Optimization Conditions of Wheat Mesophyll Protoplast Isolation

Abstract

The research object of this study is “ML7113” wheat leaf, which is used to isolate protoplast with enzyme hydrolysis method. Three main effectors—the concentration of mannitol, enzymolysis time and centrifugal force, affect the production and vitality of protoplast. While the production and vitality of wheat protoplasts were detected by the hemacytometer and the FDA staining respectively. Results showed that, with the increasing concentrations of mannitol during 0.2 M - 0.4 M, protoplast yield increases and when the concentration is 0.4 M, the protoplast vitality can be up to 95%; with the extension of enzymolysis time in 2 h to 8 h, protoplast yield reaches a maximum in 6 h, but its vitality achieves the maximum in 4 h; considering a combination of these two factors impacting on protoplast, we obtain the best time to digest for 4 h; meanwhile, with the increasing of the centrifugal force from 500 rpm - 2000 rpm, its comprehensive effect of protoplast vitality and yield is the highest when the centrifugal force is 1000 rpm for 2 min (replicated three times). So 0.4 M mannitol, 4 h enzymolysis time and 1000 rpm for 2 min centrifugal force are the best separation condition.