番木瓜幼叶原生质体分离研究

【目的】探讨番木瓜幼叶原生质体的最佳分离条件。【方法】以20～30日龄番木瓜无菌苗幼叶为材料,对原生质体分离过程中酶液组合、酶解时间、纯化离心速度（500～1500rpm）及离心时间（6～10min）进行探讨。【结果】随着酶解液中纤维素酶和果胶酶含量的提高,番木瓜原生质体产量逐渐升高,而其活力逐渐降低,其中以为2.0%纤维素酶＋0.5%果胶酶组合的效果较好,解离时间以8h为宜。随着酶解液中甘露醇浓度的升高,原生质体的产量呈现先增加后降低的趋势,在0.55mol/L时达到最大（2.48×106个/gFW）。在悬浮纯化原生质体时,以1000rpm离心6～8min的效果较好。【结论】适宜原生质体分离的酶解液为2.0%纤维素酶＋0.5%果胶酶＋25mmol/LMES＋0.55mol/L甘露醇,最适酶解时间为8h;在原生质体纯化时,使用1000rpm离心6~8min,有利于获得产量及活力较高的原生质体。

Isolation of protoplast from young leaves of papaya

Objective]The present experiment was conducted to study the optimal conditions for isolation of protoplast from young leaves of papaya. Method]The young leaves of papaya were sampled from virus-free plantlet to isolate the protoplast, and the influences of different conditions including enzymolysis solution combinations （1-3% cellulase and 0.5-0.8% pectinase）, incubation times （0-12 h）, purification centrifugal speed （500-1500 rpm） and centrifugal time （6-10 min） on isolation of protoplasts were compared and analyzed. Result]The quantity of purified protoplast increased gradually with the increasing concentration of cellulase and pectinase enzymes in enzymolysis solution, while its activity showed declining trend. The best results were obtained by incubating leaf material for 8 h in solution containing combination of 2.0% cellulase ＋0.5% pectolyase. Increasing the mannitol concentration in enzymolysis solution,the quantity of protoplasts initially increased but then showed a declining trend, and gave highest output （2.48×106 protoplast/gFW） at 0.55 mol/L mannitol concentration in enzymolysis solution. The best purification was obtained at 1000 rpm centrifuge speed for 6-8 min. Conclusion]The results revealed that incubating the leaf tissues in enzymolysis solution containing 2.0% cellulase onozuka R-10 ＋ 0.5% pectolyase Y-23 ＋ 25.0 mmol/L MES ＋ 0.55 mol/L manitol for 8 h, and purifying them at 1000 rpm centrifuge speed for 6-8 min gave best protoplast with high quality and high activity.