Efficient Somatic Embryogenesis and Plantlet Regeneration from Protoplast Culture of Crocus sativus L. R. Karamian and M. Ranjbar Department of Biology, Faculty of Sciences, Bu-Ali Sina University PO Box 65175/4161, Hamedan Iran Keywords: Crocus sativus, plantlet regeneration, protoplast culture, somatic embryogenesis Abstract The present study reports an efficient protocol for isolation and culture of protoplasts directly from embryogenic calli derived from shoot meristem culture of Crocus sativus L. Embryogenic calli were induced on Linsmaier and Skoog (1965) medium containing 4 mg/L kinetin and 1 or 4 mg/L 2,4-D. Protoplasts were isolated, embedded in Ca-alginate beads and cultured with nurse cells in Murashige and Skoog medium containing 2 mg/L kinetin, 1 mg/L 2,4-D, 100 mg/L ascorbic acid and 0.3 M mannitol at 25°C in darkness. After 4-5 weeks of culture, microcalli appeared on the surface of the Ca-alginate beads. Transferring beads to 1/2 MS medium supplemented with 0.2 mg/L kinetin and 0.1 mg/L 2,4-D increased the growth of embryogenic calli. Somatic embryo development was observed on 1/2 MS medium with 1 mg/L ABA. Maturated embryos germinated on a medium containing 25 mg/L GA 3 . Transferring germinated embryos to a medium containing 0.1 mg/L NAA and 1 mg/L BA and incubation at 20°C in a 16/8 hour light/dark cycle promoted conversion efficiency up to 88%. INTRODUCTION The culture and regeneration of protoplasts are important steps in somatic hybridization and realization of genetic manipulation of valuable plants. Indeed, plant regeneration in the genus Crocus has been usually successful when embryogenic calli were used as the source of protoplasts. Although protoplast culture in Crocus sativus L. has been attempted by using the combination of alginate entrapment and nurse culture method earlier, the regeneration frequency has not been satisfactory (Isa et al., 1990). The present study reports the high frequency of plantlet regeneration from protoplast-derived embryogenic calli of Crocus sativus L. using a nurse culture method. MATERIALS AND METHODS Bulblets were surface sterilized in 0.15% HgCl 2 solution for 10 min followed by rinsing 3 times with sterile distilled water. Shoot meristems were dissected and cultured on Linsmaier and Skoog (1965) medium (LS) containing different hormone combinations (Table 1) and incubated in dark at 20°C. After 6 weeks, two types of calli were produced from the shoot meristems. Nonembryogenic calli were soft, friable and translucent while embryogenic calli were compact with shining globular regions. For maintenance of embryogenic potential, embryogenic calli together with globular embryos were transferred to Murashige and Skoog (1962) medium (MS) supplemented with 2 mg/L kinetin, 1 mg/L 2,4-D, 100 mg/L ascorbic acid, incubated at 20°C in darkness and then used for protoplast isolation. The calli were incubated in a filter-sterilized enzyme solution consisting of MS medium with 0.1% (w/v) Pectolyase Y-23, 1% Cellulase R- 10, 1% Deriselase, 0.1% and 0.3 M mannitol. The mixture was shaken for 1 hour and then kept in a stationary position for another 1 hour. The incubation mixture was then filtered and washed twice with a washing solution to purify the isolated protoplasts. Subsequently, they were layered onto 20% sucrose and centrifuged for 5 min at 100 g. Viable and intact protoplasts floating at the interface were removed and washed twice with protoplast culture medium. The purified protoplasts were mixed gently with 2% 113 Proc. 3 rd IS on Saffron Eds.: M.Z. Tsimidou et al. Acta Hort. 850, ISHS 2010