First, the composition of the enzyme solution:
1 Cellulase 0.7% Pectinase 0.5% Sorbitol 14% Calcium chloride 5 mmol•L-1, pH 5.8
2. Composition of rinse solution: sorbitol 14%, potassium nitrate 1 mmol•L-1, potassium dihydrogen phosphate 0.2 mmol•L-1, calcium chloride 0.5 mmol•L-1,
Copper sulfate 0.01 μmol•L-1
Third, the material pretreatment: take the yellow leaves, to the midrib, put in 25% sucrose solution for 15 ~ 20min, so that the occurrence of plasmolysis,
Then, it is used for enzymolysis 4. After two times of enzymolysis, after the second enzymolysis is finished, the leaves are taken out and placed in the rinsing solution. The rinsing shake causes the protoplasts to release, and then the rinsing solution is centrifuged at 1500 rpm for 5 min. For protoplasts, experiments have shown that the highest protoplast yield is obtained at a centrifugal force of 2500 rpm for 5 min.