响应面法优化玉木耳原生质体制备条件

发布时间：2020-04-10 10:39:53 来源：文档文库

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响应面法优化玉木耳原生质体制备条件
作者：苏文英　杨和川　谭一罗　周振玲　秦裕营　李晓
来源：《南方农业学报》2020年第01期

摘要：【目的】优化玉木耳原生质体制备条件，获得高质量、高产量的原生质体，为后续玉木耳遗传育种、优良性状筛选和品种改良提供良好的试验材料。【方法】以玉木耳原生质体产量为考察指标，结合单因素试验和响应面优化法，建立原生质体制备回归方程，探究玉木耳原生质体的最佳制备条件。【结果】单因素试验结果表明，玉木耳原生质体制备的适宜条件为：酶解时间3 h、酶解液浓度2.0%、酶解温度35 ℃。响应面试验结果表明，各因素对原生质体制备影响程度的排序为：酶解温度>酶解时间>酶解液浓度，酶解温度与酶解时间的交互作用對原生质体产量有极显著影响（P<0.01），酶解时间与酶解液浓度的交互作用影响显著（P<0.05）。响应面试验优化后，获得玉木耳原生质体的制备条件为：酶解液浓度2.0%、酶解时间3.4 h、酶解温度35 ℃，此条件下玉木耳原生质体的平均产量为17.5×106 CFU/mL，与理论产量（17.77×106 CFU/mL）偏差小。【结论】利用响应面法可确立玉木耳原生质体制备的最佳条件，获得较理想的原生质体产量。

关键词：玉木耳;原生质体;响应面法;制备条件

中图分类号： S646.6; ; ; ; ; ; ; ; ; ; ; ; ; ; ;文献标志码： A 文章编号：2095-1191（2020）01-0169-07

Abstract：【Objective】 The objective of this study was to optimize the preparation conditions of protoplasts of Auricularia cornea， and to obtain high quality and high yield protoplasts， which provided good experimental materials for the subsequent genetic breeding， excellent trait screening and variety improvement of A.cornea. 【Method】The protoplast yield of A.cornea was used as an index， and the regression equation of protoplast preparation was established by combi-ning single factor experiment and response surface optimization method to study the optimal preparation conditions of protoplasts of A.cornea. 【Result】The results of single factor test showed that the suitable enzymatic hydrolysis time of protoplast preparation was 3 h， the enzymatic hydrolysis liquid concentration was 2.0%， and the enzymatic hydrolysis temperature was 35 ℃. The results of response surface methodology showed that the degree of influence of various factors on protoplast preparation was： enzymatic hydrolysis temperature>enzymatic hydrolysis time>enzymatic hydrolysis liquid concentration， and the interaction between enzymatic hydrolysis temperature and enzymatic hydrolysis time had a highly significant effect on protoplast yield（P<0.01）， and the interaction betweenenzymatic hydrolysis time and enzymatic hydrolysis liquid concentration had significant influence（P<0.05）. The results of the response surface test were as follows： enzymatic hydrolysis liquid concentration 2.0%， enzymatic hydrolysis time 3.4 h， enzymatic hydrolysis temperature 35 ℃. Under these conditions， the average yield of protoplasts of A.cornea. was 17.5×106 CFU/mL， which was lower than the theoretical yield（17.77×106 CFU/mL）. 【Conclusion】 The response surface method is used to establish the optimal conditions for the preparationof protoplasts of A. cornea， and the optimal protoplast yield is obtained.