Plant regeneration from protoplasts of immature Vigna sinensis cotyledons via somatic embryogenesis

Protoplasts were isolated from immature cotyledons of Vigna sinensis and cultured in a modified MS Liquid medium containing 0.2 mg/l 2, 4-dichlorophenoxyacetic acid (2,4-D), 1 mg/l naphthaleneacetic acid (NAA)and 0.5 mg/l 6-benzylaminopurine (BAP)in the dark at a density of 1 x 10(5)/ml. The protoplasts began to divide in 3-5 days. Sustained cell division resulted in formation of cell clusters and small calli, with the cell division frequency and plating efficiency of cell colonies reaching 27.7% and 1.7% respectively. When calli of 2 mm in size were transferred onto MSB medium (MS salts and B5 vitamins) containing 500mg/l NaCl, 500 mg/l casein hydrolysate (CH), 2 mg/l 2, 4-D and 0. 5 mg/l BAP for further growth, approximately 5% of the calli developed embryogenically. The embryogenic calli were selected and subcultured on the same composition of MSB medium and were able to maintain somatic embryogenesis capacity in subculture for a long time. When the calli were moved to MSB medium with 0.1 mg/l indole-3-acetic acid (IAA), 0.5mg/l kinetin (KT), 3-5% mannitol and 2% sucrose in the light, many somatic embryos formed from the calli. Only part of the embryoids developed further to the cotyledonary stage, and the others died at the globular, heart-shaped or torpedo stages. Finally, some cotyledonary embryoids germinated and developed into plants or shoots. The shoots were readily rooted on 1/2 strength MS medium with 0.1-0.3 mg/l indole-3-butyric acid (IBA). The plants grew well in soil and were fertile.