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目的:探讨菘蓝原生质体分离的最佳条件。方法:用纤维素酶和离析酶的混合酶液提取菘蓝无菌苗幼叶原生质体,对影响原生质体提取得率及活力的因素进行分析。结果:适宜菘蓝叶片原生质体游离的条件为:CPW+0.5% PVP+3mmol/L MES+1.0% Cellulase R-10+0.1% Macerozyme R-10+0.4mol/L甘露醇,pH5.6,25℃黑暗条件下酶解6h。在此条件下原生质体产量达1.5×10个/gFW,活力达79.14%,用“过滤-离心-漂浮法”进行纯化,蔗糖浓度21%时纯化效果最好。结论:用混合酶系统处理,能够获得大量有活性的菘蓝原生质体,为建立高效的菘蓝原生质体再生体系及其遗传操作奠定了基础。
Objective: To investigate the optimal conditions for the isolation of protoplasts of indigo. Methods: The protoplasts of young leaves of Asparagus indigotica were extracted with a mixed enzyme solution of cellulase and segregating enzymes, and the factors influencing the extraction yield and activity of protoplasts were analyzed. RESULTS: The suitable conditions for protoplast release from indigo leaves were: CPW+0.5% PVP+3 mmol/L MES+1.0% Cellulase R-10+0.1% Macerozyme R-10+0.4 mol/L mannitol, pH5.6,25 °C Enzymatic hydrolysis in dark conditions for 6 h. Under this condition, the yield of protoplasts reached 1.5×10/gFW, and the activity reached 79.14%. The purification was performed using “filter-centrifugal-floating method”. The best purification effect was obtained when the sucrose concentration was 21%. Conclusion: A large amount of active indigoplasma protoplasts can be obtained by treatment with mixed enzyme system, which lays a foundation for the establishment of efficient indigoplasma protoplast regeneration system and its genetic manipulation.