Isolation and Purification of Mesophyll Protoplasts from <i>Ginkgo biloba</i> L.

Abstract

Techniques for protoplast isolation from various plants are widely applied. The purpose of this work is to develop an efficient system for isolating and purifying mesophyll protoplasts from a gymnosperm, Ginkgo biloba L. We tried to optimize main factors influencing the isolation of mesophyll protoplasts from G. biloba, including the enzyme type and concentration, incubation time, enzyme solution pH, osmotic pressure, nylon mesh size, and centrifugation speed. The suitable enzyme digestion system consisted of digestion by 2% (w/v) cellulase R-10, 0.2% (w/v) pectolyase Y-23 and 1.5% (w/v) macerozyme R-10 for 5 h in the dark at 25°C. After filtration through a 300-mesh nylon membrane and centrifugation at 50 G, the yield and viability of the obtained protoplasts reached approximately 5.39×106 protoplasts g−1 fresh weight (FW) and 80.23%, respectively, Then, the yield and viability of protoplasts isolated via the same method in two different years were compared under the optimal conditions. It proved that the protocol was repeatable and effective.