大叶藻原生质体的分离和培养

摘要

The influences of hydrolase combination ,solution pH ,and enzymolysis time on isolation and preparation of protoplast in eelgrass Zostera marina were studied ,and the conditions for separation of pro-toplasts were optimized from the eelgrass aseptic seedlings .It was found that high protoplast at a rate of 3 .90 × 106 individuals/g eelgrass was obtained in 1 .0 g aseptic seedling leaves which were digested in 5ml enzyme solution containing 2% cellulase ,0 .5% pectinase and 0 .5mol/L sorbitol under dark conditions of pH 4 .0 for 16 hours at 17 ℃ .Neutral red staining revealed that these protoplasts had viability of 85 .0% , and the conditions of the protoplast culture were also discussed in the eelgrass .%研究了水解酶的组合、酶液pH值和酶解时间等因素对大叶藻原生质体分离制备的影响，确定了适合无菌大叶藻原生质体分离的最佳试验条件。试验结果显示，1．0g生长状态良好的大叶藻无菌幼苗叶片，在5 mL含2．0％纤维素酶、0．5％果胶酶和0．5 mol/L山梨醇混合酶液的灭菌海水中，pH 4．0，17℃黑暗条件下轻摇酶解16 h ，可获得较多的大叶藻原生质体，产量达3．90×106个/g。用中性红染色鉴定表明，原生质体存活率为85．0％。