以菜豆幼嫩叶片为材料，研究了预处理、酶液组合、酶解时间、酶解方式、甘露醇浓度及纯化时的离心转速对菜豆叶片原生质体分离的影响。结果表明：菜豆叶片质壁分离1h后，在2％纤维素酶＋1％离析酶＋10％甘露醇＋0�1％BSA＋5 mmol／L MES＋10 mmol／L CaCl2·2H2O，pH为5�8的混合液中，（25±1）℃黑暗条件下振荡酶解12 h可获得大量有活力的绿色原生质体。纯化时400 r／min离心3 min效果较好。%Young leaves of common bean were used as experiment materials to study the effects of pretreatment, enzyme combination, time of enzyme digestion, methods of enzymatic digestion, man⁃nitol concentrations and centrifugal speed during purification on protoplasts isolation of common bean leaves. The results showed that plasmolysis time was 1 h and the digestion was for 12 h, a lot of vi⁃brant green protoplasts were produced in a mixture of cellulase 2%+macerozyme 1%+10% mannitol+BSA 0�1%+MES 5 mmol/L+CaCl2·2H2O 10 mmol/L under (25±1) ℃, pH 5�8 and dark oscil⁃lation conditions. When purified, the isolated protoplasts for 400 r/min centrifugal 3 min achieved better results.
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