Isolation and Purification of Protoplasts in Roots and Leaves of Soybean Seedlings

The methods for isolation and purification of protoplast in roots and leaves of soybean seedling were investigated together in this study.The results showed that,the suitable enzyme species and concentrations for isolation of root protoplasts were CPW-13M+13%(W/V) mannotol+3% cellulose R-10+1.1% macerozyme R-10+1.0% hemicellulase,pH5.8;those for leaf were 0.15% CaCl22H2O+9% mannitol+1% cellulase R-10+0.20% pectolaseY-23,pH 5.8,and the temperature was 28℃.The reasonable enzymatic times for isolation of root and leaf protoplasts were 16 and 4 h,respectively;and the higher protoplast yield and viability were accordingly 1.46×105 protoplasts·g-1 FW and 57.8%,1.74×106 protoplasts·g-1 FW and 70.3%.The better purification for root protoplasts was the sinking method of 23% sucrose mixed with CPW-18M,that for leaf was the rising method of 25% sucrose.