TISSUE AND PROTOPLAST CULTURE AND TRANSFORMATION OF MEDICAGO HISPIDA

In the present report, we studied the organogenesis in culture in vitro of hypocotyl and cotyledon explants and cotyledon protolasts of Medicago hispida and also studied transformation of the cotyledon explants and protoplasts of M. hispida mediated by Agrobacterium tumefaciens. When the explants were cultured on MS medium supplemented with IAA 0.5—1mg/L and cytokinin (BA or ZT) 0.5—2mg/L, the shoots differentiated from the explant-derived calli in condition of light (25℃; 12h/day) at 30—40 days of culture and further developed to form intact plantlets after they were subcultured on the same medium composition. The purified protoplasts isolated from cotyledons of M. hispida were cultured in B5 liquid medium containing 2,4-D 0.5mg/L and KT 0.2mg/L. It was observed that the division frequency of the cells was about 30—41%. The shoots were induced from protoplast-derived calli after they were transferred and subcultured onto MSB medium. Using the method of co-cultivation, cotyledon explants and protoplast-derived cells of M. hispida were transformed by Agrobacterium tumefaciens strains PGV 2260 (pBI 121) or C58Cl (pBZ 6111), and transformants were abtained. Enzyme activity of GUS and NOS assay demonstrated that foreign genes expressed in the transformed plants and calli.