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Somatic emberyogenesis and plantlet regeneration in Crocus speciosus R. KARAMIAN

Department of Biology, Faculty of Sciences, Bu-Ali Sina University, Hamadan, Iran Email: R_karamian@basu.ac.ir

Abstract
Somatic embryogenesis was induced from shoot meristems of Crocus speciosus. The explants were cultivated at 20 C in darkness on Linsmaier and Skoog medium supplemented with 3% (w/v) sucrose and different growth regulator combinations. The best embryogenic rates were obtained in the presence of 4 mg/l kinetin and 1 mg/l 2,4-D, as approximately 30 % of callus formed on this medium produced somatic embryos. All phases of somatic embryogenesis were observed on half strength MS medium with 1 mg/l ABA. Maturated embryos germinated in the presence of 25 mg/l GA3. Conversion of bipolar embryos to plantlets was obtained when they were transferred to half strength Murashige and Skoog medium supplemented with 1 mg/l IAA and 1 mg/l kinetin at 20 C under a 16/8-h (light/dark) photoperiod. Key words: Crocus speciosus, plant regeneration, somatic embryogenesis

Introduction The genus Crocus includes 9 species in Iran, many of which are valuable and horticulturally important. Like other ornamental monocotyledonous species with corms, they are generally propagated vegetatively (Firoozabady & DeBoer, 1993). Numerous studies have appeared regarding in vitro propagation of Crocus sativus using corms, floral parts (Ebrahimzadeh et al., 1996) and trough somatic embryogenesis (Ebrahimzadeh et al., 2000), but the members of the genus have received little attention. In this paper, we describe for the first time how regeneration of plantlets was achieved via somatic embryogenesis from shoot meristems in C. speciosus. Materials and Methods Bulblets with a small portion of corm of C. speciosus were separated, washed with tap water and surface sterilized in 0.15 % HgCl2 solution for 10 min followed by 3 rinses with sterile distilled water. Shoot meristems were excised from corms and planted on LS medium (Linsmaier & Skoog, 1965) supplemented with 3 % (w/v) sucrose and different growth regulator combinations (Table 1). The data for callus initiation were scored after 6 weeks of culture. Callus and embryogenic callus frequencies were calculated as the percent of cultured shoot tips producing callus and embryogenic callus respectively. Embryogenic nature of culture was maintained by visual identification and selection of embryogenic sectors and removal of nonembryogenic portions at each subculture. Embryogenic calli were transferred to half strength liquid MS medium containing 1 mg/l ABA under constant agitation on a rotary shaker (80 rpm) in darkness for maturation f somatic embryos. Somatic embryos showing bipolarity were maintained in half strength solid MS medium in the presence of 25 mg GA3 until early root and shoot formation. For plantlet regeneration, germinated somatic embryos were transferred to half strength MS medium (Murashige & Skoog, 1962) supplemented with 1 mg/l kinetin and 1 mg/l IAA and incubated at 20 C under a 16/8-h (light/dark) photoperiod, provided by cool white fluorescent tubes.

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Results and Disscution Results showed that different aspects can affect the maturation and germination of somatic embryos, such as temperature and light conditions, age of explants, concentration of growth regulators (Ebrahimzadeh et. al., 2000). Embryogenic calli on half strength MS medium containing 1 mg/l IAA and 1 mg/l kin predominantly formed roots with no shoots, while calli that grew in the presence of 1 mg/l ABA showed all the stages of somatic embryo development. Although its precise action is not clear, ABA has found to induce the expression of maturation genes and to inhibit precocious germination (Ebrahimzadeh et al., 2000, George et al., 1992). However, germination of somatic embryos with well-developed root and shoot was obtained in the presence of GA3. Since the species of the genus Crocus grow from late autumn to early spring in nature, callus culture under low-temperature conditions was attempted. The data reported here demonstrated for the first the plantlet regeneration from embryogenic calli of C. speciosus. This effective somatic embryogenesis technique offers the possibility to mass multiply material that has been improved by genetic manipulation experiments. References
1. Ebrahimzadeh, H., Karamian, R. and Noori-Daloii, M.R. 1996. Regeneration of neoformant organs from organ parts of some species of Crocus. J.Sci.I.R.Iran7: 65-76. 2. Ebrahimzadeh, H., Karamian, R. and Noori-Daloii, M.R. 2000. Somatic embrogenesis and plantlet regeneration in saffron, Crocus sativus L. J.Sci.I.R.Iran 11: 169-173. 3. Firoozabady, E. and DeBoer, D.L. 1993. Plant regeneration via somatic embryogenesis in many cultivars of cotton ( Gossypium hirsutum L.). In Vitro Cell. Dev. Biol. 29P: 166-173. 4. George, P.S., Visvanath, S., Ravishankar, G.A. and Ventkataraman, L.V. 1992. Tissue culture of saffron (Crocus sativus L.): Somatic embryogenesis and shoot regeneration. Food Biotechnol. 6: 217-223. 5. Linsmaier, E.M. and Skoog, F.1965. Organic growth factor reqierments of tobacco tissue cultures. Physiol. Plant. 18: 100-127. 6. Murashige, T. and Skoog, F. 1962. A revised medium for rapid growth and bioassays with tobacco 6tissue cultures. Physiol. Plant. 15: 473-493.

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Fig. 1. Somatic embryogenesis in C. speciosus. A) Nonembryogenic callus, B) Embryogenic callus, C-E) Globular and heart-shaped somatic embryos, F) Plantlet regeneration.

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Table 3. Morphogenetic response of shoot meristem culture of C. speciosus __________________________________________ Growth regulators % Callus % Embryogenic callus Auxin + Cytokinin Initiation initiation BA + 4 4 1 NAA 4 1 4 32.5b 15c 40b 27.5b 22.5b 32.5b 2.5c 5c 0c 5d 0d 0d 10c 12.5c 0d 0d 0d 0d

BA + 2,4-D 4 4 4 1 1 4 BA + IAA 4 4 4 1 1 4 Kn + 4 4 1 NAA 4 1 4

52.5ab 47.5ab 45ab 67.5a 52.5ab 65a 15c 10c 5c

10c 16.7b 8c 17b 30a 10c 0d 0d 0d

Kn + 2,4-D 4 4 4 1 1 4 Kn + IAA 4 4 4 1 1 4

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Value is the mean for two experimentst. Data were taken 6 weeks after cultures. Mean separation within rows by DMRT, p0.05.

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