First Report of Regeneration via Somatic Embryogenesis from Shoot Apex-Derived Callus of muluense R.M. Smith Hamidou F. Sakhanokho Rowena Y. Kelley Kanniah Rajasekaran

ABSTRACT. The Hedychium consists of about 50 species, with increasing popularity as ornamentals and potential as medicinal crop , but there are no reports on somatic embryogenic regeneration of any member of this genus. The objective of this investigation was to establish an in vitro regeneration system based on somatic embryogenesis for Hedychium muluense R.M. Smith using shoot apex-derived callus. Callus was induced and proliferated on a modified Murashige and Skoog (MS) medium (CIPM) supplemented with 9.05 j.tM 2-4, D, and 4.6 p.M kinetin.

Hamidou F. Sakhanokho is affiliated with the USDA-ARS, Thad Cchran Southern Horticultural Laboratory, P.O. Box 287, 810 Hwy 26 West, Poplarville, MS39470.r . • Rowena Y. Kelley is affiliated with the USDA-ARS-C.HPRRU,81O Hwy12 E, Mississippi State, MS 39762.. . . . Kanniah Rajasekaran is affiliated with the USDAARS-SRRC, 110) Robert E. Lee Bld.Nev Orleans, LA70124. The authors thank Mr. Kermis Myrick, Ms. Lindsey Tanguis,and Ms. Alexandria Goins for technical assistance. ri Mention of trade names of commercial products in the publication is solely for the purpose of providing specific information and does not imply recommenda- tion or endorsement by the U.S. Department of Agriculture. - Address correspondence to: Hamidou F. Sakhanokho at the abo"e address (E-mail: Journal of Cop Improvement, Vol. 21(2) (#42) 2008 - Available online at http://jcrip.hworthpreSs.corn • © 2008 by The Haworth Press, Inc. All rights reserved. ., -doi:10.1080/1542752070188575 8 r . • 191 192 JOURNAL OF CROP IMPROVEMENT

Friable callus developed into somatic embryos upon transfer to liquid medium (MS basal salts and Gamborgs vitamins) that was supplemented with 0.6 !IM thidiazuron (TDZ) and 8.9 j.tM ô-benzylaminopurine (BA) and shaken for four weeks. The cultures were then transferred to three Hedych- ium embryo-development media (HEDM) of varying strengths: HEDM, 1/2 HEDM, and 1/4 HEDM. All three media contained both 0.6 tM TDZ and 8.9 M BA. Somatic embryo production was higher in full strength HEDM, which produced an average of 103 somatic embryos/explant, half of which could be converted into shoots within a month. Regenerated shoots were readily rooted on a medium supplemented with 0.6 tM 3- indoleacetic acid (IAA) and acclimatized before transfer to the greenhouse.

KEYWORDS. Ornamental ginger, somatic embryogenesis, Hedychium muluense, Borneo ginger

INTRODUCTION

Ornamental gingers are perennial monocotyledonous plants belonging to the family. They are tropical to semitropical plants compris- ing about 2,000 species and are increasingly gaining popularity because of their potential for use as flowering pot plants, both indoors and as patio and landscape plants (Kuehny et al., 2002). The genus Hedychium consists of about 50 species and is one of the most popular genera of Zingiberaceae because of its attractive foliage, diverse and showy flowers, and sweet fra- grance. With the exception of H. peregrinum, which is endemic to Madagas- car, all Hedychium species are native to central and southeastern Asia, with concentrations in southern and the Himalayan regions (Branney, 2005). Hedychiuni species are widely cultivated for their perfume essences. The scents range from the rich gardenia-like fragrance of H. coronarium to scents reminiscent of citrus, clove, and coconut (Wood, 1999). The starch of Hedychium is similar to that of arrowroot, and the aerial stems con- stitute a useful raw material for manufacturing paper (Mukherjee, 1970). In addition, Hedychium is used in ethnomedicine. For example, the essential oils in the leaves and flowers of H. gardnerianum and of H. larsenii and H. spicatum have been found to possess antimicrobial activity against both Gram-positive and Gram-negative bacteria (Madeiros et al., 2003; Gopanraj et al., 2005; Bisht et al., 2006). H. muluense or "Borneo ginger" is an epiphytic tropical plant that bears small but pretty flowers with green petals, an orange stamen, and a Sakhanokho, Kelley, and Rajasekaran 193 twisted white labellum and lateral staminodes (Branney, 2005). The main limitation to using most Hedychium as potted plants is that they grow too tall, easily reaching two meters. H. muluense is one the few dwarf Hedychium species. Therefore, it has the potential to play an important role in the development of dwarf Hedychium cultivars using either conventional or molecular breeding. In general, ornamental biotechnology has lagged behind that for agronomic crops because of limited resources generally devoted to ornamental breeding. In the last decade or so, however, some transgenic ornamental cultivars have been developed (Firoozabady et al., 1995; Marchant et al., 1998; Rosati et al., 2003; Hoshi et al., 2004). Genetic transformation requires a dependable regeneration system, prefer- ably based on somatic embryogenesis. Despite their ornamental, medici- nal, and industrial potential, very little molecular work has been done on the Hedychium species. The regeneration system available is generally based on rhizome meristem tissue culture (Tripathi and Bitaillon, 1985; Koul et al., 2005). To our knowledge, no report on regeneration based on somatic embryogenesis is available for any Hedychium species. In recalcitrant crops like monocotyledonous species, efficient and repro- ducible regeneration protocols have been developed using mostly immature cells and tissues such as immature embryos. However, the use of mature embryos, such as dry seeds, has remarkable advantages over immature tissues as explants because this eliminates the need for growing donor mate- rial in greenhouses under controlled environmental conditions, requiring intensive labor, time, and space. Additionally, dry seeds are available in large quantity year round with no problem due to seasonal influence on tis- sue culture response (Dahleen, 1999; Sharma et al., 2005). The purpose of the present study was to establish an in vitro regeneration scheme based on somatic embryogenesis for H. muluense using shoot apex-derived callus.

MATERIAL AND METHODS

Seed Sterilization and In Vitro Seed Regeneration H. muluense seeds were collected from greenhouse-grown plants, and a preliminary in vitro seed germination test showed that it took between 30 to 40 days for the seeds to germinate in a sporadic manner. To achieve a faster and more uniform germination rate, seeds were scarified in 98% sulfuric acid (H2SO4) for 30 minutes. Following the H2SO4 treatment, the seeds were rinsed promptly and thoroughly for about 5 min with running 194 JOURNAL OF CROP IMPROVEMENT

tap water to remove acid residue. Seeds were then surface sterilized under a laminar flow hood by dipping them in 100% ethanol for 5 min with gentle shaking. Thereafter, they were transferred to sterilized beakers containing 40% (v/v) sodium hypochiorite solution and one drop of TweenlM 20 and shaken for 20 min at 110 rpm. Seeds were then rinsed three times with sterile distilled water and soaked overnight in distilled sterile water on a shaker at 110 rpm and at room temperature (22°C). The following day, the seeds were rinsed again three to four times with distilled sterile water and transferred to 100 mm x 15 mm Petri dishes (5 seeds per dish maximum) containing MS (Murashige and Skoog, 1962) basal medium with 20 g sucrose, 0.75 g I_I MgCl2, and 2 g F Gelrite. The Petri dishes were then placed in an incubator where the temperature was maintained constant at 28°C, with a photoperiod regime of 16-h light (100 pmol m 2s 1 ) and 8-h dark. Following the H2SO4 scarification, in vitro seed germination rate improved to about 60% within two weeks. Callus Initiation and Proliferation

Meristematic shoot segments were removed from 3- to 5-d old in vitro-grown seedlings as described by Sharma et al. (2004), cut into 3-4 mm segments and transferred to a callus-initiation and proliferation medium consisting of MS salts, 9.05 tM 2-4,13, 4.6 p.M kinetin, I g F glutamine, 0.5 g F asparagine, 0.1 g F myo-inositol, 20 g 1 sucrose, 0.75 g F MgCl2, and 2 g 1 Gelrite. The shoot apex explants were placed into 100 mm x 20 mm Petri dishes containing the callus induction an proliferation medium and then placed in an incubator maintained at 22°C with a photoperiod regime of 16-h light (100 p.mol m 2s) and 8-h dark. Each explant from each individual seed- ling was cultured separately. After four weeks, induced callus in each Petri dish was transferred to the same but freshly prepared callus-proliferation medium. Afterwards, this step was repeated four to five times every four weeks in select- ing and transferring each time friable callus to freshly prepared medium. In Vitro Liquid Culture

After five to six months on the callus-proliferation medium, no embryos were formed, so the friable callus cultures were transferred to 125 ml flasks containing a liquid medium, which allows maximum medium-to-tissue contact. This liquid medium of MS basal medium containing macro- and micronutrients (Murashige and Skoog, 1962), vitamins (Gamborg et al., 1968), 0.6 p.M thidiazuron (1DZ), 8.9 p.M ô-benzylaminopurmne (BA), 20 g F sucrose, 0.2 g F myo-inositol, I g F casein hydrolysate, and I mg F Sakhanokho, Kelley, and Rajasekaran 195 thiamine. The cultures were placed into a shaker kept in a growth room, where the temperature was kept at 22°C, and the cultures were shaken at 110 rpm. The photopenod was 16-h light (100 imol m 2s) and 8-h dark. Somatic Embryo Development After four to five weeks in suspension culture, the calli were transferred to 100 x 20 mm Petri dishes containing somatic embryo-development media. The somatic embryo-development media were referred to as Hedychium embryo-development medium or HEDM. The HEDM medium is similar to the liquid medium described above, except that it also contains 0.75 g I_ I MgCl2 and 2 g r Gelrite. The two other media were derived from HEDM and referred to as 1/2 HEDM andl/4 HEDM, containing 1/2 and 1/4 HEDM strengths, respectively. Calli from individual explants were transferred separately to Petri dishes. There were 12 explants for each one of the three media: HEDM, 1/2 HEDM, andl/4 HEDM, and five plates per explant. The cultures were placed in an incubator kept at 22°C; the photoperiod regime was 16-h light (100 .tmol m 2 s 1 ) and 8-h dark. Four weeks after transfer, the number of somatic embryos that developed from the transferred callus from individual explants in each plate was recorded. The experiment was replicated three times, so there was a total of 36 Petri dishes for each medium treatment. Mean separation (P = 0.05) was based on Tukeys HSD test and standard errors for each treatment were calculated using SAS (SAS Institute, 2003).

Plant Regeneration and Acclimatization Four weeks after transfer to the three different embryo-development media, the number of mature somatic embryos that developed into plants in each plate was recorded. Plantlets that started to outgrow the Petri dishes were transferred to 75 ml (98.5 mm x 59 mm) culture vessels con- taining MS basal medium (Murashige and Skoog, 1962) and vitamins (Gamborg et al., 1968) supplemented with 0.6 tM IAA, 15 g l sucrose and 0.75 g l MgCl 2. The plants were kept in a growth room where the temperature was maintained at 22°C with a fluorescent light regime of 16-h light (100 imol m-2 S-1 )and 8-h dark. Most of the plants transferred to the vessels produced clumps of rooted multiple shoots. The clumps were divided into separate rooted plants, gently washed with tap water to remove any callus or gelling agent residues, transferred to premoistened Jiffy-7 peat pellets (Jiffy Products, Ltd., Canada) and covered with clear plastic bags. After three days, holes were punched in the plastic bags to 196 JOURNAL OF CROP IMPROVEMENT

allow for gradual acclimatization of the plants before transferring them to soil in the greenhouse after four to six weeks. The pH of the various media utilized was adjusted to 5.8 before autoclaving for 15 mm at 121 C.

RESULTS AND DISCUSSION

All the meristematic shoot segments cultured on CIPM (9.05 pM 2,4-D, 4.6 IIM kinetin) successfully produced callus. However, no somatic embryos were produced on this 2,4-D- and kinetin-containing medium even after 5 to 6 subcultures for a period of about 5-6 months, even though the growth reg- ulator 2,4-D has been successfully used in tissue culture of several monocots (Viertel and Hess, 1996; Ahmad et al., 2002; Kelley et al., 2002). Therefore, we transferred the callus developed on the callus-induction and callus-prolif- eration medium to a liquid medium containing the growth regulators TDZ and BA. Somatic embryos developed in about one month after the transfer from the liquid culture to HEDM, 1/2 HEDM, and 1/4 1-IEDM media (Figure IA), all of which also contained BA and TDZ. This suggested that either BA or TDZ or the combination of both growth regulators had a stimulatory effect

FIGURE 1. Somatic embryogenesis and plant regeneration in Hedychium muluense. (A): Somatic embryos developed after removal from the liquid culture and transfer to HEDM media; (B): Somatic embryos and regenerated plantlets, which were transferred into vessels (C) containing a medium for rooting and further development; (D) Clumps of multiple shoots were divided before transfer to Jiffy.

rtr , NQ II Sakhanokho, Kelley, and Rajasekaran 197

FIGURE 2. Average numbers of somatic embryos (SEs) (A) and plants (B) regenerated from H. muluense apex-derived callus culture on three embryo development media. Means with the same letter are not significantly different at the 0.05 rejection level according to Tukeys HSD test. Means are from three independent experiments with 12 explants for each determination.

120

100

80

60 0

40

20

0 HEDM ½ 1-IEDM / HELM Medium

120

100

180

60 0

J 40

20

0 HEDM ½ HEDM Vs HlDM Medium 198 JOURNAL OF CROP IMPROVEMENT

on somatic embryo initiation and/or development in H. muluense. Indeed, both BA and TDZ have been effective in tissue culti.re of wheat and barley using several types of explants (Shan et al., 2000; Ahmad et al., 2002; Gane- shan et al., 2003; Sharma et al., 2004, 2005). It is also possible that the liquid medium, which allows maximum medium-to-tissue contact, promoted initi- ation of somatic embryos. In all three media, the germinated somatic embryos generally produced clumps of multiple shoots (Figure 1 B). When these clumps were divided and transferred to vessels for rooting and further development, they produced even more clumps of multiple shoots (Figures IC and ID), which, in turn, could be divided into more plants before their transfer to the greenhouse. There were significant differences (P = 0.05) among the three media for both the number of somatic embryos and regenerated plants (Figures 2A and 2B). Both the number of somatic embryos and regenerated plants decreased as the strength of HEDM was reduced (Figures 2A and 2B). In all three media, within a month about 50% of the germinated embryos developed into plant- lets with or without roots (Figure 1 B) before transferring them to culture ves- sels containing a rooting medium (Figure IC). Overall, it took about 6-7 months frOm callus initiation to somatic embryo development. Most of this time, however, was spent in the callus initiation/proliferation medium before the transfer to the liquid medium. We were able to reduce the time taken to regenerate H. mulueñse by transferring the friable callus from the callus initi- ation/proliferation medium after 2 to 3 transfers instead of 5 to 6 transfers. To our knowledge, this is the first report of a successful regeneration via somatic embiyogenesis in Hedychium species:

REFERENCES

Ahmad, A., H. Zhong, W. Wang, and M.B. Sticklen. 2002. Shoot apical ineristem: regeneration and morphogenesis in wheat (Triticum aestivum L). In Vitro Cell. Dev. Biol.-Plant 38:163-167. Bisht, G.S., A.K. Awasthi, and T.N. Dhole. 2006. Antimicrobial activity of . Fitoterapia 77:240-242. Branney, T.M.E. 2005. Hardy gingers including Hedychiurn, , and Zingiber. Portland, OR: Timber Press Inc.. Dahleen, L.S. 1999. Donor-plant environment effects on regeneration from barley embryo-derived callus. Crop Sci. 39:682-685. Firoozabady, E., Y. Moy, W. Tucker, K. Robinson, and N. Gutterson 1995. Efficient transformation and regeneration of carnation cultivars using Agrobacteriuni. Molecular Breeding 1:283-293. Sakhanokho, Kelley, and Rajasekaran 199

Gamborg, O.L., R.A. Miller, and K. Ojima. 1968. Nutrient requirements of suspension cultures of soybean root cells. Exp. Cell Res. 50:151-158. Ganeshan, S., M. Baga, B.L. Harvey, B.G. Rossnagel, G.J. Scoles, and R.N. Chibbar. 2003. Production of multiple shoots from thidiazuron-treated mature embryos and leaf-base/apical meristem of barley (Hordeum vulgare L.). Plant Cell Tiss. Org. Cult.73:57-64. Gopanraj, G., M. Dan, S. Shihuraj, M.G. Sethuraman, and V. George. 2005. Chemical composition and antibacterial activity of the rhizome oil of Hedychium larsenii. Acta Pharm. 55:315-320. Hoshi, Y., M. Kondo, S. Mori, Y. Adachi, M. Nakano, and H. Kobayashi. 2004. Produc- tion of transgenic lily plants by Agrobacterium-mediated transformation. Plant Cell Rep. 22:359-364. Kelley, R.Y., M.S. Kang, A. Zipf, D. Wesenhurg, and G. Sharma. 2002. Genetic analysis of plant regenerahility in oats (Avena spp. L.). J. New Seeds 4:31-40. Koul, S. V. Raina, and S.K. Sharma. 2005. Conservation and propagation of high altitude medicinal and aromatic plant: Hedychium spicatum. J. Plant Biochem. & Biotechnol. 14:57-59. Kuehny, J.S., M.J. Sarmiento, and P.C. Branch. 2002. Cultural studies in ornamental ginger. In Trends in New Crops and New Uses, eds., J. Janick, and A. Whipkey. Alexandria, \ pp. 477-482, VA: ASHS Press. Màrchant, R., M.R. Davey, J.A. Lucas, C.J. Lamb, R.A. Dixon, and J.B. Power. 1998. Expression of a chitinase transgene in rose (Rosa hybrida L.) reduces development of blackspot disease (I)iplocarpon rosae Wolf). Molecular Breeding 4:187-194. Medeiros, JR., L.B. Campos, S.C. Mendonça, L.B. Davin, and N.G. Lewis. 2003. Com- position and antimicrobial activity of the essential oils from invasive species of the Azores, and Pinosporum undulatwn. Phytochemistry 64:561-565. Mukherjee, I. 1970. Chromosome studies of some species of Hedychium. Bot. Mag. Tokyo 83:237-241. Murashige, T., and F. Skoog. 1962. A revised medium for rapid growth and bioassays with tobacco tissue culture. Physiol. Plant 15:473-497. Rosati, C., P. Simoneau, D. Treutter, P. Poupard, Y. Cadot, A. Cadic, and M. Duron. 2003. Engineering of flower color in forsythia by expression of two independently-trans- formed dihydroflavonol 4-reductase and anthocyanidin synthase genes of flavonoid pathway. Molecular Breeding 12:197-208. SAS Institute. 2003. SAS 9.1.3. Cary, NC: SAS Institute, Inc. Shan, X., D. Li, and R. Qu. 2000. Thidiazuron promotes in vitro regeneration of wheat and barley. In Vitro Cell. Biol.-Plant 36:207-210. Sharma, V.K., R. Hansch, R.R. Mendel, and J. ,Schut.ze. 2004. A high efficient plant regeneration system through multiple shoot differentiation from commercial cultivars of barley (Hordeum vulgare L.) using meristematic shoot segments excised from germinated mature embryos. Plant Cell Rep. 23:9-16. Sharma, V.K., R. Hansch, R.R. Mendel, and J. Schulze. 2005. Mature embryo axis-based high frequency somatic embryogenesis and plant regeneration from multiple cultivars of barley (Hordeum vulgare L.). J. Exp. Bot. 56:1913-1922. 200 JOURNAL OF CROP IMPROVEMENT

Tripathi, B.K., and C. Bitaillon. 1985. In vitro plant regeneration of Hedychium roxburghii Blume through rhizome-meri stem culture. Plarft Cell, Tiss. Organ. Cult. 4:11-17. Viertel, K., and D. Hess. 1996. Shoot tips of wheat as an alternative source for regenerable embryogenic callus cultures. Plant Cell Tiss. Or Cult. 44:183-188. Wood, T. 1999. Ginger lilies. The American Gardener 78:40-45.

Li