Efficient somatic embryogenesis and plantlet regeneration from protoplast culture of Crocus sativus L.

摘要

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 degrees 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 GA₃. Transferring germinated embryos to a medium containing 0.1 mg/L NAA and 1 mg/L BA and incubation at 20 degrees C in a 16/8 hour light/dark cycle promoted conversion efficiency up to 88%.