This file was created by scanning the printed publication. Errors identified by the software have been corrected; however, some errors may remain.

Chapter2 Somatic En1bryogenesis From Poplar Leaf Tissue1

Young Goo Park and Sung Ho Son

for application to mass cloning. Techniques developed for poplar may also be applied to related tree species such as Introduction willow (Salix spp.).

An efficient in vitro regeneration system with cell and tissue cultures is a prerequisite for biotechnological ap­ plications to plant improvement programs. Although Literature Review whole plants have been regenerated by several techniques from various species and explants, somatic embryogen­ esis is one of the most powerful morphogenetic schemes. Studies on poplar tissue culture began in the early 1930s First reported using carrot (Steward et al. 1958}, somatic (Gautheret 1934). After 30 years, Mathes (1964}, Wmton embryogenesis was later defined by Haccius (1978) as a (1968}, and Wolter (1968) have established plant regen­ nonsexual developmental process leading to the differ­ eration methods based on callus culture. In their systems, entiation of zygotic embryo-like structures from somatic shoots and roots were induced through organogenesis cells. Two advantages of somatic embryogenesis are that: from different callus sites at different developmental 1) genetically identical propagules are provided by avoid­ stages. Since then, similar morphogenetic responses were ing genetic recombination that occurs during meiosis in reported by other authors (Chen and Huang 1980; Sellmer sexual reproduction; and 2) it provides a fast, reliable, re­ et al.1989; Whitehead and Giles 1977). Because of the po­ producible method for mass production. In contrast to or­ tential applications associated with somatic embryogen­ ganogenesis, somatic embryogenesis exhibits a bipolar esis, many studies have focused on crop, horticultural structure with a closed vascular system and differentiated species, and woody perennials (Gingas and Lineberger shoot and root meristems. 1989; Tremblay 1989). Poplars, considered an economically important species Pioneering studies on somatic embryogenesis in pop­ in many countries, are usually propagated by cuttings lar were reported by Michler and Bauer (1987, 1991), Park and/ or root suckers. Thus, cloning techniques can propa­ and Son (1988}, Cheema (1989). Somatic embryogenesis

gate superior genotypes that show heterosis in the F1 prog­ at relatively high frequency was obtained directly and/ eny from artificial hybridization. For interspecific crosses, or indirectly by leaf-tissue and cell culture of hybrid as­ some species from the Leuce (currently termed Populus) pen (Populus alba x P. grandidentata cl. 'NC5339'). With this section have superior traits such as a straight trunk and hybrid, short-term auxin treatment stimulated embryo rapid growth Giang et al. 1980). Nevertheless, most hy­ maturation and eventually allowed plants to harden un­ brids from this section have been difficult to propagate der greenhouse conditions (Michler and Bauer 1991). asexually using standard techniques. Tissue culture sys­ Cheema (1989) obtained callus and cell suspension cul­ tems, especially somatic embryogenesis are advantageous tures using semi-organized leaf culture from mature Hi­ malayan poplars (P. ciliata). Similar to most other species, somatic embryogenesis was induced in P. ciliata by expo­ sure to a high level of 2,4-dichlorophenoxyacetic acid (2,4- 0), followed by reduced auxin concentration. Callus 1 Klopfenstein, N.B.; Chun, Y. W.; Kim, M.-S.; Ahuja, M.A., eds. retained its embryonic potential over a year; however, em­ Dillon, M.C.; Carman, R.C.; Eskew, L.G., tech. eds. 1997. bryogenic suspension cultures lost this ability after 6 sub­ Micropropagation, genetic engineering, and molecular biology cultures. In another study, organogenesis and somatic of Populus. Gen. Tech. Rep. RM-GTR-297. Fort Collins, CO: U.S. embryogenesis occurred simultaneously when punctured Department of Agriculture, Forest Service, Rocky Mountain Re­ leaves of hybrid poplar (Populus nigra x P. maximowicziz) search Station. 326 p. were cultured (Park and Son 1988).

10 Somatic Embryogenesis From Poplar Leaf Tissue

With Populus, embryogenic capacity has been associ­ malt extract, and glutamine may be used to stimulate so­ ated with leaf-tissue explants. For most other species in­ matic embryogenesis. In this method, direct and indirect cluding woody plants, immature or mature zygotic embryogenesis and organogenesis can occur simulta­ embryos are commonly used as explants to initiate so­ neously. To maintain somatic embryos or embryogenic matic embryogenesis (Tremblay 1989). For many woody callus for further development, embryoids and I or em­ plant species, immature embryos are available only once bryogenic callus must be selected at an early stage. a year for a very short time. Using leaf tissue, especially that grown in vitro, for somatic embryogenesis provides Populus species with a substantial advantage. Indirect Embryogenesis Isolated embryogenic callus or embryogenic callus-de­ rived cell suspension cultures can be used for indirect so­ matic embryogenesis. A simple, reliable method for indirect somatic embryogenesis is obtained by altering the 2,4-D Poplar Somatic Embryogenesis level. A semi-solid agar medium can support further de­ velopment of embryos harvested at an early developmen­ In poplar, the developmental process, morphology, and tal stage. Overall, the culture media for maintenance of physiology of somatic embryogenesis is similar to zygotic embryogenic cell lines are similar to induction media ex­ embryogenesis. Because reports on somatic embryogen­ cept that plant growth regulator levels are lower. esis of poplar are limited, summarizing a general method is difficult. However, based on our research and other re­ Maturation and Germination ports, some critical steps for somatic embryogenesis in Populus spp. are described. The steps in somatic embryo­ Harvested embryos require several washes in MS liq­ genesis from leaf explants of poplar are: 1) explant prepa­ uid medium without plant growth regulators. Somatic ration and adjustment to culture conditions; 2) induction embryos originating directly or indirectly from poplar leaf of direct and/ or indirect embryogenesis; and 3) matura­ cultures typically do not exhibit dormancy. Maturation of tion and germination. somatic embryos in poplar can be triggered by the chang­ ing plant growth regulators. In 2 cases, the maturation medium included BA, alone or combined with a­ Explant Preparation and Culture Conditions naphthaleneacetic acid. Increased levels of osmoticum As donor plants, greenhouse- or field-grown plants can may effectively stimulate maturation. provide explants to initiate in vitro plantlets or callus. Typi­ cally, stem-node sections with or without buds are disinfested using 0.5 to 1 percent of sodium hypochlorite for 5 to 10 min. Murashigeand Skoog (1962) (MS)culture medium with a low concentration of cytokinin (6-benzyladenine; BA), Limitations alone and in combination with high levels of auxin (2,4-D), is used to induce in vitro bud break and callus induction, Although somatic embryogenesis was successfully respectively. To multiply the shoots for source material, api­ demonstrated in Populus species, the induced somatic ces are excised from shoots before subculturing on prolif­ embryo is strikingly plastic in its development. Many of eration medium containing 0.88 J.lM BA. A 16-h photoperiod the induced embryos exhibited abnormal growth charac­ provided by cool-white, fluorescent light is used for shoot teristics including: 1) bearing multiple secondary embry­ elongation and direct embryogenesis. Complete darkness is oids among the embryonic axes; 2) rapid growth of used to establish and maintain callus and embryogenic cell nonembryonic callus during embryogenesis; 3) abnormal suspension cultures. Suspension cultures are routinely agi­ cotyledons growth; and 4) growth failure of either cotyle­ tated at 100 to 120 rpm using a gyratory shaker, and subcul­ don or root radical. This plasticity may result from the tured at 2-week intervals. altered gene expression during development. Another problem associated with poplar embryogenesis is its low Direct Embryogenesis success rate and poor reproducibility in comparison with carrot. Because all stages from induction to maturation Isolated leaves (< 2 em in diameter) from in vitro cul­ are very labor intensive, large-scale propagation through tured plantlets are cultured on MS medium supplemented somatic embryogenesis is impractical without improving with BA and 2,4-D. Leaves are further prepared by punc­ overall efficiency. A critical limitation is lack of knowl­ turing with a pin or slicing into leaf disks with a cork borer edge on the somatic embryogenic processes of poplar. (1 em in diameter). Amendments such as coconut milk, Continued intensive studies are needed.

USDA Forest Service Gen. Tech. Rep. RM-GTR-297. 1997. 11 Section I In Vitro Culture

Conclusion and Prospects Literature Cited

Cheema, G.S. 1989. Somatic embryogenesis and plant re­ Somatic embryogenesis can be induced from leaf ex­ generation from cell suspension and tissue culture of plants of poplar. Many factors such as basal media com­ mature Himalayan poplar (Populus ciliata). Plant Cell position, undefined substances, amino acids, plant growth Rep. 8: 124-127. regulator dosage, and treatment methods influence so­ Chen, D.; Huang, M. 1980. Culture of the apical tissue of matic embryogenesis. Among these factors, 2,4-0 plays a Populus nigra cv. Blanc de Garonne and the variation of paramount role as an auxin source. However, a phytohor­ their isozymes. J. Nanjing Tech. Coli. For. Products. 3: mone imbalance usually causes the formation of abnor­ 104-107. mal or incomplete structures. Because the role of auxins Gautheret, R.J. 1934. Culture du tissu cambial. CR Acad. in embryoid formation is complex, purification and iden­ Sci. (Paris). 198: 2195-2196. tifi~ation of various auxin receptors and understanding Ging~s, V.M.; Lineberger, R.D. 1989. Asexual embryogen­ the1r molecular action will contribute to our knowledge esis and plant regeneration in Quercus. Plant Cell Tiss. of cellular differentiation mechanisms related to somatic Org. Cult. 17: 191-203. embryogenesis (LoSchiavo 1995). Using leaf explants of Haccius, B. 1978. Question of unicellular origin on non­ poplar, the somatic embryogenic response was examined zygotic embryos in callus cultures. Phytomorphology. using inverted stereo microscopy (Michler and Bauer 1991; 28: 74-81. Park and Son 1988). When embryos were produced from Jiang, H.; Han, Y.; Zhao, H.; Gu, W.; Yang, C.; Fan, K.; Xing, cultured leaves, they appeared to arise directly or via X. 1980. Breeding of the superior hybrid clone 741 of callogenesis from wounded portions or from sliced leaf poplar. Hebei Linye Keji. 1: 72-75. ends. LoSchiavo, F. 1995. Early events in embryogenesis. In: In recent years, embryogenesis through cell suspension Bajaj, Y.P.S., ed. Biotechnology in agriculture and for­ culture has provided opportunities for gene transforma­ estry 30. Berlin: Springer-Verlag Publication: 20-29. tion by microparticle bombardment of intact cells or di­ Mathes, M.C. 1964. The culture of isolated triploid aspen rect gene transfer using protoplasts. Liquid plating of tissue. For. Sci. 10: 35-38. protoplast allows stringent selection of transformed cells. Mi~hler, C.H.; Bauer, E.O. 1987. Somatic embryogenesis Cell suspension culture-based somatic embryogenesis is m plant cell culture of Populus. In vitro. Cell Oev. Bioi. also applicable to germplasm preservation and mutant se­ 23 {3), Partll: 46A. lection. If successfully developed, these technologies can Michler, C.H.; Bauer, E.O. 1991. High frequency somatic be employed in bioreactor systems for mass production. e~bryogenesis from leaf tissue of Populus spp. Plant Atte~pts at. large-scale embryogenesis are being applied Set. 77: 111-118. to vanous btoreactor systems such as air-lift, impeller, and Murashige, T.; Skoog, F. 1962. A revised medium for rapid hybrid types. growth and bio assays with tobacco tissue cultures. Induction of somatic embryogenesis and organogenesis Physiol. Plant. 15: 473-479. have usually occurred simultaneously, but embryoid for­ Park, Y.G.; Son, S.H. 1988. In vitro organogenesis and somatic mation efficiency was relatively low. Abnormalities in embryogenesis from punctured leaf of Populus nigra x P. m?rphology and growth were evident. It is our hope that maximowiczii. Plant Cell Tiss. Org. Cult. 15: 95-105. th1s s~mmary of somatic embryogenesis methods for pop­ Sellmer, J.C.; McCown, B.H.; Haissig, B.E. 1989. Shoot culture lar wtll be useful to future studies. Continued studies are dynamics of six clones. Tree Physiol. 5: 219-227. needed to improve culture systems and for further under­ Populus Steward, F.C.; Mapes, M.O.; Mears, K. 1958. Growth and standing of somatic embryogenesis for application to pop­ organized development of cultured cells II. Organiza­ lar and other woody plants. tion in cultures grown from freely suspended cells. Amer. J. Bot. 45: 705-708. Tremblay, F.~. 1989. Somatic embryogenesis and plantlet regeneration from embryos isolated from stored seeds of Picea glauca. Can. J. Bot. 68: 236-242. Acknowledgments Whitehead, H.C.M.; Giles, K.L. 1977. Rapid propagation of pop­ lar by tissue culture methods. N. Z. J. For. Sci. 7: 40-43. Winton, L.L. 1968. Plantlet formation from aspen tissue Part of this manuscript was reported in: Bajaj, Y.P.S., 1995. culture. Science. 160: 1234-1235. ed ..Biotechnology in agriculture and forestry 30. Berlin: Wolter, K.E. 1968. Root and shoot initiation in aspen cal­ Spnnger-Verlag Publication: 446-454. lus culture. Nature. 219: 503-510.

12 USDA Forest Service Gen. Tech. Rep. RM-GTR-297. 1997.