Preparation and purification of protoplast from Nervilia fordii (Hance) Schltr. leaves.

[Objective] To study the preparation and purification method of protoplast from Nervilia fordii(Hance) Schltr.leaves.[Method] Taking the yield and survival rate of protoplast as index,the effects of different factors such as pretreatment,enzymolysis method,enzymolysis temperature,rotate speed of centrifugation,illumination,and sieve mesh number on the separation and purification of protoplast from N.fordii leaves were studied.[Result] The optimum process for protoplast isolation was obtained as follows: N.fordii leaves were first pretreated for 1 h in CPW solution containing 13% mannitol,and then incubated at(25±1)℃ in enzyme solution under dark conditions;after the enzymolysis,the enzyme liquid was filtrated through 200 mesh screen and centrifuged for 8 min at the speed of 800 r/min.Repeat the filtration and centrifugation once before microscopic examination.Under the above conditions,a large amount of complete and clear protoplast with few cell debris was obtained.[Conclusion] The preparation system has been successfully established and laid a good foundation for establishing protoplast regeneration system from N.fordii leaves.