Isolation and culture of mesophyll protoplast from apricot

摘要

Yields of 10~6-10~7 apricot mesophyll protoplasts g fw~(-1) were obtained depending on factors such as plasmolysing pretreatment, digesting enzymes and digestion time. Onozuka R-10 (1 percent) in combination with Pectolyase Y-23 (0.1 percent) and Hemicellulase (1 percent) was found best for protoplast isolation among several enzyme combinations tested. Viability was 83 percent with this enzyme combination. Plasmolysis of leaves for 90 min in a 13 percent sorbitol solution greatly increased the number of protoplasts obtained. Optimum incubation time of 13-16 h produced the best combination of yield and viability. During the purification of protoplasts a critical step was the centrifugation of the sucrose gradient at 75 g with a dramatic decrease inthe recovery of protoplasts at lower and higher centrifugation speeds. Protoplasts were cultured under different media composition and growth regulator combinations. Limited growth and division of protoplasts embedded in agarose drops were obtained.