PLANT REGENERATION FROM CALLUS PROTOPLASTS OF MEDICAGO SATIVA SSP. SATIVA

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.