Plantlet Regeneration via Somatic Embryogenesis in Four Species of Crocus

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1 Plantlet Regeneration via Somatic Embryogenesis in Four Species of Crocus Roya Karamian Department of Biology, Faculty of Science Bu-Ali Sina University Hamadan Iran Keywords: meristems, micropropagation, plant growth regulators, somatic embryos Abstract Somatic embryogenesis was initiated in four species of Crocus, C. sativus, C. cancellatus, C. michelsonii and C. caspius using shoot meristem culture on LS medium containing 4 mg/l NAA and 4 mg/l BA or 1 mg/l 2,4-D and 4 mg/l kinetin. Somatic embryogenesis was asynchronous in all of four species and various stages of somatic embryo development were observed when embryogenic calli with globular somatic embryos were transferred into half strength MS medium containing 1 mg/l abscisic acid. Maturated embryos could be germinated on half strength MS medium supplemented with 2 mg/l GA. Complete plantlets were obtained by transferring of germinated embryos into half strength MS medium supplemented with 1 mg/l NAA and 1 mg/l BA at 2 ºC under a 16/ h (light/dark) cycle. INTRODUCTION Crocus is an important genus belonging to Iridaceae. It has nine species in Iran, many of which are valuable and horticulturally important. Like other ornamental monocotyledonous species with corms, they are generally propagated vegetatively. Numerous studies have appeared regarding in vitro propagation of Crocus sativus (George et al., 1992; Ebrahimzadeh et al., 1996; Ebrahimzadeh et al. 2) but other members of the genus have received little attention (Ebrahimzadeh et al., 1996). The present study reports how regeneration of plantlets was achieved via somatic embryogenesis from shoot meristems culture in C. sativus, C. cancellatus, C. michelsonii and C. caspius. MATERIALS AND METHODS Corms were collected from different areas of Iran during two annual flowering seasons. Sprouted corms were utilized as source of explants. Bulblets with small portion of corm attached were separated and washed with tap water and surface sterilized in.1% HgCl 2 solution for 1 min followed by rinsing times with sterile distilled water. Shoot meristems were dissected and cultured on LS medium (Linsmaier and Skoog, 196) containing % sucrose and different concentration of cytokinins (BA, Kn) and auxins (2,4-D, IAA, NAA) (Tables 1-). All media were solidified by.7% agar after adjusting the ph to. and autoclaved for 1 min at 121 C at 14 kpa. Cultures were incubated in dark at 2 C. The experiment was repeated twice using 2 explants per treatment. The data for callus initiation were scored after 6 weeks of culture. Callus and embryogenic callus induction frequencies were calculated as the percent of cultured shoot tips producing callus and embryogenic callus respectively. Data from a total of two replications were statistically analyzed using a SAS program and separated by DMRT. Embryogenic nature of cultures was maintained by visual identification and selection of embryogenic sectors and removal of soft and translucent nonembryogenic portions at the time of subculturing. Embryogenic calli with globular embryos were transferred to a halfstrength liquid MS medium (Murashige and Skoog, 1962) without growth regulator or with 1 mg/l ABA for maturation of somatic embryos. Maturated somatic embryos were transferred to half strength MS medium containing 2 mg/l GA for germination. For plant regeneration, germinated somatic embryos were transferred to half strength MS Proc. I st IS on Saffron Eds: J.-A. Fernández & F. Abdullaev Acta Hort 6, ISHS 24 2

2 medium supplemented with 1 mg/l NAA and 1 mg/l BA and incubated under a 16/ h (light/dark) photoperiod with a temperature of 2 C. RESULTS AND DISCUSSION After 6- weeks of culture, shoot meristems on LS medium containing different growth regulators formed calli. Frequency of callus initiation, embryogenic callus production and morphogenetic nature of callus varied with kind of growth regulator used (Tables 1-). In the studied species, the highest frequency of callus initiation and embryogenic callus induction were recorded on LS medium with NAA and BA or 2,4-D and kin. During initial stages of callus development yellowish and soft calli grew from cultured explants, which after - transfers were visually distinguishable to be of two types: yellow to brownish friable and nodular embryogenic calli with shining globular structures and off-white to yellow soft calli (Figures 1-4). Production of embryogenic calli increased considerably with careful selection of embryogenic regions and frequent subculturing on the maintenance MS medium. Somatic embryo development proceeded through various stages of globular, heart-shaped and bipolar embryos were observed in half strength liquid MS basal medium growth regulator free or with 1 mg/l abscisic acid. Somatic embryos showing bipolarity could be germinated on half strength MS medium containing 2 mg/l GA. Germinated somatic embryos transferred into half strength MS medium containing 1 mg/l of each NAA and BA resulted in shoot and root development and plantlet regeneration after -1 weeks of inoculation under a 16/ h (light/dark) photoperiod with a temperature of 2 ºC (Figures 1-4). Some plantlets were left to grow in culture under the same conditions without further subcultures produced corm. Then regenerated plantlets were kept on a medium with low nutrient levels to harden them before transfer to pots. The results reported here showed that two types of calli grew from cultured shoot meristems. Production of embryogenic callus was initially slow but careful selection of embryogenic regions and frequent subculturing on the maintenance MS medium resulted in vigorous proliferation of embryogenic callus. Somatic embryo development in all of studied species was asynchronous and different stages could be observed simultaneously in the same embryogenic callus. This protocol agrees with general mode of plant regeneration through somatic embryogenesis in number of plants. It was observed that 2ºC temperature was suitable for the regeneration of calli. This result agrees with earlier reports on this genus (Ebrahimzadeh et al., 2). Many aspects can affect the maturation and germination process, such as temperature and light condition, age of explants and concentration of growth regulators (Firoozabady and DeBoer, 199). The present study where callus cultures derived from meristems, facilitates the proliferation of embryogenic callus and subsequent regeneration of plantlets from somatic embryos and proved to be a useful alternate method for micropropagation. Literature Cited Ebrahimzadeh, H., Karamian, R. and Noori-Daloii, M.R Regeneration of neoformant organs from organ parts of some species of Crocus. J. Sci. I. R. Iran 7: Ebrahimzadeh, H., Karamian, R. and Noori-Daloii, M.R. 2. Somatic embryogenesis and plantlet regeneration in saffron, Crocus sativus L. J. Sci. I. R. Iran 11: Firoozabady, E. and DeBoer, D.L Plant regeneration via somatic embryogenesis in many cultivars of cotton (Gossypium hirsutum L.). In Vitro Cell. Dev. Biol. 29P: George, P.S., Visvanath, S., Ravishankar, G.A. and Ventkataraman, L.V Tissue culture of saffron (Crocus sativus L.): Somatic embryogenesis and shoot regeneration. Food Biotechnol. 6: Linsmaier, E.M. and Skoog, F.196. Organic growth factor requirements of tobacco tissue cultures. Physiol. Plant. 1: Murashige, T. and Skoog, F A revised medium for rapid growth and bioassays 24

3 with tobacco tissue cultures. Physiol. Plant. 1: Tables Table1. Morphogenic response of shoot meristem culture of C. sativus Growth regulators Auxin + Cytokinin BA + NAA BA + 2,4-D No. of Responsive explants % Callus Initiation 7.a 6ab 6b bc 2.d 27.de % Embryogenic callus initiation 4a 2.ab ab 2.ab 27.b b Morphogenic nature Compact,nodular embryogenic Yellowish soft friable and compact nodular BA + IAA 7.e 12.e 7.e Very slow growing compact callus with growing buds Kn + NAA Kn + 2,4-D Kn + IAA d 6b 4cd 2.bc 47.c 2de 2de 2de 1de 2 b 17.c 1c 2bc 2bc 22.bc 1c 12.c 1c Compact hard callus with green buds Brownish soft friable and compact nodular Whitish hard callus with growing buds Explants were cultured on LS medium supplemented with growth regulators in different combinations. Value is the mean for two experiments, 2 explants in each experiment. Data were taken 6 weeks after cultures. Mean separation within rows by DMRT, p.. 2

4 Table 2. Morphogenic response of shoot meristem culture of C. cancellatus Growth regulators Auxin + Cytokinin 2,4-D + BA No. of Responsive explants 6 4 % Callus initiation e 2 de 2e % Embryogenic callus initiation c c c Morphogenetic nature Yellow to brownish, compact, nodular IAA + BA e 1 de e c Off-white to yellow, compact, nodular NAA + BA a 6 ab bc 1 1 c Yellowish, soft callus 2,4-D + Kn ab 6 abc 4d 4 ab 4 a b Off-white, friable, nodular embryogenic callus IAA + Kn 4 2 de 2e 1 de 1 c 1 c White, soft callus with leaf-like structures NAA + Kn White, soft callus 2 de 4d 2 de Explants were cultured on LS medium supplemented with growth regulators in different combinations. Value is the mean for two experiments, 2 explants in each experiment. Data were taken 6 weeks after cultures. Mean separation within rows by DMRT, p.. 26

5 Table. Morphogenic response of shoot meristem culture of C. michelsonii Growth regulators Auxin + Cytokinin BA + NAA BA + 2,4-D No. of Responsive explants % Callus Initiation b c 7. b 2 b 1 b b % Embryogenic callus initiation. cd c Morphogenetic nature Off-white, soft callus Brownish, compact, nodular callus BA + IAA Kn + NAA ab 4 ab 4 ab 2 b 16.7 bc Off-white, soft, friable, nodular callus Kn + 2,4-D a ab 62. a 27 b 4 a 2 b Yellow to brownish, friable, nodular embryogenic callus Kn + IAA c Brownish, soft callus Explants were cultured on LS medium supplemented with growth regulators in different combinations. Value is the mean for two experiments, 2 explants in each experiment. Data were taken 6 weeks after cultures. Mean separation within rows by DMRT, p.. 27

6 Fig. 1. Plantlet regeneration via somatic embryogenesis in C. sativus. Fig. 2. Plantlet regeneration via somatic embryogenesis in C. cancellatus 2

7 . Fig.. Plantlet regeneration via somatic embryogenesis in C. michelsonii. Fig. 4. Plantlet regeneration via somatic embryogenesis in C. caspicus. 29