普通小麦幼苗叶肉细胞原生质体分离方法的优化

The Optimization of Isolation Method for Mesophyll Protoplast from Common Wheat (Triticum aestivum) Seedlings

以7日龄小麦幼苗的叶肉细胞为材料,采用正交试验分析了纤维素酶浓度、离析酶浓度、酶解时间、甘露醇浓度对原生质体分离时的产量和活力的影响;采用瞬时表达技术通过PEG-Ca^(2+)介导法转化小麦的原生质体,分析了PEG浓度对转化效率的影响。结果表明,小麦叶片原生质体分离的最佳条件为纤维素酶1.5%,离析酶0.75%,甘露醇0.4 mol/L,酶解时间3 h,得到的原生质体的产量可达到7.2×10~6个/g·FW,活力超过95%;瞬时表达实验结果表明,在25%PEG浓度下,小麦原生质体的转化效率最高。

By orthogonal test,we analyzed the effects of concentrations of cellulose,macerozyme,and mannitiol as well as the time of enzyme digestion on the productivity and vitality during the isolation of protoplast from 7 days of wheat seedling mesophyll. Further,the transient expression technology was employed to analyze the effects of PEG concentration on the transformation efficiency by PEG-Ca^（2＋）-mediated transformation of mesophyll. The results showed that the optimal condition for isolating the mesophyll from wheat leaves was ：1.5% of cellulase,0.75% of macerozyme,0.4 mol/L mannitol,and 3 h of enzyme digestion;the protoplast yield was 7.2×10~6 cells/g·FW,the vitality was more than 95%. The results from the transient expression technology revealed that transformation efficiency was the highest under 25% PEG.