基于蛹虫草单核芽生孢子的原生质体制备条件的优化

Optimization of the Preparation Conditions of Protoplasts from Mononuclear Blastospores of Cordyceps militaris

为了获得单细胞核的蛹虫草材料和制备原生质体的最适酶解条件,以蛹虫草芽生孢子为试验材料,通过荧光染色观察其细胞核数,以溶壁酶质量分数、酶解温度、酶解时间为试验因子,原生质体得率为响应值,采用单因素和Box-Behnken试验设计进行优化。荧光显微镜观察结果表明蛹虫草芽生孢子为单个细胞核。响应面优化试验结果表明蛹虫草原生质体最适制备条件为:溶壁酶质量分数2.06%,酶解温度26.1℃,酶解时间2.57 h。在此条件下,原生质体得率的预测值为8.97×10^6个/mL,验证试验所得原生质体得率为(9.17±0.29)×10^6个/mL,预测值和试验值差异不显著,回归模型拟合良好。在优化条件下制备的原生质体在含有0.8 mol/L甘露醇的再生培养基上的再生率达43.33%±2.08%。

The aim of this paper is to obtain the single nucleus of Cordyceps militaris and the optimal preparation conditions of protoplasts.The number of cell nuclei of blastospores was observed by fluorescent staining.The optimized conditions for preparing protoplasts were studied by the single factor test and the Box-Behnken design with three variables as following:lywallzyme concentration,temperature,and digestion time.The yield of protoplasts was the evaluation index.The observation of fluorescence microscopy showed that the blastospores of Cordyceps militaris were single nuclei.The results of response surface optimization indicated that the optimal condition for preparing protoplasts was performed with 2.06%lywallzyme under 26.1℃for 2.57 h.Under the optimal conditions,the predicted yield of protoplasts was 8.97×10^6 protoplasts per milliliter,whereas,the experimental yield of protoplasts was(9.17±0.29)×10^6 protoplasts per milliliter.The predicted yield and the experimental yield of protoplasts were not significantly different from each other.The regeneration rate of protoplasts prepared under optimized conditions was up to 43.33%±2.08%on the regeneration medium containing 0.8 mol/L mannitol.