响应面法优化罂粟科植物叶片原生质体的制备条件

Optimization of the Preparation Conditions of Leaf Protoplasts of Papaveraceae by Response Surface Methodology

【目的】系统优化罂粟科(Papaveraceae)植物叶片原生质体提取的影响因素,建立其高效的原生质体提取体系。【方法】以花菱草(Eschscholtzia californica Cham.)幼苗为罂粟科模式植物,通过单因素试验对影响原生质体产量和活力的预制液浓度、酶解液组成、酶解时间进行考察,借助响应面Box-Behnken设计预测最佳提取方案,并将最佳方案在罂粟亚科(Papaveroideae)、角茴香亚科(Hypecooideae)和荷包牡丹亚科(Fumarioideae)的代表性植物中进行验证。【结果】当预制液浓度为0.8 mol/L、酶解时间6 h、酶解液组成为1.5%纤维素酶+0.4%离析酶+0.3%果胶酶时,花菱草原生质体的产量和活力最为理想,与响应面法的预测结果一致,能够成功获取罂粟科植物叶片的原生质体。【结论】本研究方法可实现罂粟科植物叶片原生质体的高效提取,为后续瞬时表达体系、亚细胞定位、单细胞测序等研究提供技术支撑。

[Purpose]To optimize the influencing factors of protoplast extraction from Papaveraceae leaves and establish an efficient protoplast extraction system.[Methods]Taking the seedlings of Eschscholtzia californica Cham.as the model plant of Papaveraceae,the concentration of preparation solution,the composition of enzymolysis solution,and the time of enzymolysis were examined by single factor experiments,which affected the yield and vitality of protoplasts;the optimal extraction protocol was predicted by response surface Box-Behnken design.The optimum protocol was validated in representative plants of Papaveroideae,Hypecooideae and Fumarioideae.[Results]Wh-en the concentration of preparation solution was 0.8 mol/L,the time of enzymolysis was six hours,and the composition of enzymolysis solution was 1.5%cellulase+0.4%macerozyme+0.3%pectinase,the yield and vitality of protoplasts in E.californica were ideal,which was consistent with the predicted values from response surface methodology,and it was able to obtain the leaves protoplasts from the family of Papaveraceae.[Conclusion]This research method can realize the efficient at extraction of protoplasts from leaves of Papaveraceae plants,providing effective technical support for subsequent studies such as transient expression system,subcellular location and single-cell sequencing.