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

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 When 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.