冬虫夏草无性型原生质体再生条件研究

Study on Protoplast Regeneration Conditions of Anamorphic Strains of Cordyceps sinensis

[目的]优化冬虫夏草原生质体再生条件。[方法]考察纤维素酶浓度、蜗牛酶浓度、两者混合体积配比、酶解温度、酶解时间、菌龄、稳定剂及培养基等条件对冬虫夏草无性型原生质体再生率的影响。[结果]冬虫夏草无性型原生质体再生的最佳条件为:纤维素酶浓度0.5%,蜗牛酶浓度0.5%,纤维素酶与蜗牛酶体积比1:1,酶解温度35℃,酶解时间1.5h,菌龄6d,甘露醇0.5mol,③号再生培养基(蔗糖10g,葡萄糖4g,酵母粉4g,甘露醇0.5mol,pH值6.2,琼脂18g,水加至1000ml),在该最佳条件下原生质体再生率为0.43%。[结论]优化的冬虫夏草无性型原生质体再生条件简便、合理、可行。

[ Objective ] The research aimed to select the optimum extracting procedure for regenerations of protoplast of auamorphie strains of Cordyceps sinensis. [ Method] The optimum extracting procedure was studied through one-factor test. The effects of cellulose enzyme concentration, snailase concentration, volumetric ratio of them, hydrolytic temperature, hydrolytic time, mycelia age, stabilizer and culture medium on the regeneration rate of protoplast of anamorphic strains were studied. [ Result] The optimum extracting procedure of the regeneration conditions of protoplast were as follow： Concentration of cellulose enzyme was 0.5% , concentraction of snailase was 0.5 % , volumetric ratio of them was 1 ： 1, hydrolytic temperature was 35 ℃, hydrolytic time was 1.5 h, mycelia age was 6 d, mannitol was 0.5 mol, the No. 3 medium （sugar 10 g, glucose 4 g, yeast power 4 g, mannitol 0.5 tool, pH value 6.2, agar 18 g, the total volume 1000 ml）. Under the best conditions, the protoplast regeneration rate was 0.43%. [ Conclusion ] The optimum extracting procedure was simple, reasonable and reliable.