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Hedychium Muluense R.M. Smith Hamidou F

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Hedychium Muluense R.M. Smith Hamidou F First Report of Plant Regeneration via Somatic Embryogenesis from Shoot Apex-Derived Callus of Hedychium muluense R.M. Smith Hamidou F. Sakhanokho Rowena Y. Kelley Kanniah Rajasekaran ABSTRACT. The genus Hedychium consists of about 50 species, with increasing popularity as ornamentals and potential as medicinal crop plants, 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 Zingiberaceae 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 China 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 rhizome 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 rhizomes 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 cultivars 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.
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