响应面法优化平菇单核原生质体的制备及再生技术

Optimiztion Technology of Preparation and Regeneration of Mononuclear Protoplast of Pleurotus ostreatus through Response Surface Method

为了提高平菇原生质体融合育种效率,本研究通过构建基于原生质体产量及单核原生质体再生率的响应面模型,优化平菇原生质体制备体系。以平菇菌株黑平16-1为试材,分别通过单因素试验考察菌丝菌龄(3~6 d)、酶液浓度(0.5%~2.0%)、酶解温度(28~34℃)和酶解时间(3~6 h)对原生质体制备及再生率的影响,并在单因素试验基础上,设计4因素3水平的响应面试验,建立各因素与响应值(原生质体产量和单核原生质体再生率)之间的数学模型,确定原生质体制备及单核原生质体再生的最佳提取工艺。结果表明,最佳提取工艺为菌龄4 d、酶液浓度1.51%、酶解温度28.3℃、酶解时间3.7 h,在此条件下平菇原生质体产量和单核原生质体再生率分别为(2.25±0.21)×10^7个·mL^-1和(6.80±0.42)%,与模型预测值接近(2.27×10^7个·mL^-1和6.83%),方程拟合度良好,该方法适用于对平菇原生质体的制备及单核原生质体再生条件的参数优化。

In order to improve the efficiency of Pleurotus ostreatus protoplast fusion breeding,a response surface model based on protoplast yield and mononuclear protoplast regeneration rate was constructed to optimize the protoplast preparation system of P.ostreatus.The effects of mycelium age(3~6 d),enzyme concentration(0.5%~2.0%),enzyme solution temperature(28~34℃)and enzymatic hydrolysis time(3~6 h)on protoplast preparation and regeneration rate was studied by using P.ostreatus strain Heiping 16-1 as material.On the basis of single factor experiment,the response surface test of 4 factors and 3 levels was designed,and the mathematical model between each factor and response value(protoplast yield and mononuclear protoplast regeneration rate)was established to determine the optimal extraction process of protoplast preparation and single nuclear protoplast regeneration.The results showed that the optimal extraction process was as follows:the mycelium age was 4 d,the enzyme solution concentration was 1.51%,the enzyme solution temperature was 28.3°C,and the enzymatic hydrolysis time was 3.7 h.Under the above condition,the preparation rate of P.ostreatus protoplasts was(2.25±0.21)×10^7 cells·mL^-1,and the regeneration rate of mononuclear protoplasts was(6.80±0.42)%,which was closed to the predicted value of the model(2.27×10^7 cells·mL^-1,6.83%).In conclusion,the response surface equation was well fitted,and this method is suitable for the preparation of P.ostreatus protoplasts,and the parameter optimization of mononuclear protoplast regeneration conditions.