997 R † Hopea odorata Embryonic –40°C, –196°C control 6 15% 1994 axis freezing R Lagestroemia Whole seed –196°C, direct plunge 9.8 87% 1996 floribunda† A Lagestroemia speciosa Whole seed –20°C, deep freeze A 12.8 24% 1996 † Leucaena Whole seed –196°C, direct plunge 9.6 79% 1997 leucocephylla† A Melia azaderach Whole seed –20°C, deep freeze A 12.4 43% 1997 Pterocarpus indicus Whole seed –196°C, direct plunge 4 – 6 90% 1994 A Shorea ovalis Embryonic –40°C, –196°C control 8 7% 1994 axis freezing R Shorea parvifolia Embryonic –40°C, –196°C control 5 – 7 10% 1994 axis freezing R Shorea macrophylla Embryonic –40°C, –196°C control 10 5% 1993 axis freezing R Swietenia macrophylla Whole seed –196°C, direct plunge 5 – 6 63% 1995 † A Swietenia macrophylla Embryonic –196°C, encap., direct 5 – 8 63% 1995 axis plunge A † Tectona grandis Whole seed –20°C, deep freeze A 6 – 9 97% 1996 Tectona grandis Whole seed –196°C, direct plunge 7 – 9 90% 1997 A Thyrsostachys Whole seed –196°C, direct plunge 7.8 86% 1993 482 Cryopreservation of Tropical Plant Germplasm

siamensis A † = also being planted for urban forestry. ‡ A = ambient thawing; R = rapid thawing. Posters 483

Present activities and perspectives on in vitro conservation in the MAFF Genebank, Japan Susumu Miyashita, Yasufumi Kunihiro, Tsukasa Nagamine and Kazuto Shirata National Institute of Agrobiological Resources, Kannondai, Tsukuba, Ibaraki, Japan

The Ministry of Agriculture Forestry and Fisheries (MAFF) Japan is conserving 210 000 plant germplasm accessions. Accessions conserved as seeds represent more than 80% of the accessions and the rest are 40 000 vegetatively propagated accessions. Seed accessions are conserved in storage rooms maintained at –1 and –10°C. As most crop seeds are safely conserved for relatively long periods under these conditions, there are few problems in their preservation. Most of the vegetatively propagated plant accessions are maintained in the field. Field conservation requires a lot of labour and space. In addition, environmental factors such as disease and insect damage could kill this germplasm. Furthermore, crops requiring repeated planting could get mixed or contaminated. In vitro conservation involving successive subculturing and cryopreservation could replace field conservation for some species. The MAFF Genebank uses this method for only a few crop species at present and experiments to apply these techniques more broadly are now in progress. The samples cryopreserved in the MAFF Genebank include winter buds of mulberry (Morus sp.) and apple, and apple pollen. In vitro conservation using successive subculturing is performed for many species, and represents a practical means of preservation. In vitro conservation which allows long intervals between subcultures such as for stem tip cultures of sweet potato and citrus, plantlet cultures of taro, strawberry and chrysanthemum, represents a practical method for in vitro conservation of plant genetic resources (Table 1). However, it is necessary to carefully check that no mutants occur during subculturing. Field maintenance is likely to be the main conservation method for vegetatively propagated plants in the MAFF Genebank in the future. However, it is necessary that in vitro preservation using subculturing and/or cryopreservation techniques be applied for some crop species such as potato and sweet potato, which otherwise have high maintenance costs and high labour requirements. Further, the relationship between the central bank and sub-banks needs clarification. For example, the central bank can conduct cryopreservation that requires special facilities, and sub-banks can take charge of subculturing that requires specific protocols for each crop. 484 Cryopreservation of Tropical Plant Germplasm

Table 1. Current status of utilization of in vitro conservation and cryopreservation for conservation of crops in MAFF organizations Practical use level Renewal for PGR preser- Crop Material Method interval vation† Potato Small plant Subculture 1–2 mo c Sweet potato Shoot apex Subculture 3–6 mo b Sweet potato Small plant Subculture 2 mo c Kiwi fruits Shoot apex Subculture 1 mo c Apple Shoot apex Subculture 1 mo c Apple Pollen Cryopreservation 10 y a Apple Winter bud Cryopreservation – a Citrus Shoot apex Subculture 6 mo b Citrus Callus Subculture 2 mo c Taro Callus Subculture 1 y a Taro Small plant Subculture 4 mo b Taro Lateral bud Subculture 2 mo c Strawberry Small plant Subculture 3 y a Chrysanthemum Small plant Subculture 3–6 mo b Mulberry Shoot apex Subculture 2 mo c Mulberry Winter bud Cryopreservation 5 y a † PGR = Plant Genetic Resources; a: already in use; b: minor technical improvements needed; c: major technical improvements needed. Posters 485

Cryopreservation: an in vitro method for conserving Ribes germplasm in international genebanks Barbara M. Reed¹, R.M. Brennan² and Erica E. Benson³ ¹ USDA-ARS, Corvallis, OR 97333-2521, USA ² Soft Fruit Genetics Dept. SCRI, Invergowrie, Dundee, DD2 5DA, Scotland, UK ³ School of Molecular and Life Sciences, University of Abertay-Dundee, Dundee, DD1 1HG, Scotland, UK

Introduction Cryopreservation, storage of living cells and tissues at liquid nitrogen (LN) temperatures, is now widely applied to a diverse range of plant species and tissue systems (Bajaj 1995). The development of novel cryoprotection methods has also increased the application of cryogenic storage to plant germplasm. Inter-genebank validation of storage protocols for important germplasm collections will aid in the successful implementation of cryopreservation in plant genebanks worldwide. The objective of this study was to validate three different cryopreservation methods (controlled freezing, chemical vitrification and encapsulation-dehydration) in genetic resources laboratories based in two different locations [the USDA–ARS National Clonal Germplasm Repository (NCGR), Corvallis, OR and the Scottish Crop Research Institute (SCRI)/University of Abertay-Dundee (UAD), Dundee Scotland] for the long- term storage of Ribes meristems in LN.

Materials and methods

Plant materials Micropropagated plantlets of black currant Ribes nigrum L. cv. Ojebyn and red currant R. aureum Pursh cv. Red Lake were multiplied and shoot-tip meristems recovered on NCGR–Ribes medium (RIB) (Reed and Yu 1995). Plants were -2 -1 grown at 25°C with 16-h days (25 µmol m s ). All cultures were cold acclimated for 1 week in an incubator (8-h days at 22°C and 16-h nights at – 1°C) before 0.8-mm shoot-tip meristems were excised.

Vitrification A technique for clover (Yamada et al. 1991) modified for Ribes was used (Reed and Yu 1995). Shoot-tip meristems from cold-acclimated plantlets were pretreated for 2 d in the incubator under the cold-acclimating conditions described above on RIB medium containing 5% DMSO (v/v). PVS2 cryoprotectant was dispensed into cryotubes on ice and meristems were added and stirred. After 20 min the vials were submerged in LN. Samples were rewarmed for 1 min in a 45°C water-bath and then transferred to a 22°C water-bath for 2 min. The shoot-tips were immediately rinsed twice in liquid RIB medium with 1.2M sucrose, and transferred to RIB medium for recovery. 486 Cryopreservation of Tropical Plant Germplasm

Encapsulation-dehydration A method developed for pear (Dereuddre et al. 1990) was modified for Ribes. Shoot-tip meristems were dissected onto agar plates, individually encased in alginate beads [3% (w/v) low-viscosity alginic acid with 0.75M sucrose in liquid RIB medium without calcium, pH 5.7] and pretreated for 18 h in liquid RIB medium with 0.75M sucrose. Beads were then air-dried for 3 or 4 h, placed in cryotubes, and plunged into LN. Vials were rewarmed at room temperature for 15 min, and plated.

Controlled freezing The method used was developed for Ribes (Reed and Yu 1995). Meristems were pretreated as for vitrification, transferred to 0.25 ml liquid RIB medium in 1.2-ml plastic cryotubes and 1 ml of the cryoprotectant PGD was added over 30 min. A further 30 min equilibration at 4°C was followed by cooling at 0.5°C/min to –40°C and plunging into LN. Samples were thawed for 1 min in a 45°C water-bath, transferred to a 22°C water-bath for 2 min, rinsed in liquid RIB medium and plated. A Cryomed 1000 freezer was used at NCGR and a Planar Kryo 10 freezer at UAD. The Cryomed freezer initiated crystallization of the cryoprotectant by quickly dropping the chamber temperature from –10 to –50°C, then rewarming, while in the Planar freezer the vials were touched with a forceps chilled in LN to seed the vials.

Data analysis Each experiment included 20 meristems in each of three cryovials (n = 60) with 5–15 control (not frozen) meristems per treatment. Each experiment was done twice. Recovery was defined as greening, leaf expansion and shoot production.

Results and discussion Vitrification was moderately successful (25–68%) for both Red Lake and Ojebyn at both locations. The toxicity of the vitrification solutions probably contributed to low survival of controls and reduced the regrowth of frozen meristems. These results may be further improved if recent advances in pretreatment which decrease vitrification solution toxicity are incorporated into the protocols (Luo and Reed 1997). Encapsulation-dehydration produced excellent results (>90%) for both genotypes at NCGR and for Ojebyn at UAD. Controlled-freezing results were improved by a lower freezing rate but were low for both genotypes. Differences in freezers and familiarity with slow freezing also may have affected the results. Controlled freezing was improved at NCGR with a lower freezing rate (0.3°C/min) and further improvements may be possible with some additional adjustments. Earlier studies indicated a definite genotype-related response of Ribes to cryopreservation techniques (Reed and Yu 1995). Some differences in results are evident between the two laboratories, so more precise reporting or utilization of protocols may be necessary to standardize the procedures. Much of the success of tissue culture and cryopreservation protocols is dependent on familiarity with the procedures. The condition of the stock plants from which meristems are taken has a major Posters 487 bearing on cryopreservation results and growth room conditions at each facility will be unique. The major variables in this study were the differences in personnel, growth rooms and cryopreservation equipment at the two facilities. These differences are to be expected and the success of the protocols despite the effects of different facilities is encouraging. Work is now under way to apply these protocols to a wide range of germplasm of the genus Ribes at both NCGR and UAD/SCRI.

Acknowledgements Partial funding for this collaboration from NATO (grant no. CRG 940164) is gratefully acknowledged, as is technical support from Isobel Pimberley and Mike Black (Abertay), and Sandra Gordon (SCRI). Work at NCGR–Corvallis was supported by CRIS 5358–21000–014.

References Bajaj, Y.P.S. 1995. Cryopreservation of plant cell, tissue and organ culture for the conservation of germplasm and biodiversity. Pp. 3–18 in Biotechnology in Agriculture and Forestry – Cryopreservation of Plant Germplasm I, Vol. 32. Y.P.S. Bajaj (ed.). Springer– Verlag, New York. Dereuddre, J., C. Scottez, Y. Arnaud, and M. Duron. 1990. Resistance of alginate-coated axillary shoot tips of pear tree (Pyrus communis L. cv. Beurre Hardy) in vitro plantlets to dehydration and subsequent freezing in liquid nitrogen. Comptes Rendus de l’Académie des Sciences, Paris, 310:317–323. Luo, J. and B.M. Reed. 1997. Abscisic acid-responsive protein, bovine serum albumin, and proline pretreatments improve recovery of in vitro currant shoot-tip meristems and callus cryopreserved by vitrification. Cryobiology 34:240–250. Reed, B.M. and X. Yu. 1995. Cryopreservation of in vitro-grown gooseberry and currant meristems. Cryo–Letters 16:131–136. Yamada, T., A. Sakai, T. Matsumura and S. Higuchi. 1991. Cryopreservation of apical meristems of white clover (Trifolium repens L.) by vitrification. Plant Science 78:81–87. 488 Cryopreservation of Tropical Plant Germplasm

Cryopreservation of apple in vitro germplasm in China Yongjie Wu¹, Florent Engelmann² and Yanhua Zhao¹ ¹ Changli Institute of Pomology, Heibei Academy of Agricultural and Forestry Sciences, Changli Town 066600, Qin Huang Dao City, Heibei Province, P.R. China ² IPGRI, 00145 Rome, Italy

Introduction The Changli Institute of Pomology initiated in the 1980s the establishment of an in vitro collection of apple germplasm, in order to progressively duplicate their field collection. Today, 147 cultivars belonging to seven apple species are conserved in vitro under slow growth. The intervals between subcultures at standard temperature have been extended from 3–4 months to 10–12 months by increasing the sucrose concentration and by adding mannitol or abscisic acid in the medium. Cryopreservation research started eight years ago in the institute, with the aim to establish freezing protocols which could be used for the long-term storage of the germplasm collections of apple and other fruit trees maintained by the institute.

Results and discussion Four different methods were experimented for cryopreserving apple in vitro shoot-tips.

Two-step freezing (Chang et al. 1992) The experiments showed that the cryoprotective treatment, the prefreezing temperature and the freezing rate had an effect on the survival rate of shoot- tips after cryopreservation. By using a combination of 5% DMSO + 5% glycerol for cryoprotection, prefreezing apices at 0.2°C/min from 0 to –40°C before immersion in liquid nitrogen, the survival rates achieved were around 53% (Table 1).

Table 1. Regrowth rate (%) of control (–LN) and cryopreserved (+LN) apices of four apple cultivars frozen using four different cryopreservation techniques (two-step freezing; vitrification; encapsulation-dehydration; droplet freezing) Regrowth (%) Two-step Vitrification Encaps. Droplet freezing Cultivar –LN +LN –LN +LN –LN +LN –LN +LN Jonagold 29 20 75 53 100 66 98 70 Baleng Haitang 100 57 100 81 100 69 100 88 Fuji 75 69 100 58 100 67 100 76 Jinbiao 83 67 100 88 100 75 95 87 Average 72 53 94 70 100 69 98 80 Posters 489

Vitrification (Zhao et al. 1994) The critical factor was the choice of the vitrification solution. Only the PVS3 vitrification solution (Nishiwaza et al. 1993) allowed survival with all cultivars tested. The duration of preculture and pretreatment could also influence the survival rate. However, washing explants with a 1.2M sucrose solution after rewarming did not increase survival, in contrast with what is generally observed with most species (Engelmann 1997).

Encapsulation-dehydration (Wu et al. 1998) The water content of beads was the most important parameter to control in order to achieve high survival. When the bead water content was 60%, the survival rate was around 10% only, while it increased to 66–75% when water content was around 30%.

Droplet freezing (Zhao et al. 1998) Preculturing apices on culture medium supplemented with DMSO (0.5–5%) increased survival after cryopreservation. Survival of cryopreserved apices was achieved only when a slow, two-step freezing protocol was employed.

Whatever the cryopreservation technique employed, cold-hardening mother-plants for 3 weeks at 5°C had a positive effect on survival. It was also observed that using apices sampled on mother-plants which had not been subcultured for long periods (up to 26 weeks) drastically increased survival after cryopreservation. This could be related to the lower water content of apices after an extended period without subculture. The cryopreservation method used had an effect on the regrowth pattern and speed of shoot-tips. Callusing was always observed with two-step freezing and vitrification. Callusing was lowest with the encapsulation-dehydration method. The speed of regrowth of cryopreserved apices was higher using encapsulation-dehydration than with two-step and droplet freezing. Based on all the observations performed during the above experiments, the encapsulation-dehydration technique has been selected for routine cryopreservation of apple germplasm in the Changli Institute of Pomology.

Acknowledgement Some of the experiments described in this paper have been performed in the framework of IPGRI projects.

References Chang, Y., S. Chen and Y. Zhao. 1992. Cryopreservation of apple in vitro shoot tips by two- step freezing method. Pp. 461–464 in Proceedings of the China Association for Science and Technology First Academic Annual Meeting of Youths, Oct. 1992, Beijing, China. Engelmann, F. 1997. In vitro conservation methods. Pp. 119–162 in Biotechnology and Plant Genetic Resources: Conservation and Use. Ford–Lloyd B.V., H.J. Newbury and J.A. Callow (eds.). CABI, Wallingford, UK. Nishizawa, S., A. Sakai, Y., Amano and T. Matsuzawa. 1993. Cryopreservation of asparagus (Asparagus officinalis L.) embryogenic suspension cells and subsequent plant regeneration by vitrification. Plant Science 91: 67–73. 490 Cryopreservation of Tropical Plant Germplasm

Wu, Y., Y. Zhao, F. Engelmann, M. Zhou, S. Chen, L.A. Withers and D. Zhang. 1998. The cryopreservation of in vitro cultured apple shoot tips: the effect of water content of material shoot tips. P. 1136 in Abstracts of the 1998 Congress on In Vitro Biology, May 1998, Las Vegas, USA. Zhao, Y., S. Chen and Y. Wu. 1994. Cryopreservation of apple in vitro shoot tips by vitrification. Pp. 581–589 in Proceedings of the China Association for Science and Technology Second Academic Annual Meeting of Youths, May 1998, Beijing, China. Zhao, Y., Y. Wu, F. Engelmann, M. Zhou, S. Chen, L.A. Withers and D. Zhang. 1998. The cryopreservation of in vitro cultured apple shoot tips by simple encapsulation- dehydration. P. 1135 in Abstracts of the 1998 Congress on In Vitro Biology, May 1998, Las Vegas, USA. Posters 491

Conclusions and recommendations 477

Conclusions and recommendations The meeting allowed participants to obtain a very comprehensive overview of the current knowledge concerning the biological and physical mechanisms involved in cryopreservation, and of the status of the development of protocols for new species, with a strong focus on tropical species, as well as of their application in genebanks. The workshop was unanimous in making the following recommendations. • More basic research is needed to improve our understanding of the biological and physical mechanisms involved in cryopreservation processes. This will allow us to broaden the applicability of cryopreservation by extending it to problem species, to achieve higher survival rates and to simplify the freezing protocols. • It is also essential to systematically assess the stability of the plants regenerated from cryopreservation using all available detection techniques including morphological, physiological, biochemical and molecular methods. • More cryopreservation research should be performed on tropical plants, especially on species with recalcitrant seeds, which are often difficult to cryopreserve and for which cryopreservation finds its main application. • Development research should be emphasized to facilitate the scaling-up of cryopreservation. This implies that a whole of range of important issues should be looked at, including notably survival criteria, number of propagules stored per accession, recovery testing over time, pathogen status, data management, type of information, storage procedures, recovery data and controls. Increased contacts with institutes which are already routinely using cryopreservation on a large scale (for plant, microorganism and animal germplasm conservation) will be necessary. • There is an urgent need for improving communication between cryopreservation researchers, and it is suggested to establish an informal network. • Finally, more public awareness is needed to “sell” cryopreservation research, to obtain more sustainable funding, since cryopreservation research requires a long-term commitment.

478 Cryopreservation of Tropical Plant Germplasm

Programme

[Day 1] October 20 (Tue)

08:30-09:00 Registration

Opening Ceremony 09:00-09:15 Opening address: Nobuyoshi Maeno (Director General, Japan International Research Center for Agricultural Sciences, Japan)

Keynote addresses Chairperson: Tsuguhiro Hoshino (Japan International Research Center for Agricultural Sciences, Japan)

09:15-09:45 Development of cryopreservation techniques – Akira Sakai (Japan) 09:45-10:15 Importance of cryopreservation for the conservation of plant genetic resources – Florent Engelmann (IPGRI) 10:15-10:35 Coffee break

Session 1. Fundamental aspects of cryopreservation Chairperson: David Cyr (BC Research Inc., Canada)

10:35-11:05 Freezing behaviours in plant tissues as visualized by NMR microscopy and their regulatory mechanisms – Masaya Ishikawa (National Institute of Agrobiological Resources, Japan) 11:05-11:35 Ultrastructural aspects for freezing adaptation of cells by vitrification – Seizo Fujikawa (Hokkaido University, Japan) 11:35-11:50 Discussion 11:50-13:30 Lunch

Chairperson: Patricia Berjak (University of Natal, South Africa)

13:30-14:00 The use of physical and biochemical studies to elucidate and reduce cryopreservation-induced damage in hydrated/desiccated plant germplasm – Dominique Dumet (University of Abertay-Dundee, UK) 14:00-14:30 Physiological and molecular changes in tobacco suspension cells during development of tolerance to cryopreservation by vitrification – Poula Reinhoud

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(Leiden University, The Netherlands)

480 Cryopreservation of Tropical Plant Germplasm

14:30-14:45 Coffee break 14:45-15:15 Molecular mechanisms of freezing and drought tolerance in plants – Kazuko Yamaguchi-Shinozaki (Japan International Research Center for Agricultural Sciences, Japan) 15:15-15:45 Cryopreservation of medicinal plant resources: retention of bio-synthetic capabilities in transformed cultures – Kayo Yoshimatsu (National Institute of Health Sciences, Japan) 15:45-16:00 Discussion

Reception

[Day 2] October 21 (Wed)

Session 2. Cryopreservation techniques Cryopreservation of plant cells Chairperson: Masaya Ishikawa (National Institute of Agrobiological Resources, Japan)

09:00-09:30 Cryopreservation of undifferentiated plant cells – Poula Reinhoud (Leiden University, The Netherlands) 09:30-09:50 Cryopreservation of banana embryogenic cell suspensions: a tool for genetic engineering – Bart Panis (Catholic University of Leuven, Belgium) 09:50-10:10 Cryopreservation of sugarcane embryogenic callus using a simplified freezing process – Florent Engelmann (IPGRI) 10:10-10:20 Discussion 10:20-10:40 Coffee break

Cryopreservation of pollen Chairperson: William Roca (CIAT)

10:40-11:00 Use of stored pollen for wide crosses in wheat haploid production – Masanori Inagaki (Japan International Research Center for Agricultural Sciences, Japan ) 11:00-11:20 Storage of pollen for long-term conservation of yam genetic resources – Nyat Quat Ng (IITA) 11:20-11:30 Discussion

Cryopreservation of embryos Chairperson: H.F. Chin (IPGRI/APO) 11:30-12:00 Cryopreservation of embryonic axes – Patricia Berjak

Programme 481

(University of Natal, South Africa)

482 Cryopreservation of Tropical Plant Germplasm

12:00-13:00 Lunch 13:00-13:20 Cryopreservation of zygotic embryos of tropical fruit trees – a study on Lansium domesticum and Baccaurea species – M. Noor Normah (University Kebangsaan Malaysia, Malaysia) 13:20-13:40 Cryopreservation of coffee (Coffea arabica L.) seeds: Towards a simplified protocol for routine use in coffee genebanks – Stéphane Dussert (ORSTOM, France) 13:40-14:00 Cryopreservation of melon somatic embryos by desiccation method – Kei Shimonishi (Kagoshima Biotechnology Institute, Japan) 14:00-14:20 Cryopreservation of oil-palm (Elaeis guineensis Jacq.) polyembryonic cultures – Dominique Dumet (University of Abertay-Dundee, UK) 14:20-14:30 Discussion

Visit to genebank in Tsukuba and practical demonstrations

[Day 3] October 22 (Thur)

Session 2. Cryopreservation techniques (Cont.) Cryopreservation of apices Chairperson: Florent Engelmann (IPGRI)

09:00-09:30 Recent development in cryopreservation of shoot apices of tropical species – Hiroko Takagi (Japan International Research Center for Agricultural Sciences, Japan) 09:30-09:50 Cryopreservation of vegetatively propagated species (mainly mulberry) – Takao Niino (Tohoku National Agricultural Experimental Station, Japan) 09:50-10:10 Genotype considerations in temperate fruit crop cryopreservation – Barbara M. Reed (USAD-ARS, USA) 10:10-10:30 Coffee

Chairperson: Tsukasa Nagamine (National Institute for Agrobiological Resources, Japan)

10:30-10:50 Cryopreservation of in vitro-grown meristems of potato (Solanum tuberosum L.) by encapsulation-vitrification – Dai Hirai (Hokkaido Prefectural Plant Genetic Resources Center, Japan) 10:50-11:10 Cryopreservation of in vitro-cultured meristems of wasabi – Toshikazu Matsumoto (Shimane Agricultural Experiment Station, Japan)

Programme 483

11:10-11:30 Cryopreservation of citrus apices using the encapsulation- dehydration technique – Florent Engelmann (IPGRI) 11:30-11:50 Development of cassava cryopreservation – Roosevelt H. Escobar (CIAT) 11:50-13:00 Lunch

Chairperson: Mary Taylor (Secretariat of the Pacific Community, Fiji)

13:00-13:20 Cryopreservation of in vitro-grown shoot tips of five vegetatively propagated tropical monocots by vitrification technique – Nguyen Tien Thin (Nuclear Research Institute, Vietnam) 13:20-13:40 Cryopreservation of yam apices: a comparative study with three different techniques – Binay B. Mandal (National Bureau of Plant Genetic Resources, India) 13:40-14:00 Cryopreservation of proliferating meristem cultures of banana – Bart Panis (Catholic University of Leuven, Belgium)

14:00-14:10 Discussion

14:10-15:30 Poster viewing (& Coffee)

15:30-17:00 Discussion on cryopreservation techniques, incorporating both oral and poster presentations

Chairpersons: Barbara M. Reed (USDA-ARS, USA) M. Noor Normah (Universiti Kebangsaan Malaysia, Malaysia) Bart Panis (Catholic University of Leuven, Belgium)

Buffet Party

[Day 4] October 23 (Fri)

Session 3. Ongoing cryopreservation projects – Research and its application Chairperson: Shou Yong Ng (IITA)

09:00-09:20 Application of cryopreservation protocols at a clonal genebank – Barbara Reed (USDA-ARS USA) 09:20-09:40 Advances in potato cryopreservation at the International Potato Center, Peru – Ali Golmirzaie (CIP) 09:40-10:00 The in vitro germplasm collection at the Musa INIBAP Transit Centre and the importance of cryopreservation –

484 Cryopreservation of Tropical Plant Germplasm

Bart Panis (Catholic University of Leuven, Belgium) 10:00-10:20 Coffee break 10:20-10:40 Cryopreservation: Roles in clonal propagation and germplasm conservation of conifers – David Cyr (BC Research Inc., Canada) 10:40-11:00 Conservation of threatened flora by cryopreservation of shoot apices – Daren Touchell (USAD-ARS, USA) 11:00-11:20 Conservation and cryopreservation of tropical tree species – Chalermpol Kirdmanee (National Center for Genetic Engineering and Biotechnology, Thailand) 11:20-11:40 Cryopreervation and cassava germplasm conservation at CIAT – William Roca (CIAT) 11:40-12:00 Discussion 12:00-13:10 Lunch

Session 4. Current status of cryopreservation research and future perspectives of its application in national programmes Chairperson: H.F. Chin (IPGRI-APO)

13:10-13:20 [India]: Cryopreservation research in India: current status and future perspectives – Binay B. Mandal (National Bureau of Plant Genetic Resources, India) 13:20-13:30 [Malaysia]: Current status of cryopreservation research and future perspectives of its application in Malaysia – Normah M. Noor (University Kebangsaan Malaysia, Malaysia) 13:30-13:40 [Indonesia]: Cryopreservation of tropical plants: current research status in Indonesia – Enny Sudarmonowati (Indonesian Institute of Sciences, Indonesia) 13:40-13:50 [Thailand]: Status and future prospects of cryopreservation in Thailand – Chalermpol Kirdmanee (National Center for Genetic Engineering and Biotechnology, Thailand) 13:50-14:00 [Philippines]: Phillipines: Conservation and cryopreservation research – Alfinetta Zamora (University of the Philippines Los Baños, Philippines) 14:00-14:10 [Vietnam]: Current status and future perspectives of plant cryopreservation in Viet Nam – Thinh Tien Nguyen (Nuclear Research Institute, Viet Nam) 14:10-14:20 [China]: Cryopreservation of plant germplasm in China – Tie-gang Lu (Chinese Academy of Sciences, China ) 14:20-14:30 [South Africa]: Current status of cryopreservation research and future perspectives of its application in South Africa – Patricia Berjak (University of Natal, South

Programme 485

Africa)

486 Cryopreservation of Tropical Plant Germplasm

14:30-14:40 [Nigeria]: Applying cryopreservation techniques in national programmes in Nigeria – Pius M. Kyesmu (University of Jos, Nigeria) 14:40-14:50 [Costa Rica]: Current status of cryopreservation research and future perspectives for its application in Costa Rica – Ana Abdelnour Esquivel (Costa Rica Institute of Technology, Costa Rica) 14:50-15:00 [South Pacific]: Current status of cryopreservation research and future perspectives of its application in the South Pacific – Mary Taylor (Secretariat for the Pacific Community, Fiji) 15:00-15:20 Coffee break

15:20-16:20 General Discussion and Summarization

Closing address Florent Engelmann (IPGRI)

Participants 487

Participants

Dr Ana ABDELNOUR-ESQUIVEL Fax: 81-047-363-1221 Costa Rica Institute of Technology [email protected] Cartago, Costa Rica 179-7050 COSTA RICA Present address: Tel: 506-552-53-33 Marilyn M. Belarmino Fax: 506-551-53-48 Dept. of Horticulture, ViSCA [email protected] Baybay, Leyte 6521-A Philippines Dr Maria Elena AGUILAR e-mail:[email protected] CATIE-Unidad de Biotecnologia Turrialba 7170 Prof. Patricia BERJAK COSTA RICA School of Life & Environmental Sciences Tel: 506-55-66-455 University of Natal Fax: 506-55-61-533 Durban 4041 [email protected] SOUTH AFRICA Tel: 27-31-260-3197 Dr Sarah E. ASHMORE Fax: 27-31-2601195/2029 School of BBS [email protected] Griffith University Nathan QLD 4111 Ms Rommanee CHAROENSUB AUSTRALIA Scientific Equipment Center, KURDI Tel: 61-7-3875-7346 Kasetsart University Fax: 61-7-3875-7656 Bangkok 10900 [email protected] THAILAND Tel: 66-2-579-5538 Ms Khin AYE Fax: 66-2-579-9617 Seed Bank [email protected] Central Agriculture Research Institute Yein, Yyinmana Dr Rekha CHAUDHURY MYANMAR National Bureau of Plant Genetic Tel: 095-067-2118 Resources (NBPGR), Pusa Campus New Delhi 110012 Ms Margarita C. BARTICEVIC INDIA INIA CRI Tel: 91-11-5783697 La Platina Fax: 91-11-5731495, 5785619 Santa Rosa 11610 La Pintana [email protected] Santiago CHILE Prof. H.F. CHIN IPGRI-APO Dr Marilyn M. BELARMINO Regional Office APO Laboratory of Plant Cell Technology PO Box 236 UPM Post Office Faculty of Horticulture Serdang 43400 Selangor Chiba University MALAYSIA 648 Matsudo, Matsudo City Fax: 603-9487655 Chiba 271-8510 [email protected] JAPAN

488 Cryopreservation of Tropical Plant Germplasm

Dr Mohammed K. CHOWDHURY Mr Stéphane DUSSERT International Paper ORSTOM 719 Southlands Road 911 Av.d'Agropolis Bainbridge GA 31717 34032 Montpellier BP5045 USA FRANCE Tel: 912-246-3642 ext. 283 Tel: 33-467-416185 Fax: 912-243-0766 Fax: 33-467-547800 [email protected] [email protected]

Dr Richard J. CROSS Dr Florent ENGELMANN Crop & Food Research IPGRI Private Bag 4704 Via delle Sette Chiese 142 Christchurch 00145 Rome NEW ZEALAND ITALY Tel: 64-3-325-6400 Tel: 39-06-51892224 Fax: 64-3-3252074 Fax: 39-06-5750309 [email protected] [email protected]

Dr David CYR Dr Roosevelt ESCOBAR Clone Bank Development CIAT BC Research Inc. SILUAGEN INC. Km 17 Via Cali-Palmira 3650 Wesbrook Mall A.A. 6713 Vancouver V6S2L2 COLOMBIA CANADA Tel: 57-2-4450055 Tel: 404-224-4331 Fax: 57-2-4450073 Fax: 604-224-0540 [email protected] [email protected] Dr Bruno FLORIN Ms Guiomar S. DAVILA Centre de Recherche Nestlé-Tours Saitama Ornamental Plants Research 101 Avenue Gustave Eiffel Center Notre Dame d' Oé BP9716 24 Ohaza Kushibiki 37097 Tours Cedex 02 Fukaya, Saitama 366-0815 FRANCE JAPAN Tel: 33-247-62-83-83 Tel: 81- 0485-72-1220 Fax: 33-247-49-14-14 Fax: 81-0485-72-1223 [email protected]

Dr Dominique DUMET Prof. Seizo FUJIKAWA University of Abertay-Dundee Institute of Low Temperature Science School of Molecular and Life Sciences Hokkaido University Kydd Building Sapporo, Kita-ku, Kita 19 Nishi 8 Bell St. Dundee, Scotland, DD1 HG Hokkaido 060-0819 UK JAPAN Tel: 44-1-382-308-651 Tel: 81-011-706-5471 Fax: 44-1-382-308-663 Fax: 81-011-706-7142 [email protected] [email protected]

Participants 489

Mr Eiji FUJIYOSHI Dr Kazumasa HIRATA Seikai National Fish Research Institute Graduate School of Pharmaceutical 49 Kokubu-machi Sciences Nagasaki 850-0951 Osaka University JAPAN 1-6 Yamadaoka, Suita Tel: 81-095-822-8158 Osaka 565-0871 Fax: 81-095-821-4494 JAPAN Tel: 81-06-879-8236 Dr Seiichi FUKAI Fax: 81-06-879-8239 Kagawa University [email protected] Miki-cho, Kita-gun Kagawa 761-0795 Dr Tsuguhiro HOSHINO JAPAN Japan International Research Center for Tel: 81-087-891-3072 Agricultural Sciences (JIRCAS) Fax: 81-087-891-3072 1-2 Ohwashi, Tsukuba [email protected] Ibaraki 305-8686 JAPAN Dr Ali M. GOLMIRZAIE Tel: 81-0298-38-6305 International Potato Center (CIP) Fax: 81-0298-38-6650 Apartado 5969 [email protected] Lima 100 PERU Prof. Xue-Lin HUANG Tel: 51-14-349-6017 Department of Biology Fax: 51-14-35-1570 Zhongshan University [email protected] Guangzhou, 135 west Xingang Road 510275 Dr Claudia L. GUEVARA CHINA Centro Internacional de Agricultura Fax: 86-20-84184592 Tropical (CIAT) [email protected] A.A. 67-13 Cali Dr Masanori INAGAKI COLOMBIA Japan International Research Center for Tel: 57-2-4450000 Agricultural Sciences (JIRCAS) Fax: 57-2-4450073 1-2 Ohwashi, Tsukuba [email protected] Ibaraki 305-8686 JAPAN Mr Dai HIRAI Tel: 81-0298-38-6313 Hokkaido Plant Genetic Resources Fax: 81-0298-38-6316 Center [email protected] 363-2 Minami Takinokawa Takikawa Hokkaido 073-0013 Dr Katsuaki ISHII JAPAN Forestry and Forest Products Research Tel: 81-0125-23-3195 Institute Fax: 81-0125-24-3877 Matsunosato-1, Kukisaki, Inashiki-gun [email protected] Ibaraki 305-8687 JAPAN Tel: 81-0298-73-3211 (ext 451) Fax: 81-0298-73-3795 [email protected]

490 Cryopreservation of Tropical Plant Germplasm

Dr Masaya ISHIKAWA Dr Manabu KATANO Dept. Genetic Resources, NIAR Department of Agronomy Kannondai 2-1-2,Tsukuba School of Agriculture Ibaraki 305-8602 Kyushu Tokai University JAPAN Choyo-son, Aso-gun Tel: 81-0298-38-7449 Kumanoto 869-1404 Fax: 81-0298-38-7408 JAPAN [email protected] Tel: 81-09676-7-0611 Fax: 81-09676-7-2659 Dr Keiko ISHIKAWA Mkatano@aa-1. ktokai-u.ac.jp Laboratory of Plant Cell Technology Faculty of Horticulture Dr Chalermpol KIRDMANEE Chiba University National Centre for Genetic Engineering 648 Matsudo, Matsudo City & Biotechnology Chiba 271-8510 National Science and Technology JAPAN Development Agency Tel: 81-047-363-1221 731 Rama VI Road, Rajdhevee Fax: 81-047-366-2230 Bangkok 10400 [email protected] THAILAND Tel: 66-2-6448150-4 Ms Miwako ITO Fax: 66-2-6448107 Saitama Ornamental Plants Research [email protected] Center 24 Ohaza Kushibiki Mr Takasi KOBAYASHI Fukaya Laboratory of Horticultural Plant Saitama 366-0815 Breeding JAPAN Faculty of Agriculture Tel: 81-0485-72-1220 Meiji University Fax: 81-0485-72-1223 1-1-1, Higashi-mita, Tama-ku [email protected] Kawasaki, Kanagawa 214-8571 JAPAN Ms Rie ITO-OGAWA Tel: 81-44-934-7096 Aichi Agricultural Research Center Fax: 81-44-934-7092 Sagamine Yazako Nagakute-cho [email protected] Aichi 480-1103 JAPAN Mr Yasufumi KUNIHIRO Tel: 81-0561-62-0085 National Institute of Agrobiological Fax: 81-0561-63-0815 Resources [email protected] Kannondai 2-1-2,Tsukuba Ibaraki 305-8602 Mr Yutaka JITSUYAMA JAPAN Hokkaido University Tel: 81-0298-7050 Nishi 9, Kitakyujo, Kita-ku Fax: 81-0298-38-7054 Sapporo [email protected] Hokkaido 060-0809 JAPAN Tel: 81-011-706-5471 Fax: 81-011-706-7142 [email protected]

Participants 491

Dr Pius M. KYESMU Dr Mari MARUTANI Department of Botany and Microbiology College of Agriculture and Life Sciences University of Jos Uniuversity of Guam P.M.B.2048s UOG Station Jos Mangilao Guam 96923 NIGERIA USA Tel: 234-73-460740 Tel: 671-735-2131 [email protected] Fax: 671-734-6842 [email protected] Dr Tie Gang LU Institute of Botany Dr M. MARZALINA Chinese Academy of Sciences Seed Technology Section Beijing 100093, China Forest Plantation Division Forest Research Institute Malaysia Present address: (FRIM) Laboratory of Genome Function Kepong National Institute of Agrobiological Kuala Lumpur 52109 Resouces MALAYSIA Kannondai 2-1-2, Tsukuba Tel: 6-03-6342633 Ibaraki 305-8602 Fax: 6-03-6367753 JAPAN [email protected] Tel: 81-0298-38-7006 Fax: 81-0298-38-7006 Dr Toshikazu MATSUMOTO [email protected] Shimane Agricultural Experiment Station Dr Nobuyoshi MAENO Ashiwata 2440, Izumo Japan International Research Center for Shimane 693-0035 Agricultural Sciences (JIRCAS) JAPAN 1-2 Ohwashi, Tsukuba Tel: 81-0853-22-6650 Ibaraki 305-8686 Fax: 81-0853-21-8380 JAPAN [email protected] Tel: 81-0298-38-6301 [email protected] Ms Than MAY Seed Bank, Cenral Agriculture Research Dr Binay B. MANDAL Institute National Bureau of Plant Genetic Yein, Pyinmana Resources (NBPGR), Pusa Campus MYANMAR New Delhi 110012 Tel: 095-067-21118 INDIA Tel: 91-11-5818343 Mr Tomohiro MINAMI Fax: 91-11-5785619 Kagoshima Agricultural Experiment [email protected] Station Osumi Branch 4938 Hosoyamada, Kushira Kagoshima 893-1601 JAPAN Tel: 81-994-62-4354 Fax: 81-994-62-2973 [email protected]

492 Cryopreservation of Tropical Plant Germplasm

Mr Susumu MIYASHITA Dr S.Y.C. NG National Institute of Agrobiological International Institute of Tropical Resources Agriculture (IITA), Nigeria Kannondai, 2-1-2,Tsukuba c/o Lambourn & Co. Ibaraki 305-8602 Carolyn House JAPAN 26 Dingwall Rd Tel: 81-0298-38-7050 Croydon CR9 3EE Fax: 81-0298-38-7054 ENGLAND [email protected] Tel: 234-2-241-2626 Fax: 234-2-241-2221 Dr Nobuo MURATA [email protected] Agriculture, Forestry and Fisheries Technology Society (AFFTIS) Dr Takao NIINO 2-1-2 Kannondai, Tsukuba Tohoku National Agricultural Ibaraki 305-0856 Experiment Station JAPAN MAFF Tel: 81-0298-36-6543 50 Harajukuminami, Arai Fax: 81-0298-36-5206 Fukushima 960-2156 [email protected], JAPAN [email protected] Tel: 81-024-593-5151 Fax: 81-024-593-2155 Dr Tsukasa NAGAMINE [email protected] National Institute of Agrobiological Resources Ms Eiko NIWATA 2-1-2 Kannondai, Tsukuba Aomori Pref. Green Biocenter Ibaraki 305-8602 221-10 Yamaguchi, Nogi JAPAN Aomori 030-0142 Tel: 81-0298-38-7449 JAPAN Fax: 81-0298-38-7408 Tel: 81-177-28-1015 [email protected] Fax: 81-177-28-1017 [email protected] Dr N. Quat NG International Institute of Tropical Dr Akinori NOGUCHI Agriculture (IITA), Nigeria Japan International Research Center for c/o Lambourn & Co. Agricultural Sciences (JIRCAS) Carolyn House 1-2 Ohwashi, Tsukuba 26 Dingwall Rd Ibaraki 305-8686 Croydon CR9 3EE JAPAN ENGLAND Tel: 81-0298-38-6307 Tel: 234-2-241-2626 Fax: 81-0298-38-6652 Fax: 234-2-241-2221 [email protected] [email protected]

Participants 493

Prof. M.N. NORMAH Prof. Young-Goo PARK Department of Botany Kyungpook National University Faculty of Life Sciences Department of Forestry University Kebangsaan Malaysia Taegu 702-701 Bangi 43600 REPUBLIC of KOREA MALAYSIA Tel: 82-53-950-5747 Tel: 603-8292867 Fax: 82-53-950-6708 Fax: 603-8293244 [email protected] [email protected] Ms Gabriela B. QUIRAGA Ms Rieko OGURA Faculty of Agricultural Sciences Laboratory of Horticultural Plant Cordoba National University Breeding Av. Valparaiso s/n cc 509-5000 Faculty of Agriculture, Meiji University Cordoba 1-1-1, Higashi-mita, Tama-ku ARGENTINE Kawasaki, Kanagawa 214-8571 JAPAN Mr Punathil E. RAJASEKARAN Tel: 81-044-934-7096 Indian Institute of Horticultural Fax: 81-044-934-7092 Research [email protected] Hessaraghatta Lake (PO) Bangalore 590089 Dr Seibi OKA INDIA National Institute of Agrobiological Tel: 91-80-8496634 Resources Fax: 91-80-5282619 2-1-2 Kannondai, Tsukuba [email protected] Ibaraki 305-8602 JAPAN Dr Barbara REED Tel: 81-0298-38-8388 US Dept. of Agriculture Fax: 81-0298-38-8397 National Clonal Germplasm Repository [email protected] 33447 Pegria Rd Corvallis OR 97333-2521 Dr Kiyoharu OONO USA National Institute of Agrobiological Tel: 1-541-750-8712 ext.111 Resources Fax: 1-541-750-8717 2-1-2 Kannondai, Tsukuba [email protected] Ibaraki 305-8602 JAPAN Dr Poula J. REINHOUD Tel: 81-0298-38-7461 Institute of Molecular Plant Sciences Fax: 81-0298-38-7408 PO Box 9502 2300 [email protected] RA Leiden THE NETHERLANDS Dr Bart PANIS Tel: 31-71-5274752 Catholic University of Leuven Fax: 31-71-5274469 Kard. Mercierlaan 92 [email protected] 3001 Heverlee BELGIUM Tel: 016-32-14-81 Fax: 016-32-19-93 [email protected]

494 Cryopreservation of Tropical Plant Germplasm

Dr William ROCA Dr Kei SHIMONISHI Centro Internacional de Agricultura Kagoshima Biotechnology Institute Tropical (CIAT) 4938 Hosoyamada, Kushira Km 17 via Cali-Palmira Kagoshima 893-1601 A.A. 6713 JAPAN COLOMBIA Tel: 81-0994-62-4112 Tel: 57-2-4450055 Fax: 81-0994-62-4114 Fax: 57-2-4450073 [email protected] [email protected] Dr Enny SUDARMONOWATI Mr Mustapha B. A. ROUAISSI Researcher of R&D Laboratory of Genetics Centre for Biotechnology-LIPI Agronomic National Institute of Tunisia Jl.Raya Bogor KM.46 Cité Maharajene Cibinong 16911 43 Avenue Charles Nicolle INDONESIA 1082 Tunis Tel: 62-21-875-4625 TUNISIA Fax: 62-21-875-4588 [email protected] Prof. Akira SAKAI Asabucho 1-5-23 Mr SYAFARUDDIN Kitaku Research Institute for Spice and Sapporo 001-0045 Medicinal Crops JAPAN JL. Tentara Pelajar No.3 Tel: 81-011-716-7711 Bogor Fax: 81-011-716-7711 INDONESIA [email protected] Dr Hiroko TAKAGI Dr Satoshi SAKAMOTO Japan International Research Center for JICA EXPERT Agricultural Sciences (JIRCAS) JI. Perkutut No.13 1-2 Ohwashi, Tsukuba Tanah Sareal Ibaraki 305-8686 Bogor 16161 JAPAN INDONESIA Tel: 81-0298-38-6384 Tel: 62-251-327615 Fax: 81-0298-38-6342 Fax: 62-251-327615 [email protected] [email protected] Prof. Isao TARUMOTO Dr Koichi SHIMOMURA College of Agriculture Tsukuba Medical Plant Research Station Osaka Prefecture University NIHS 1-1 Gakuen-cho, Sakai 1 Hachimandai, Tsukuba Osaka 599-8231 Ibaraki 305-0843 JAPAN JAPAN Tel: 81-0722-54-9404 Tel: 81-0298-38-0573 Fax: 81-0722-54-9404 Fax: 81-0298-38-0575 [email protected]

Participants 495

Dr Mary TAYLOR Dr Kunio TSUBOTA Secretariat of the Pacific Community Japan International Research Center for Private Mail Bag Agricultural Sciences (JIRCAS) Suva 1-2 Ohwashi, Tsukuba FIJI Ibaraki 305-8686 Tel: 679-370733 JAPAN Fax: 679-370021 Tel: 81-0298-38-6304 [email protected] Fax: 81-0298-38-6342 [email protected] Mr Hiroyoshi TERUYA Okinawa Agricultural Experiment Mr Hikaru TSUKAZAKI Station National Research Institute of 76 Kanekadan Gushikawa Vegetable, Ornamental Plants and Tea Okinawa 904-2241 360 Knsawa, Ano-Town JAPAN Mie 514-2392 Tel: 81-098-973-5530 JAPAN Fax: 81-098-973-6333 Tel: 81-059-268-4654 [email protected] Dr Tien Nguyen THINH Nuclear Research Institute Mr Cuong Pham VAN 01 Nguyen Tuluc St. Department of Food Crops Dalat Faculty of Agronomy VIET NAM Hanoi Agricultural University Tel: 84-63-8-25107 Gialam Hanoi Fax: 84-63-8-21122 VIETNAM

Mr Tin TIN Dr Tuan Dhinh VUONA Germplasm Conservation Section Cuu Long Delta Rice Research Institute, c/o Seed Bank CARI Vietnam Yezin Pynmana Present address: MYANMAR Biological Res. Division JIRCAS Dr Darren H. TOUCHELL 1-2 Ohwashi, Tsukuba USDA-ARS Ibaraki 305-8686 National Seed Storage Laboratory JAPAN 1111 South Mason St [email protected] Ft Collins, Colorado 80521 USA Mr Reyes B. WALTEROSWALDO Tel: 1-970-495-3217 Experiment Station Fax: 1-970-221-1427 PICHILINGUE, INIAP [email protected] Km 5 Road Quevedo-EL Empalme EQUADOR Ms Kaori TOUNO University of Chiba Dr Masahiko WATANABE 1 Hachimandai Tsukuba NISES Ibaraki 305-0843 4-11-1-416-303, Matsushiro, Tsukuba JAPAN Ibaraki 305-0035 Tel: 81-0298-38-0573 JAPAN Fax: 81-0298-38-0575

496 Cryopreservation of Tropical Plant Germplasm

Mr Yongjie WU Dr Kayo YOSHIMATSU Changli Institute of Pomology Tsukuba Medical Plant Research Station Hebei Academy of Agricultural and National Institute of Health Sciences Forestry Sciences 1 Hachimandai, Tsukuba Hebei 066600 Ibaraki 305-0843 Changli Town JAPAN CHINA Tel: 81-0298-38-0573 Fax: 335-20-23417, 23228 Fax: 81-0298-38-0575 [email protected] [email protected]

Mr Eiji YAMAGUCHI Ms Alfinetta B. ZAMORA National Center for Seeds and Seedlings University of the Philippines at Los Unzen Station Baños Saigo-bo 1494-34, Mizuho Los Baños Minami Takaki Laguna 4037 Nagasaki 859-1211 PHILIPPINES JAPAN Tel: 049-536-3528 Tel: 81-0957-77-2100 Fax: 049-536-3438 Fax: 81-0957-77-2154 [email protected] [email protected]

Dr Kazuko YAMAGUCHI-SHINOZAKI Japan International Research Center for Agricultural Sciences (JIRCAS) 1-2 Ohwashi, Tsukuba Ibaraki 305-8686 JAPAN Tel: 81-0298-38-6641 Fax: 81-0298-38-6643 [email protected]

478 Cryopreservation of Tropical Plant Germplasm

Programme

[Day 1] October 20 (Tue)

08:30-09:00 Registration

Opening Ceremony 09:00-09:15 Opening address: Nobuyoshi Maeno (Director General, Japan International Research Center for Agricultural Sciences, Japan)

Keynote addresses Chairperson: Tsuguhiro Hoshino (Japan International Research Center for Agricultural Sciences, Japan)

09:15-09:45 Development of cryopreservation techniques – Akira Sakai (Japan) 09:45-10:15 Importance of cryopreservation for the conservation of plant genetic resources – Florent Engelmann (IPGRI) 10:15-10:35 Coffee break

Session 1. Fundamental aspects of cryopreservation Chairperson: David Cyr (BC Research Inc., Canada)

10:35-11:05 Freezing behaviours in plant tissues as visualized by NMR microscopy and their regulatory mechanisms – Masaya Ishikawa (National Institute of Agrobiological Resources, Japan) 11:05-11:35 Ultrastructural aspects for freezing adaptation of cells by vitrification – Seizo Fujikawa (Hokkaido University, Japan) 11:35-11:50 Discussion 11:50-13:30 Lunch

Chairperson: Patricia Berjak (University of Natal, South Africa)

13:30-14:00 The use of physical and biochemical studies to elucidate and reduce cryopreservation-induced damage in hydrated/desiccated plant germplasm – Dominique Dumet (University of Abertay-Dundee, UK) 14:00-14:30 Physiological and molecular changes in tobacco suspension cells during development of tolerance to cryopreservation by vitrification – Poula Reinhoud (Leiden University, The Netherlands) Programme 479

14:30-14:45 Coffee break 14:45-15:15 Molecular mechanisms of freezing and drought tolerance in plants – Kazuko Yamaguchi-Shinozaki (Japan International Research Center for Agricultural Sciences, Japan) 15:15-15:45 Cryopreservation of medicinal plant resources: retention of bio-synthetic capabilities in transformed cultures – Kayo Yoshimatsu (National Institute of Health Sciences, Japan) 15:45-16:00 Discussion

Reception

[Day 2] October 21 (Wed)

Session 2. Cryopreservation techniques Cryopreservation of plant cells Chairperson: Masaya Ishikawa (National Institute of Agrobiological Resources, Japan)

09:00-09:30 Cryopreservation of undifferentiated plant cells – Poula Reinhoud (Leiden University, The Netherlands) 09:30-09:50 Cryopreservation of banana embryogenic cell suspensions: a tool for genetic engineering – Bart Panis (Catholic University of Leuven, Belgium) 09:50-10:10 Cryopreservation of sugarcane embryogenic callus using a simplified freezing process – Florent Engelmann (IPGRI) 10:10-10:20 Discussion 10:20-10:40 Coffee break

Cryopreservation of pollen Chairperson: William Roca (CIAT)

10:40-11:00 Use of stored pollen for wide crosses in wheat haploid production – Masanori Inagaki (Japan International Research Center for Agricultural Sciences, Japan ) 11:00-11:20 Storage of pollen for long-term conservation of yam genetic resources – Nyat Quat Ng (IITA) 11:20-11:30 Discussion

Cryopreservation of embryos Chairperson: H.F. Chin (IPGRI/APO) 11:30-12:00 Cryopreservation of embryonic axes – Patricia Berjak (University of Natal, South Africa) 480 Cryopreservation of Tropical Plant Germplasm

12:00-13:00 Lunch 13:00-13:20 Cryopreservation of zygotic embryos of tropical fruit trees – a study on Lansium domesticum and Baccaurea species – M. Noor Normah (University Kebangsaan Malaysia, Malaysia) 13:20-13:40 Cryopreservation of coffee (Coffea arabica L.) seeds: Towards a simplified protocol for routine use in coffee genebanks – Stéphane Dussert (ORSTOM, France) 13:40-14:00 Cryopreservation of melon somatic embryos by desiccation method – Kei Shimonishi (Kagoshima Biotechnology Institute, Japan) 14:00-14:20 Cryopreservation of oil-palm (Elaeis guineensis Jacq.) polyembryonic cultures – Dominique Dumet (University of Abertay-Dundee, UK) 14:20-14:30 Discussion

Visit to genebank in Tsukuba and practical demonstrations

[Day 3] October 22 (Thur)

Session 2. Cryopreservation techniques (Cont.) Cryopreservation of apices Chairperson: Florent Engelmann (IPGRI)

09:00-09:30 Recent development in cryopreservation of shoot apices of tropical species – Hiroko Takagi (Japan International Research Center for Agricultural Sciences, Japan) 09:30-09:50 Cryopreservation of vegetatively propagated species (mainly mulberry) – Takao Niino (Tohoku National Agricultural Experimental Station, Japan) 09:50-10:10 Genotype considerations in temperate fruit crop cryopreservation – Barbara M. Reed (USAD-ARS, USA) 10:10-10:30 Coffee

Chairperson: Tsukasa Nagamine (National Institute for Agrobiological Resources, Japan)

10:30-10:50 Cryopreservation of in vitro-grown meristems of potato (Solanum tuberosum L.) by encapsulation-vitrification – Dai Hirai (Hokkaido Prefectural Plant Genetic Resources Center, Japan) 10:50-11:10 Cryopreservation of in vitro-cultured meristems of wasabi – Toshikazu Matsumoto (Shimane Agricultural Experiment Station, Japan) Programme 481

11:10-11:30 Cryopreservation of citrus apices using the encapsulation- dehydration technique – Florent Engelmann (IPGRI) 11:30-11:50 Development of cassava cryopreservation – Roosevelt H. Escobar (CIAT) 11:50-13:00 Lunch

Chairperson: Mary Taylor (Secretariat of the Pacific Community, Fiji)

13:00-13:20 Cryopreservation of in vitro-grown shoot tips of five vegetatively propagated tropical monocots by vitrification technique – Nguyen Tien Thin (Nuclear Research Institute, Vietnam) 13:20-13:40 Cryopreservation of yam apices: a comparative study with three different techniques – Binay B. Mandal (National Bureau of Plant Genetic Resources, India) 13:40-14:00 Cryopreservation of proliferating meristem cultures of banana – Bart Panis (Catholic University of Leuven, Belgium)

14:00-14:10 Discussion

14:10-15:30 Poster viewing (& Coffee)

15:30-17:00 Discussion on cryopreservation techniques, incorporating both oral and poster presentations

Chairpersons: Barbara M. Reed (USDA-ARS, USA) M. Noor Normah (Universiti Kebangsaan Malaysia, Malaysia) Bart Panis (Catholic University of Leuven, Belgium)

Buffet Party

[Day 4] October 23 (Fri)

Session 3. Ongoing cryopreservation projects – Research and its application Chairperson: Shou Yong Ng (IITA)

09:00-09:20 Application of cryopreservation protocols at a clonal genebank – Barbara Reed (USDA-ARS USA) 09:20-09:40 Advances in potato cryopreservation at the International Potato Center, Peru – Ali Golmirzaie (CIP) 09:40-10:00 The in vitro germplasm collection at the Musa INIBAP Transit Centre and the importance of cryopreservation – Bart Panis (Catholic University of Leuven, Belgium) 482 Cryopreservation of Tropical Plant Germplasm

10:00-10:20 Coffee break 10:20-10:40 Cryopreservation: Roles in clonal propagation and germplasm conservation of conifers – David Cyr (BC Research Inc., Canada) 10:40-11:00 Conservation of threatened flora by cryopreservation of shoot apices – Daren Touchell (USAD-ARS, USA) 11:00-11:20 Conservation and cryopreservation of tropical tree species – Chalermpol Kirdmanee (National Center for Genetic Engineering and Biotechnology, Thailand) 11:20-11:40 Cryopreervation and cassava germplasm conservation at CIAT – William Roca (CIAT) 11:40-12:00 Discussion 12:00-13:10 Lunch

Session 4. Current status of cryopreservation research and future perspectives of its application in national programmes Chairperson: H.F. Chin (IPGRI-APO)

13:10-13:20 [India]: Cryopreservation research in India: current status and future perspectives – Binay B. Mandal (National Bureau of Plant Genetic Resources, India ) 13:20-13:30 [Malaysia]: Current status of cryopreservation research and future perspectives of its application in Malaysia – Normah M. Noor (University Kebangsaan Malaysia, Malaysia) 13:30-13:40 [Indonesia]: Cryopreservation of tropical plants: current research status in Indonesia – Enny Sudarmonowati (Indonesian Institute of Sciences, Indonesia ) 13:40-13:50 [Thailand]: Status and future prospects of cryopreservation in Thailand – Chalermpol Kirdmanee (National Center for Genetic Engineering and Biotechnology, Thailand) 13:50-14:00 [Philippines]: Phillipines: Conservation and cryopreservation research – Alfinetta Zamora (University of the Philippines Los Ba ños, Philippines) 14:00-14:10 [Vietnam]: Current status and future perspectives of plant cryopreservation in Viet Nam – Thinh Tien Nguyen (Nuclear Research Institute, Viet Nam) 14:10-14:20 [China]: Cryopreservation of plant germplasm in China – Tie-gang Lu (Chinese Academy of Sciences, China ) 14:20-14:30 [South Africa]: Current status of cryopreservation research and future perspectives of its application in South Africa – Patricia Berjak (University of Natal, South Africa) Programme 483

14:30-14:40 [Nigeria]: Applying cryopreservation techniques in national programmes in Nigeria – Pius M. Kyesmu (University of Jos, Nigeria) 14:40-14:50 [Costa Rica]: Current status of cryopreservation research and future perspectives for its application in Costa Rica – Ana Abdelnour Esquivel (Costa Rica Institute of Technology, Costa Rica) 14:50-15:00 [South Pacific]: Current status of cryopreservation research and future perspectives of its application in the South Pacific – Mary Taylor (Secretariat for the Pacific Community, Fiji) 15:00-15:20 Coffee break

15:20-16:20 General Discussion and Summarization

Closing address Florent Engelmann (IPGRI) 484 Cryopreservation of Tropical Plant Germplasm

Participants

Dr Ana ABDELNOUR-ESQUIVEL Fax: 81-047-363-1221 Costa Rica Institute of Technology [email protected] Cartago, Costa Rica 179-7050 COSTA RICA Present address: Tel: 506-552-53-33 Marilyn M. Belarmino Fax: 506-551-53-48 Dept. of Horticulture, ViSCA [email protected] Baybay, Leyte 6521-A Philippines Dr Maria Elena AGUILAR e-mail:[email protected] CATIE-Unidad de Biotecnologia Turrialba 7170 Prof. Patricia BERJAK COSTA RICA School of Life & Environmental Sciences Tel: 506-55-66-455 University of Natal Fax: 506-55-61-533 Durban 4041 [email protected] SOUTH AFRICA Tel: 27-31-260-3197 Dr Sarah E. ASHMORE Fax: 27-31-2601195/2029 School of BBS [email protected] Griffith University Nathan QLD 4111 Ms Rommanee CHAROENSUB AUSTRALIA Scientific Equipment Center, KURDI Tel: 61-7-3875-7346 Kasetsart University Fax: 61-7-3875-7656 Bangkok 10900 [email protected] THAILAND Tel: 66-2-579-5538 Ms Khin AYE Fax: 66-2-579-9617 Seed Bank [email protected] Central Agriculture Research Institute Yein, Yyinmana Dr Rekha CHAUDHURY MYANMAR National Bureau of Plant Genetic Tel: 095-067-2118 Resources (NBPGR), Pusa Campus New Delhi 110012 Ms Margarita C. BARTICEVIC INDIA INIA CRI Tel: 91-11-5783697 La Platina Fax: 91-11-5731495, 5785619 Santa Rosa 11610 La Pintana [email protected] Santiago CHILE Prof. H.F. CHIN IPGRI-APO Dr Marilyn M. BELARMINO Regional Office APO Laboratory of Plant Cell Technology PO Box 236 UPM Post Office Faculty of Horticulture Serdang 43400 Selangor Chiba University MALAYSIA 648 Matsudo, Matsudo City Fax: 603-9487655 Chiba 271-8510 [email protected] JAPAN Participants 485

Dr Mohammed K. CHOWDHURY Mr Stéphane DUSSERT International Paper ORSTOM 719 Southlands Road 911 Av.d'Agropolis Bainbridge GA 31717 34032 Montpellier BP5045 USA FRANCE Tel: 912-246-3642 ext. 283 Tel: 33-467-416185 Fax: 912-243-0766 Fax: 33-467-547800 [email protected] [email protected]

Dr Richard J. CROSS Dr Florent ENGELMANN Crop & Food Research IPGRI Private Bag 4704 Via delle Sette Chiese 142 Christchurch 00145 Rome NEW ZEALAND ITALY Tel: 64-3-325-6400 Tel: 39-06-51892224 Fax: 64-3-3252074 Fax: 39-06-5750309 [email protected] [email protected]

Dr David CYR Dr Roosevelt ESCOBAR Clone Bank Development CIAT BC Research Inc. SILUAGEN INC. Km 17 Via Cali-Palmira 3650 Wesbrook Mall A.A. 6713 Vancouver V6S2L2 COLOMBIA CANADA Tel: 57-2-4450055 Tel: 404-224-4331 Fax: 57-2-4450073 Fax: 604-224-0540 [email protected] [email protected] Dr Bruno FLORIN Ms Guiomar S. DAVILA Centre de Recherche Nestlé-Tours Saitama Ornamental Plants Research 101 Avenue Gustave Eiffel Center Notre Dame d' Oé BP9716 24 Ohaza Kushibiki 37097 Tours Cedex 02 Fukaya, Saitama 366-0815 FRANCE JAPAN Tel: 33-247-62-83-83 Tel: 81- 0485-72-1220 Fax: 33-247-49-14-14 Fax: 81-0485-72-1223 [email protected]

Dr Dominique DUMET Prof. Seizo FUJIKAWA University of Abertay-Dundee Institute of Low Temperature Science School of Molecular and Life Sciences Hokkaido University Kydd Building Sapporo, Kita-ku, Kita 19 Nishi 8 Bell St. Dundee, Scotland, DD1 HG Hokkaido 060-0819 UK JAPAN Tel: 44-1-382-308-651 Tel: 81-011-706-5471 Fax: 44-1-382-308-663 Fax: 81-011-706-7142 [email protected] [email protected] 486 Cryopreservation of Tropical Plant Germplasm

Mr Eiji FUJIYOSHI Dr Kazumasa HIRATA Seikai National Fish Research Institute Graduate School of Pharmaceutical 49 Kokubu-machi Sciences Nagasaki 850-0951 Osaka University JAPAN 1-6 Yamadaoka, Suita Tel: 81-095-822-8158 Osaka 565-0871 Fax: 81-095-821-4494 JAPAN Tel: 81-06-879-8236 Dr Seiichi FUKAI Fax: 81-06-879-8239 Kagawa University [email protected] Miki-cho, Kita-gun Kagawa 761-0795 Dr Tsuguhiro HOSHINO JAPAN Japan International Research Center for Tel: 81-087-891-3072 Agricultural Sciences (JIRCAS) Fax: 81-087-891-3072 1-2 Ohwashi, Tsukuba [email protected] Ibaraki 305-8686 JAPAN Dr Ali M. GOLMIRZAIE Tel: 81-0298-38-6305 International Potato Center (CIP) Fax: 81-0298-38-6650 Apartado 5969 [email protected] Lima 100 PERU Prof. Xue-Lin HUANG Tel: 51-14-349-6017 Department of Biology Fax: 51-14-35-1570 Zhongshan University [email protected] Guangzhou, 135 west Xingang Road 510275 Dr Claudia L. GUEVARA CHINA Centro Internacional de Agricultura Fax: 86-20-84184592 Tropical (CIAT) [email protected] A.A. 67-13 Cali Dr Masanori INAGAKI COLOMBIA Japan International Research Center for Tel: 57-2-4450000 Agricultural Sciences (JIRCAS) Fax: 57-2-4450073 1-2 Ohwashi, Tsukuba [email protected] Ibaraki 305-8686 JAPAN Mr Dai HIRAI Tel: 81-0298-38-6313 Hokkaido Plant Genetic Resources Fax: 81-0298-38-6316 Center [email protected] 363-2 Minami Takinokawa Takikawa Hokkaido 073-0013 Dr Katsuaki ISHII JAPAN Forestry and Forest Products Research Tel: 81-0125-23-3195 Institute Fax: 81-0125-24-3877 Matsunosato-1, Kukisaki, Inashiki-gun [email protected] Ibaraki 305-8687 JAPAN Tel: 81-0298-73-3211 (ext 451) Fax: 81-0298-73-3795 [email protected] Participants 487

Dr Masaya ISHIKAWA Dr Manabu KATANO Dept. Genetic Resources, NIAR Department of Agronomy Kannondai 2-1-2,Tsukuba School of Agriculture Ibaraki 305-8602 Kyushu Tokai University JAPAN Choyo-son, Aso-gun Tel: 81-0298-38-7449 Kumanoto 869-1404 Fax: 81-0298-38-7408 JAPAN [email protected] Tel: 81-09676-7-0611 Fax: 81-09676-7-2659 Dr Keiko ISHIKAWA Mkatano@aa-1. ktokai-u.ac.jp Laboratory of Plant Cell Technology Faculty of Horticulture Dr Chalermpol KIRDMANEE Chiba University National Centre for Genetic Engineering 648 Matsudo, Matsudo City & Biotechnology Chiba 271-8510 National Science and Technology JAPAN Development Agency Tel: 81-047-363-1221 731 Rama VI Road, Rajdhevee Fax: 81-047-366-2230 Bangkok 10400 [email protected] THAILAND Tel: 66-2-6448150-4 Ms Miwako ITO Fax: 66-2-6448107 Saitama Ornamental Plants Research [email protected] Center 24 Ohaza Kushibiki Mr Takasi KOBAYASHI Fukaya Laboratory of Horticultural Plant Saitama 366-0815 Breeding JAPAN Faculty of Agriculture Tel: 81-0485-72-1220 Meiji University Fax: 81-0485-72-1223 1-1-1, Higashi-mita, Tama-ku [email protected] Kawasaki, Kanagawa 214-8571 JAPAN Ms Rie ITO-OGAWA Tel: 81-44-934-7096 Aichi Agricultural Research Center Fax: 81-44-934-7092 Sagamine Yazako Nagakute-cho [email protected] Aichi 480-1103 JAPAN Mr Yasufumi KUNIHIRO Tel: 81-0561-62-0085 National Institute of Agrobiological Fax: 81-0561-63-0815 Resources [email protected] Kannondai 2-1-2,Tsukuba Ibaraki 305-8602 Mr Yutaka JITSUYAMA JAPAN Hokkaido University Tel: 81-0298-7050 Nishi 9, Kitakyujo, Kita-ku Fax: 81-0298-38-7054 Sapporo [email protected] Hokkaido 060-0809 JAPAN Tel: 81-011-706-5471 Fax: 81-011-706-7142 [email protected] 488 Cryopreservation of Tropical Plant Germplasm

Dr Pius M. KYESMU Dr Mari MARUTANI Department of Botany and Microbiology College of Agriculture and Life Sciences University of Jos Uniuversity of Guam P.M.B.2048s UOG Station Jos Mangilao Guam 96923 NIGERIA USA Tel: 234-73-460740 Tel: 671-735-2131 [email protected] Fax: 671-734-6842 [email protected] Dr Tie Gang LU Institute of Botany Dr M. MARZALINA Chinese Academy of Sciences Seed Technology Section Beijing 100093, China Forest Plantation Division Forest Research Institute Malaysia Present address: (FRIM) Laboratory of Genome Function Kepong National Institute of Agrobiological Kuala Lumpur 52109 Resouces MALAYSIA Kannondai 2-1-2, Tsukuba Tel: 6-03-6342633 Ibaraki 305-8602 Fax: 6-03-6367753 JAPAN [email protected] Tel: 81-0298-38-7006 Fax: 81-0298-38-7006 Dr Toshikazu MATSUMOTO [email protected] Shimane Agricultural Experiment Station Dr Nobuyoshi MAENO Ashiwata 2440, Izumo Japan International Research Center for Shimane 693-0035 Agricultural Sciences (JIRCAS) JAPAN 1-2 Ohwashi, Tsukuba Tel: 81-0853-22-6650 Ibaraki 305-8686 Fax: 81-0853-21-8380 JAPAN [email protected] Tel: 81-0298-38-6301 [email protected] Ms Than MAY Seed Bank, Cenral Agriculture Research Dr Binay B. MANDAL Institute National Bureau of Plant Genetic Yein, Pyinmana Resources (NBPGR), Pusa Campus MYANMAR New Delhi 110012 Tel: 095-067-21118 INDIA Tel: 91-11-5818343 Mr Tomohiro MINAMI Fax: 91-11-5785619 Kagoshima Agricultural Experiment [email protected] Station Osumi Branch 4938 Hosoyamada, Kushira Kagoshima 893-1601 JAPAN Tel: 81-994-62-4354 Fax: 81-994-62-2973 [email protected] Participants 489

Mr Susumu MIYASHITA Dr S.Y.C. NG National Institute of Agrobiological International Institute of Tropical Resources Agriculture (IITA), Nigeria Kannondai, 2-1-2,Tsukuba c/o Lambourn & Co. Ibaraki 305-8602 Carolyn House JAPAN 26 Dingwall Rd Tel: 81-0298-38-7050 Croydon CR9 3EE Fax: 81-0298-38-7054 ENGLAND [email protected] Tel: 234-2-241-2626 Fax: 234-2-241-2221 Dr Nobuo MURATA [email protected] Agriculture, Forestry and Fisheries Technology Society (AFFTIS) Dr Takao NIINO 2-1-2 Kannondai, Tsukuba Tohoku National Agricultural Ibaraki 305-0856 Experiment Station JAPAN MAFF Tel: 81-0298-36-6543 50 Harajukuminami, Arai Fax: 81-0298-36-5206 Fukushima 960-2156 [email protected], JAPAN [email protected] Tel: 81-024-593-5151 Fax: 81-024-593-2155 Dr Tsukasa NAGAMINE [email protected] National Institute of Agrobiological Resources Ms Eiko NIWATA 2-1-2 Kannondai, Tsukuba Aomori Pref. Green Biocenter Ibaraki 305-8602 221-10 Yamaguchi, Nogi JAPAN Aomori 030-0142 Tel: 81-0298-38-7449 JAPAN Fax: 81-0298-38-7408 Tel: 81-177-28-1015 [email protected] Fax: 81-177-28-1017 [email protected] Dr N. Quat NG International Institute of Tropical Dr Akinori NOGUCHI Agriculture (IITA), Nigeria Japan International Research Center for c/o Lambourn & Co. Agricultural Sciences (JIRCAS) Carolyn House 1-2 Ohwashi, Tsukuba 26 Dingwall Rd Ibaraki 305-8686 Croydon CR9 3EE JAPAN ENGLAND Tel: 81-0298-38-6307 Tel: 234-2-241-2626 Fax: 81-0298-38-6652 Fax: 234-2-241-2221 [email protected] [email protected] 490 Cryopreservation of Tropical Plant Germplasm

Prof. M.N. NORMAH Prof. Young-Goo PARK Department of Botany Kyungpook National University Faculty of Life Sciences Department of Forestry University Kebangsaan Malaysia Taegu 702-701 Bangi 43600 REPUBLIC of KOREA MALAYSIA Tel: 82-53-950-5747 Tel: 603-8292867 Fax: 82-53-950-6708 Fax: 603-8293244 [email protected] [email protected] Ms Gabriela B. QUIRAGA Ms Rieko OGURA Faculty of Agricultural Sciences Laboratory of Horticultural Plant Cordoba National University Breeding Av. Valparaiso s/n cc 509-5000 Faculty of Agriculture, Meiji University Cordoba 1-1-1, Higashi-mita, Tama-ku ARGENTIN E Kawasaki, Kanagawa 214-8571 JAPAN Mr Punathil E. RAJASEKARAN Tel: 81-044-934-7096 Indian Institute of Horticultural Fax: 81-044-934-7092 Research [email protected] Hessaraghatta Lake (PO) Bangalore 590089 Dr Seibi OKA INDIA National Institute of Agrobiological Tel: 91-80-8496634 Resources Fax: 91-80-5282619 2-1-2 Kannondai, Tsukuba [email protected] Ibaraki 305-8602 JAPAN Dr Barbara REED Tel: 81-0298-38-8388 US Dept. of Agriculture Fax: 81-0298-38-8397 National Clonal Germplasm Repository [email protected] 33447 Pegria Rd Corvallis OR 97333-2521 Dr Kiyoharu OONO USA National Institute of Agrobiological Tel: 1-541-750-8712 ext.111 Resources Fax: 1-541-750-8717 2-1-2 Kannondai, Tsukuba [email protected] Ibaraki 305-8602 JAPAN Dr Poula J. REINHOUD Tel: 81-0298-38-7461 Institute of Molecular Plant Sciences Fax: 81-0298-38-7408 PO Box 9502 2300 [email protected] RA Leiden THE NETHERLANDS Dr Bart PANIS Tel: 31-71-5274752 Catholic University of Leuven Fax: 31-71-5274469 Kard. Mercierlaan 92 [email protected] 3001 Heverlee BELGIUM Tel: 016-32-14-81 Fax: 016-32-19-93 [email protected] Participants 491

Dr William ROCA Dr Kei SHIMONISHI Centro Internacional de Agricultura Kagoshima Biotechnology Institute Tropical (CIAT) 4938 Hosoyamada, Kushira Km 17 via Cali-Palmira Kagoshima 893-1601 A.A. 6713 JAPAN COLOMBIA Tel: 81-0994-62-4112 Tel: 57-2-4450055 Fax: 81-0994-62-4114 Fax: 57-2-4450073 [email protected] [email protected] Dr Enny SUDARMONOWATI Mr Mustapha B. A. ROUAISSI Researcher of R&D Laboratory of Genetics Centre for Biotechnology-LIPI Agronomic National Institute of Tunisia Jl.Raya Bogor KM.46 Cité Maharajene Cibinong 16911 43 Avenue Charles Nicolle INDONESIA 1082 Tunis Tel: 62-21-875-4625 TUNISIA Fax: 62-21-875-4588 [email protected] Prof. Akira SAKAI Asabucho 1-5-23 Mr SYAFARUDDIN Kitaku Research Institute for Spice and Sapporo 001-0045 Medicinal Crops JAPAN JL. Tentara Pelajar No.3 Tel: 81-011-716-7711 Bogor Fax: 81-011-716-7711 INDONESIA [email protected] Dr Hiroko TAKAGI Dr Satoshi SAKAMOTO Japan International Research Center for JICA EXPERT Agricultural Sciences (JIRCAS) JI. Perkutut No.13 1-2 Ohwashi, Tsukuba Tanah Sareal Ibaraki 305-8686 Bogor 16161 JAPAN INDONESIA Tel: 81-0298-38-6384 Tel: 62-251-327615 Fax: 81-0298-38-6342 Fax: 62-251-327615 [email protected] [email protected] Prof. Isao TARUMOTO Dr Koichi SHIMOMURA College of Agriculture Tsukuba Medical Plant Research Station Osaka Prefecture University NIHS 1-1 Gakuen-cho, Sakai 1 Hachimandai, Tsukuba Osaka 599-8231 Ibaraki 305-0843 JAPAN JAPAN Tel: 81-0722-54-9404 Tel: 81-0298-38-0573 Fax: 81-0722-54-9404 Fax: 81-0298-38-0575 [email protected] 492 Cryopreservation of Tropical Plant Germplasm

Dr Mary TAYLOR Dr Kunio TSUBOTA Secretariat of the Pacific Community Japan International Research Center for Private Mail Bag Agricultural Sciences (JIRCAS) Suva 1-2 Ohwashi, Tsukuba FIJI Ibaraki 305-8686 Tel: 679-370733 JAPAN Fax: 679-370021 Tel: 81-0298-38-6304 [email protected] Fax: 81-0298-38-6342 [email protected] Mr Hiroyoshi TERUYA Okinawa Agricultural Experiment Mr Hikaru TSUKAZAKI Station National Research Institute of 76 Kanekadan Gushikawa Vegetable, Ornamental Plants and Tea Okinawa 904-2241 360 Knsawa, Ano-Town JAPAN Mie 514-2392 Tel: 81-098-973-5530 JAPAN Fax: 81-098-973-6333 Tel: 81-059-268-4654 [email protected] Dr Tien Nguyen THINH Nuclear Research Institute Mr Cuong Pham VAN 01 Nguyen Tuluc St. Department of Food Crops Dalat Faculty of Agronomy VIET NAM Hanoi Agricultural University Tel: 84-63-8-25107 Gialam Hanoi Fax: 84-63-8-21122 VIETNAM

Mr Tin TIN Dr Tuan Dhinh VUONA Germplasm Conservation Section Cuu Long Delta Rice Research Institute, c/o Seed Bank CARI Vietnam Yezin Pynmana Present address: MYANMAR Biological Res. Division JIRCAS Dr Darren H. TOUCHELL 1-2 Ohwashi, Tsukuba USDA-ARS Ibaraki 305-8686 National Seed Storage Laboratory JAPAN 1111 South Mason St [email protected] Ft Collins, Colorado 80521 USA Mr Reyes B. WALTEROSWALDO Tel: 1-970-495-3217 Experiment Station Fax: 1-970-221-1427 PICHILINGUE, INIAP [email protected] Km 5 Road Quevedo-EL Empalme EQUADOR Ms Kaori TOUNO University of Chiba Dr Masahiko WATANABE 1 Hachimandai Tsukuba NISES Ibaraki 305-0843 4-11-1-416-303, Matsushiro, Tsukuba JAPAN Ibaraki 305-0035 Tel: 81-0298-38-0573 JAPAN Fax: 81-0298-38-0575 Participants 493

Mr Yongjie WU Dr Kayo YOSHIMATSU Changli Institute of Pomology Tsukuba Medical Plant Research Station Hebei Academy of Agricultural and National Institute of Health Sciences Forestry Sciences 1 Hachimandai, Tsukuba Hebei 066600 Ibaraki 305-0843 Changli Town JAPAN CHINA Tel: 81-0298-38-0573 Fax: 335-20-23417, 23228 Fax: 81-0298-38-0575 [email protected] [email protected]

Mr Eiji YAMAGUCHI Ms Alfinetta B. ZAMORA National Center for Seeds and Seedlings University of the Philippines at Los Unzen Station Baños Saigo-bo 1494-34, Mizuho Los Baños Minami Takaki Laguna 4037 Nagasaki 859-1211 PHILIPPINES JAPAN Tel: 049-536-3528 Tel: 81-0957-77-2100 Fax: 049-536-3438 Fax: 81-0957-77-2154 [email protected] [email protected]

Dr Kazuko YAMAGUCHI-SHINOZAKI Japan International Research Center for Agricultural Sciences (JIRCAS) 1-2 Ohwashi, Tsukuba Ibaraki 305-8686 JAPAN Tel: 81-0298-38-6641 Fax: 81-0298-38-6643 [email protected] 484 Cryopreservation of Tropical Plant Germplasm

Participants

Dr Ana ABDELNOUR-ESQUIVEL Fax: 81-047-363-1221 Costa Rica Institute of Technology [email protected] Cartago, Costa Rica 179-7050 COSTA RICA Present address: Tel: 506-552-53-33 Marilyn M. Belarmino Fax: 506-551-53-48 Dept. of Horticulture, ViSCA [email protected] Baybay, Leyte 6521-A Philippines Dr Maria Elena AGUILAR e-mail:[email protected] CATIE-Unidad de Biotecnologia Turrialba 7170 Prof. Patricia BERJAK COSTA RICA School of Life & Environmental Sciences Tel: 506-55-66-455 University of Natal Fax: 506-55-61-533 Durban 4041 [email protected] SOUTH AFRICA Tel: 27-31-260-3197 Dr Sarah E. ASHMORE Fax: 27-31-2601195/2029 School of BBS [email protected] Griffith University Nathan QLD 4111 Ms Rommanee CHAROENSUB AUSTRALIA Scientific Equipment Center, KURDI Tel: 61-7-3875-7346 Kasetsart University Fax: 61-7-3875-7656 Bangkok 10900 [email protected] THAILAND Tel: 66-2-579-5538 Ms Khin AYE Fax: 66-2-579-9617 Seed Bank [email protected] Central Agriculture Research Institute Yein, Yyinmana Dr Rekha CHAUDHURY MYANMAR National Bureau of Plant Genetic Tel: 095-067-2118 Resources (NBPGR), Pusa Campus New Delhi 110012 Ms Margarita C. BARTICEVIC INDIA INIA CRI Tel: 91-11-5783697 La Platina Fax: 91-11-5731495, 5785619 Santa Rosa 11610 La Pintana [email protected] Santiago CHILE Prof. H.F. CHIN IPGRI-APO Dr Marilyn M. BELARMINO Regional Office APO Laboratory of Plant Cell Technology PO Box 236 UPM Post Office Faculty of Horticulture Serdang 43400 Selangor Chiba University MALAYSIA 648 Matsudo, Matsudo City Fax: 603-9487655 Chiba 271-8510 [email protected] JAPAN Participants 485

Dr Mohammed K. CHOWDHURY Mr Stéphane DUSSERT International Paper ORSTOM 719 Southlands Road 911 Av.d'Agropolis Bainbridge GA 31717 34032 Montpellier BP5045 USA FRANCE Tel: 912-246-3642 ext. 283 Tel: 33-467-416185 Fax: 912-243-0766 Fax: 33-467-547800 [email protected] [email protected]

Dr Richard J. CROSS Dr Florent ENGELMANN Crop & Food Research IPGRI Private Bag 4704 Via delle Sette Chiese 142 Christchurch 00145 Rome NEW ZEALAND ITALY Tel: 64-3-325-6400 Tel: 39-06-51892224 Fax: 64-3-3252074 Fax: 39-06-5750309 [email protected] [email protected]

Dr David CYR Dr Roosevelt ESCOBAR Clone Bank Development CIAT BC Research Inc. SILUAGEN INC. Km 17 Via Cali-Palmira 3650 Wesbrook Mall A.A. 6713 Vancouver V6S2L2 COLOMBIA CANADA Tel: 57-2-4450055 Tel: 404-224-4331 Fax: 57-2-4450073 Fax: 604-224-0540 [email protected] [email protected] Dr Bruno FLORIN Ms Guiomar S. DAVILA Centre de Recherche Nestlé-Tours Saitama Ornamental Plants Research 101 Avenue Gustave Eiffel Center Notre Dame d' Oé BP9716 24 Ohaza Kushibiki 37097 Tours Cedex 02 Fukaya, Saitama 366-0815 FRANCE JAPAN Tel: 33-247-62-83-83 Tel: 81- 0485-72-1220 Fax: 33-247-49-14-14 Fax: 81-0485-72-1223 [email protected]

Dr Dominique DUMET Prof. Seizo FUJIKAWA University of Abertay-Dundee Institute of Low Temperature Science School of Molecular and Life Sciences Hokkaido University Kydd Building Sapporo, Kita-ku, Kita 19 Nishi 8 Bell St. Dundee, Scotland, DD1 HG Hokkaido 060-0819 UK JAPAN Tel: 44-1-382-308-651 Tel: 81-011-706-5471 Fax: 44-1-382-308-663 Fax: 81-011-706-7142 [email protected] [email protected] 486 Cryopreservation of Tropical Plant Germplasm

Mr Eiji FUJIYOSHI Dr Kazumasa HIRATA Seikai National Fish Research Institute Graduate School of Pharmaceutical 49 Kokubu-machi Sciences Nagasaki 850-0951 Osaka University JAPAN 1-6 Yamadaoka, Suita Tel: 81-095-822-8158 Osaka 565-0871 Fax: 81-095-821-4494 JAPAN Tel: 81-06-879-8236 Dr Seiichi FUKAI Fax: 81-06-879-8239 Kagawa University [email protected] Miki-cho, Kita-gun Kagawa 761-0795 Dr Tsuguhiro HOSHINO JAPAN Japan International Research Center for Tel: 81-087-891-3072 Agricultural Sciences (JIRCAS) Fax: 81-087-891-3072 1-2 Ohwashi, Tsukuba [email protected] Ibaraki 305-8686 JAPAN Dr Ali M. GOLMIRZAIE Tel: 81-0298-38-6305 International Potato Center (CIP) Fax: 81-0298-38-6650 Apartado 5969 [email protected] Lima 100 PERU Prof. Xue-Lin HUANG Tel: 51-14-349-6017 Department of Biology Fax: 51-14-35-1570 Zhongshan University [email protected] Guangzhou, 135 west Xingang Road 510275 Dr Claudia L. GUEVARA CHINA Centro Internacional de Agricultura Fax: 86-20-84184592 Tropical (CIAT) [email protected] A.A. 67-13 Cali Dr Masanori INAGAKI COLOMBIA Japan International Research Center for Tel: 57-2-4450000 Agricultural Sciences (JIRCAS) Fax: 57-2-4450073 1-2 Ohwashi, Tsukuba [email protected] Ibaraki 305-8686 JAPAN Mr Dai HIRAI Tel: 81-0298-38-6313 Hokkaido Plant Genetic Resources Fax: 81-0298-38-6316 Center [email protected] 363-2 Minami Takinokawa Takikawa Hokkaido 073-0013 Dr Katsuaki ISHII JAPAN Forestry and Forest Products Research Tel: 81-0125-23-3195 Institute Fax: 81-0125-24-3877 Matsunosato-1, Kukisaki, Inashiki-gun [email protected] Ibaraki 305-8687 JAPAN Tel: 81-0298-73-3211 (ext 451) Fax: 81-0298-73-3795 [email protected] Participants 487

Dr Masaya ISHIKAWA Dr Manabu KATANO Dept. Genetic Resources, NIAR Department of Agronomy Kannondai 2-1-2,Tsukuba School of Agriculture Ibaraki 305-8602 Kyushu Tokai University JAPAN Choyo-son, Aso-gun Tel: 81-0298-38-7449 Kumanoto 869-1404 Fax: 81-0298-38-7408 JAPAN [email protected] Tel: 81-09676-7-0611 Fax: 81-09676-7-2659 Dr Keiko ISHIKAWA Mkatano@aa-1. ktokai-u.ac.jp Laboratory of Plant Cell Technology Faculty of Horticulture Dr Chalermpol KIRDMANEE Chiba University National Centre for Genetic Engineering 648 Matsudo, Matsudo City & Biotechnology Chiba 271-8510 National Science and Technology JAPAN Development Agency Tel: 81-047-363-1221 731 Rama VI Road, Rajdhevee Fax: 81-047-366-2230 Bangkok 10400 [email protected] THAILAND Tel: 66-2-6448150-4 Ms Miwako ITO Fax: 66-2-6448107 Saitama Ornamental Plants Research [email protected] Center 24 Ohaza Kushibiki Mr Takasi KOBAYASHI Fukaya Laboratory of Horticultural Plant Saitama 366-0815 Breeding JAPAN Faculty of Agriculture Tel: 81-0485-72-1220 Meiji University Fax: 81-0485-72-1223 1-1-1, Higashi-mita, Tama-ku [email protected] Kawasaki, Kanagawa 214-8571 JAPAN Ms Rie ITO-OGAWA Tel: 81-44-934-7096 Aichi Agricultural Research Center Fax: 81-44-934-7092 Sagamine Yazako Nagakute-cho [email protected] Aichi 480-1103 JAPAN Mr Yasufumi KUNIHIRO Tel: 81-0561-62-0085 National Institute of Agrobiological Fax: 81-0561-63-0815 Resources [email protected] Kannondai 2-1-2,Tsukuba Ibaraki 305-8602 Mr Yutaka JITSUYAMA JAPAN Hokkaido University Tel: 81-0298-7050 Nishi 9, Kitakyujo, Kita-ku Fax: 81-0298-38-7054 Sapporo [email protected] Hokkaido 060-0809 JAPAN Tel: 81-011-706-5471 Fax: 81-011-706-7142 [email protected] 488 Cryopreservation of Tropical Plant Germplasm

Dr Pius M. KYESMU Dr Mari MARUTANI Department of Botany and Microbiology College of Agriculture and Life Sciences University of Jos Uniuversity of Guam P.M.B.2048s UOG Station Jos Mangilao Guam 96923 NIGERIA USA Tel: 234-73-460740 Tel: 671-735-2131 [email protected] Fax: 671-734-6842 [email protected] Dr Tie Gang LU Institute of Botany Dr M. MARZALINA Chinese Academy of Sciences Seed Technology Section Beijing 100093, China Forest Plantation Division Forest Research Institute Malaysia Present address: (FRIM) Laboratory of Genome Function Kepong National Institute of Agrobiological Kuala Lumpur 52109 Resouces MALAYSIA Kannondai 2-1-2, Tsukuba Tel: 6-03-6342633 Ibaraki 305-8602 Fax: 6-03-6367753 JAPAN [email protected] Tel: 81-0298-38-7006 Fax: 81-0298-38-7006 Dr Toshikazu MATSUMOTO [email protected] Shimane Agricultural Experiment Station Dr Nobuyoshi MAENO Ashiwata 2440, Izumo Japan International Research Center for Shimane 693-0035 Agricultural Sciences (JIRCAS) JAPAN 1-2 Ohwashi, Tsukuba Tel: 81-0853-22-6650 Ibaraki 305-8686 Fax: 81-0853-21-8380 JAPAN [email protected] Tel: 81-0298-38-6301 [email protected] Ms Than MAY Seed Bank, Cenral Agriculture Research Dr Binay B. MANDAL Institute National Bureau of Plant Genetic Yein, Pyinmana Resources (NBPGR), Pusa Campus MYANMAR New Delhi 110012 Tel: 095-067-21118 INDIA Tel: 91-11-5818343 Mr Tomohiro MINAMI Fax: 91-11-5785619 Kagoshima Agricultural Experiment [email protected] Station Osumi Branch 4938 Hosoyamada, Kushira Kagoshima 893-1601 JAPAN Tel: 81-994-62-4354 Fax: 81-994-62-2973 [email protected] Participants 489

Mr Susumu MIYASHITA Dr S.Y.C. NG National Institute of Agrobiological International Institute of Tropical Resources Agriculture (IITA), Nigeria Kannondai, 2-1-2,Tsukuba c/o Lambourn & Co. Ibaraki 305-8602 Carolyn House JAPAN 26 Dingwall Rd Tel: 81-0298-38-7050 Croydon CR9 3EE Fax: 81-0298-38-7054 ENGLAND [email protected] Tel: 234-2-241-2626 Fax: 234-2-241-2221 Dr Nobuo MURATA [email protected] Agriculture, Forestry and Fisheries Technology Society (AFFTIS) Dr Takao NIINO 2-1-2 Kannondai, Tsukuba Tohoku National Agricultural Ibaraki 305-0856 Experiment Station JAPAN MAFF Tel: 81-0298-36-6543 50 Harajukuminami, Arai Fax: 81-0298-36-5206 Fukushima 960-2156 [email protected], JAPAN [email protected] Tel: 81-024-593-5151 Fax: 81-024-593-2155 Dr Tsukasa NAGAMINE [email protected] National Institute of Agrobiological Resources Ms Eiko NIWATA 2-1-2 Kannondai, Tsukuba Aomori Pref. Green Biocenter Ibaraki 305-8602 221-10 Yamaguchi, Nogi JAPAN Aomori 030-0142 Tel: 81-0298-38-7449 JAPAN Fax: 81-0298-38-7408 Tel: 81-177-28-1015 [email protected] Fax: 81-177-28-1017 [email protected] Dr N. Quat NG International Institute of Tropical Dr Akinori NOGUCHI Agriculture (IITA), Nigeria Japan International Research Center for c/o Lambourn & Co. Agricultural Sciences (JIRCAS) Carolyn House 1-2 Ohwashi, Tsukuba 26 Dingwall Rd Ibaraki 305-8686 Croydon CR9 3EE JAPAN ENGLAND Tel: 81-0298-38-6307 Tel: 234-2-241-2626 Fax: 81-0298-38-6652 Fax: 234-2-241-2221 [email protected] [email protected] 490 Cryopreservation of Tropical Plant Germplasm

Prof. M.N. NORMAH Prof. Young-Goo PARK Department of Botany Kyungpook National University Faculty of Life Sciences Department of Forestry University Kebangsaan Malaysia Taegu 702-701 Bangi 43600 REPUBLIC of KOREA MALAYSIA Tel: 82-53-950-5747 Tel: 603-8292867 Fax: 82-53-950-6708 Fax: 603-8293244 [email protected] [email protected] Ms Gabriela B. QUIRAGA Ms Rieko OGURA Faculty of Agricultural Sciences Laboratory of Horticultural Plant Cordoba National University Breeding Av. Valparaiso s/n cc 509-5000 Faculty of Agriculture, Meiji University Cordoba 1-1-1, Higashi-mita, Tama-ku ARGENTIN E Kawasaki, Kanagawa 214-8571 JAPAN Mr Punathil E. RAJASEKARAN Tel: 81-044-934-7096 Indian Institute of Horticultural Fax: 81-044-934-7092 Research [email protected] Hessaraghatta Lake (PO) Bangalore 590089 Dr Seibi OKA INDIA National Institute of Agrobiological Tel: 91-80-8496634 Resources Fax: 91-80-5282619 2-1-2 Kannondai, Tsukuba [email protected] Ibaraki 305-8602 JAPAN Dr Barbara REED Tel: 81-0298-38-8388 US Dept. of Agriculture Fax: 81-0298-38-8397 National Clonal Germplasm Repository [email protected] 33447 Pegria Rd Corvallis OR 97333-2521 Dr Kiyoharu OONO USA National Institute of Agrobiological Tel: 1-541-750-8712 ext.111 Resources Fax: 1-541-750-8717 2-1-2 Kannondai, Tsukuba [email protected] Ibaraki 305-8602 JAPAN Dr Poula J. REINHOUD Tel: 81-0298-38-7461 Institute of Molecular Plant Sciences Fax: 81-0298-38-7408 PO Box 9502 2300 [email protected] RA Leiden THE NETHERLANDS Dr Bart PANIS Tel: 31-71-5274752 Catholic University of Leuven Fax: 31-71-5274469 Kard. Mercierlaan 92 [email protected] 3001 Heverlee BELGIUM Tel: 016-32-14-81 Fax: 016-32-19-93 [email protected] Participants 491

Dr William ROCA Dr Kei SHIMONISHI Centro Internacional de Agricultura Kagoshima Biotechnology Institute Tropical (CIAT) 4938 Hosoyamada, Kushira Km 17 via Cali-Palmira Kagoshima 893-1601 A.A. 6713 JAPAN COLOMBIA Tel: 81-0994-62-4112 Tel: 57-2-4450055 Fax: 81-0994-62-4114 Fax: 57-2-4450073 [email protected] [email protected] Dr Enny SUDARMONOWATI Mr Mustapha B. A. ROUAISSI Researcher of R&D Laboratory of Genetics Centre for Biotechnology-LIPI Agronomic National Institute of Tunisia Jl.Raya Bogor KM.46 Cité Maharajene Cibinong 16911 43 Avenue Charles Nicolle INDONESIA 1082 Tunis Tel: 62-21-875-4625 TUNISIA Fax: 62-21-875-4588 [email protected] Prof. Akira SAKAI Asabucho 1-5-23 Mr SYAFARUDDIN Kitaku Research Institute for Spice and Sapporo 001-0045 Medicinal Crops JAPAN JL. Tentara Pelajar No.3 Tel: 81-011-716-7711 Bogor Fax: 81-011-716-7711 INDONESIA [email protected] Dr Hiroko TAKAGI Dr Satoshi SAKAMOTO Japan International Research Center for JICA EXPERT Agricultural Sciences (JIRCAS) JI. Perkutut No.13 1-2 Ohwashi, Tsukuba Tanah Sareal Ibaraki 305-8686 Bogor 16161 JAPAN INDONESIA Tel: 81-0298-38-6384 Tel: 62-251-327615 Fax: 81-0298-38-6342 Fax: 62-251-327615 [email protected] [email protected] Prof. Isao TARUMOTO Dr Koichi SHIMOMURA College of Agriculture Tsukuba Medical Plant Research Station Osaka Prefecture University NIHS 1-1 Gakuen-cho, Sakai 1 Hachimandai, Tsukuba Osaka 599-8231 Ibaraki 305-0843 JAPAN JAPAN Tel: 81-0722-54-9404 Tel: 81-0298-38-0573 Fax: 81-0722-54-9404 Fax: 81-0298-38-0575 [email protected] 492 Cryopreservation of Tropical Plant Germplasm

Dr Mary TAYLOR Dr Kunio TSUBOTA Secretariat of the Pacific Community Japan International Research Center for Private Mail Bag Agricultural Sciences (JIRCAS) Suva 1-2 Ohwashi, Tsukuba FIJI Ibaraki 305-8686 Tel: 679-370733 JAPAN Fax: 679-370021 Tel: 81-0298-38-6304 [email protected] Fax: 81-0298-38-6342 [email protected] Mr Hiroyoshi TERUYA Okinawa Agricultural Experiment Mr Hikaru TSUKAZAKI Station National Research Institute of 76 Kanekadan Gushikawa Vegetable, Ornamental Plants and Tea Okinawa 904-2241 360 Knsawa, Ano-Town JAPAN Mie 514-2392 Tel: 81-098-973-5530 JAPAN Fax: 81-098-973-6333 Tel: 81-059-268-4654 [email protected] Dr Tien Nguyen THINH Nuclear Research Institute Mr Cuong Pham VAN 01 Nguyen Tuluc St. Department of Food Crops Dalat Faculty of Agronomy VIET NAM Hanoi Agricultural University Tel: 84-63-8-25107 Gialam Hanoi Fax: 84-63-8-21122 VIETNAM

Mr Tin TIN Dr Tuan Dhinh VUONA Germplasm Conservation Section Cuu Long Delta Rice Research Institute, c/o Seed Bank CARI Vietnam Yezin Pynmana Present address: MYANMAR Biological Res. Division JIRCAS Dr Darren H. TOUCHELL 1-2 Ohwashi, Tsukuba USDA-ARS Ibaraki 305-8686 National Seed Storage Laboratory JAPAN 1111 South Mason St [email protected] Ft Collins, Colorado 80521 USA Mr Reyes B. WALTEROSWALDO Tel: 1-970-495-3217 Experiment Station Fax: 1-970-221-1427 PICHILINGUE, INIAP [email protected] Km 5 Road Quevedo-EL Empalme EQUADOR Ms Kaori TOUNO University of Chiba Dr Masahiko WATANABE 1 Hachimandai Tsukuba NISES Ibaraki 305-0843 4-11-1-416-303, Matsushiro, Tsukuba JAPAN Ibaraki 305-0035 Tel: 81-0298-38-0573 JAPAN Fax: 81-0298-38-0575 Participants 493

Mr Yongjie WU Dr Kayo YOSHIMATSU Changli Institute of Pomology Tsukuba Medical Plant Research Station Hebei Academy of Agricultural and National Institute of Health Sciences Forestry Sciences 1 Hachimandai, Tsukuba Hebei 066600 Ibaraki 305-0843 Changli Town JAPAN CHINA Tel: 81-0298-38-0573 Fax: 335-20-23417, 23228 Fax: 81-0298-38-0575 [email protected] [email protected]

Mr Eiji YAMAGUCHI Ms Alfinetta B. ZAMORA National Center for Seeds and Seedlings University of the Philippines at Los Unzen Station Baños Saigo-bo 1494-34, Mizuho Los Baños Minami Takaki Laguna 4037 Nagasaki 859-1211 PHILIPPINES JAPAN Tel: 049-536-3528 Tel: 81-0957-77-2100 Fax: 049-536-3438 Fax: 81-0957-77-2154 [email protected] [email protected]

Dr Kazuko YAMAGUCHI-SHINOZAKI Japan International Research Center for Agricultural Sciences (JIRCAS) 1-2 Ohwashi, Tsukuba Ibaraki 305-8686 JAPAN Tel: 81-0298-38-6641 Fax: 81-0298-38-6643 [email protected] 494 Cryopreservation of Tropical Plant Germplasm

Author Index

Abdelnour-Esquivel, Ana ...... 325 Frattarelli, Andrea ...... 357, 434 Abe, Hiroshi ...... 67 Fregene, M...... 273 Aberlenc-Bertossi, F...... 341 Fu, Jia-Rui...... 393 Aguilar, María Elena ...... 449 Fujikawa, Seizo...... 36, 446 Anthony, F...... 161 Fukuda, Hiroo...... 352 Arata, Yoji...... 22 Ganeshan, S...... 360 Ashmore, Sarah ...... 453 Golmirzaie, Ali M...... 250, 388, 460 Barney, V...... 378 González-Arnao, María Teresa ...... Benson, Erica E...... 43, 385, 470 ...... 110, 217, 390 Berjak, Patricia ...... 140, 315, 371 Guevara, C.L...... 378 Borroto-Nordelo, C...... 110 Hamon, S...... 161 Brennan, R.M...... 470 Harada, T...... 446 Brulard, Eric ...... 344, 348 Higo, Kenichi ...... 463 Caicedo, L.E...... 378 Hirai, Dai...... 205 Chabrillange, Nathalie .....161, 172, Hirata, Kazumasa ...... 444 ...... 341, 414 Huang, Xue-Lin...... 393 Chang, Yongjian ...... 246, 382, 385 Ide, Hiroyuki ...... 22 Charoensub, R...... 401 Inagaki, Masanori...... 130 Chaudhury, Rekha ...... 457 Ishii, Katsuaki...... 421 Cheng, Yun-Feng...... 393 Ishikawa, Keiko...... 355 Côte, François ...... 449 Ishikawa, Masaya...... 22, 167, 338, Cyr, David R...... 261 ...... 352, 366, 368, 396, 429, 463 Damasco, Olivia P...... 299 Ito, Miwako ...... 338 Damiano, Carmine ...... 357, 434 Ito-Ogawa, Rie...... 396 Daniel, I.O...... 136 Jian, Ling-Cheng ...... 310 Daws, Matthew ...... 371 Jitsuyama, Yutaka ...... 36, 446 Debouck, D...... 222, 273 Kars, Ilona ...... 57 dela Cruz, Felipe...... 299 Karube, Minoru ...... 368 Delgado, Cecilia...... 388 Kasuga, Mie...... 67 DeNoma, Jeanine...... 246 Katano, Manabu...... 399 Diaz, Sandra ...... 460 Kato, Satoshi...... 338 Drew, Roderick...... 453 Kijne, Jan W...... 57, 91 Ducos, Jean Paul...... 348 Kioko, Joseph ...... 371 Dumet, Dominique...... 43, 172, 385 Kirdmanee, Chalermpol...... 297 Dussert, S...... 161, 172 Kitashima, Tomomi...... 22 Duval, Y...... 172, 341 Komanine, Atsushi...... 352 Engelmann, Florent ...... 8, 110, 161, Kunihiro, Yasufumi...... 468 ...... 172, 217, 341, 357, 390, Kyesmu, P.M...... 320, 411 ...... 414, 434, 449, 473 Li, Xiao-Ju...... 393 Escobar, Roosevelt H...... 222, 273, Liu, Qiang...... 67 ...... 404, 408 Lu, Tie-Gang...... 310 Florin, Bruno ...... 341, 348 Mafla, Graciela ...... 273, 404 Author index 495

Mainah, G...... 156 Reed, Barbara M...... 200, 246, 382, Malaurie, Bernard ...... 414 ...... 385, 470 Mandal, B.B...... 233, 282 Reinhoud, Poula J...... 57, 91 Márquez Ravelo, Manfred ...... 390 Remy, Serge...... 103 Martínez Montero, Marcos E...... Rios, Alba ...... 217 ...... 110, 390 Roca, William M. 222, 273, 404, 408 Maruyama, Emilio...... 421 Rosmithayani ...... 437 Marzalina, M...... 465 Sági, László...... 103 Masuya, Yoshihiro...... 399 Sakai, Akira...... 1, 205, 227, 401, Matsumoto, Tatsuo...... 338 ...... 424, 426, 437 Matsumoto, Toshikazu...... 212, 424 Sakuma, Yoh ...... 67 Mii, M...... 355 Saraswathy, R...... 156 Minami, Tomohiro ...... 426 Saunders, Rodney ...... 453 Miura, Setsuko...... 67 Schoofs, Hilde...... 103, 238 Miyamoto, Kazuhisa ...... 444 Seguel, Ivette ...... 194 Miyashita, Susumu...... 468 Shatnawi, Mohamad A...... 434 Morenza, Marlene ...... 217 Shimomura, Koichiro...... 77 Mulyaningsih, E.S...... 437 Shimonishi, Kei ...... 167, 368 Murayama, Toru ...... 194 Shinozaki, Kazuo ...... 67 Murikami, Takako...... 330 Shirata, Kazuto...... 468 Mycock, D.J...... 140 Sudarmonowati, Enny ...... 291, 437 Nagamine, Tsukasa ...... 468 Supaibulwatana, Kanyarat...... 297 Nashatul, Z.N.A...... 465 Sun, Ching-San...... 310 Ng, Shou Yong Choy...... 418 Suzuki, Masahiko...... 429 Ng, Nyat Quat ...... 136, 418 Suzuki, Seiichi...... 167 Niino, Takao...... 194 Suzuki, T...... 446 Niwata, Eiko ...... 396, 429 Swennen, Rony...... 103, 238, 255 Noirot, M...... 341 Takagi, Hiroko ... 178, 227, 411, 441 Normah, M.N...... 156, 287, 431 Tan, B.S...... 431 Oosawa, Katsuji...... 167, 396 Tandon, Pramod ...... 352 Ospina, J.A...... 378 Taylor, Mary...... 330 Paet, Cynthia N...... 299 Tessereau, Hervé...... 348 Palacio, Juan D...... 408 Thinh, Nguyen Tien...... 227, 238, Pammenter, N.W...... 140 ...... 306, 441 Panis, Bart ...... 103, 238, 255 Touchell, Darren H...... 269 Panta, Ana ...... 250, 388, 460 Touno, Kaori ...... 77 Pétiard, Vincent...... 344, 348 Towill, Leigh E...... 115 Phansiri, S...... 401 Trouslot, Marie-France...... 414 Priadi, D...... 437 Tsukazaki, H...... 355 Price, William S...... 22 Urra Villavicencio, Caridad...... Pritchard, Hugh...... 371 ...... 217, 390 Puentes-Díaz, C...... 110 Van den houwe, Ines...... 255 Rajashekaran, R.K...... 360 Van Iren, Frank ...... 57, 91 Rangel, Maria P...... 408 Vásquez, Nelly ...... 449 496 Cryopreservation of Tropical Plant Germplasm

Versteege, Isabella ...... 57 Walker, Marieanne ...... 140 Walters, Christina...... 115 Watt, Paula ...... 140 Wesley-Smith,, J...... 140 Wu, Yongjie ...... 357, 473 Xiao, Jie-Ning ...... 393 Yah, Emelda ...... 449 Yamaguchi, Eiji...... 366 Yamaguchi–Shinozaki, Kazuko .67 Yokoyama, Tadashi...... 463 Yongmanitchai, W...... 401 Yoshimatsu, Kayo...... 77 Zamora, Alfinetta B...... 299 Zhao, Yanhua ...... 473