Calli derived from hypocotyledon of Medicago sativa ssp. sativa grew rapidly and dispersed easily on subculture media, Protoplasts isolated from calli of l2 days after subculture at a high yield of 6.5×l07
/g fresh weigh. Protoplasts were cultured on modified SHmedium containing l.0mgl/L 2, 4-D, 0.5mg/L BA, 2.0g/L CH, 2%sucrose, 6% glucose, 5 mmol/L 2-(N-morpholino)-ethanesulfonic acid with plating density of 1.0×105
/mL. Protoplast plating efficiency was 3.7%after 12 days in culture. Microcalli derived from protoplasts multiplicated on solid MSmedium containing 2.0mg/L 2, 4-D. Somatic embryos and plant regeneration were obtained when these calli were transferred to B5
medium containing 2.0mg/L 2-ip and 0.1mg/L NAA.