秦巴蛹虫草原生质体的制备及再生条件研究
Study on isolation and regeneration of Qinba Cordyceps militaris protoplast

【目的】研究秦巴蛹虫草原生质体制备及再生的最适条件，建立高效制备和再生原生质体的方法。【方法】以秦巴蛹虫草无性型菌株CM-16为材料，研究细胞壁裂解酶种类、酶解时间、酶解温度、菌丝体菌龄及稳渗剂种类对原生质体制备及再生效果的影响，并在单因素试验结果基础上进行正交优化试验。【结果】(1)以0.6 mol/L NaCl为稳渗剂，用混合酶（质量分数1%纤维素酶和质量分数0.5%蜗牛酶按1∶1体积比混合）在(26±1) ℃对菌龄6 d的菌丝酶解3 h，原生质体的产量最高。(2)以0.6 mol/L甘露醇为稳渗剂，用混合酶（质量分数1%纤维素酶和质量分数0.5%蜗牛酶按1∶1体积比混合）在(26±1) ℃对菌龄5 d的菌丝酶解2 h，原生质体的再生率最高；验证试验结果表明，在此条件下，原生质体再生率平均为25.7%，此时原生质体平均产量为83.42×105 mL－1。【结论】对酶解温度、酶解时间、菌丝体菌龄、稳渗剂及酶系种类等条件的优化，可提高秦巴蛹虫草原生质体的产量及再生率。
【Objective】The purpose of this paper was to study the optimal conditions for isolation and regeneration of Qinba Cordyceps militaris (C.militaris) protoplasts,and to establish efficient methods for protoplast regenerated strains.【Method】Taking CM-16 strains from anamorph of Qinba C.militaris as test material,type of lyase,mycelia age,osmotic pressure stabilizer,enzymolysis time and temperature were studied on isolation and regeneration of protoplasts,and based on the results,the single factor conditions were optimized through orthogonal test.【Result】(1) Using 0.6 mol/L NaCl,mixed enzymes(1% （wt） cellulase and 0.5% （wt） snailase was mixed by 1∶1 volume ratio),6 d age of mycelia,(26±1) ℃,and 3 h,the protoplasts yield was the highest.(2) Using 0.6 mol/L mannitol,mixed enzymes(1% （wt） cellulase and 0.5% （wt） snailase was mixed by 1∶1 volume ratio),5 d age of mycelia,(26±1) ℃,and 2 h,the protoplasts regeneration rate was the highest.The verification test results showed that the average rate of regenerated protoplasts was 25.7% and the average yield of protoplasts was 83.42×105 mL-1.【Conclusion】The optimization of enzymolysis temperature and time,mycelia age,osmotic pressure stabilizer and enzyme type was essential to increase the protoplasts yield and regeneration rate of Qinba C.militaris