Somatic Embryogenesis and Plant Regeneration from Protoplast Culture of Crocus pallasii Subsp. haussknechtii

Abstract

A protocol has been developed for plant regeneration from protoplast culture of Crocus pallasii subsp. haussknechtii using regenerable embryogenic calli obtained from shoot meristem culture on MS+9.28 microM kinetin+4.52 microM 2,4-D. Protoplasts were isolated directly from embryogenic calli, embedded in Ca-alginate beads and cultured with nurse cells in MS+4.64 microM kinetin+4.52 microM 2,4-D+5.68 microM ascorbic acid+0.3 M mannitol at 20 +/- 2 degrees C in darkness. After appearing ofmicrocalli on the surface of the beads, they were transferred onto 1/2MS+2.32 microM kinetin+2.26 microM 2,4-D+5.68 microM ascorbic acid for growth of embryogenic calli. Somatic embryos matured on MS medium growth regulator free and germinated on 1/2MS+14.45 microM GA3 +4.43 microM BA at 20 +/- 2 degrees C in a 16/8 h light/dark cycle.