I have some suspensions and i want to isolate protoplast from it but on treatment with cellulase and macerozyme the cell wall is not getting ruptured properly. As a result I am not able to isolate protoplast from it. I have also tried to isolate protoplast from callus but the same problem persists. Although plasmolysis takes place but on digestion with enzymes cell wall is not removed. What should I do to solve this problem?
You can try using four layer mira cloth and then a two layer mira cloth along with wasing the protoplast with wash buffer. but remember getting the cell wall digestion is the key, try different duration for getting the cell wall digestion and follow with the above stated procedure
Thanks for reply Gazala but i have already tried different concentration of enzymes for different time intervals but protoplast is not obtained even after 48 hours
Hello Morris, I have used a gradient from 0.5-6% cellulase Onozuka RS, 0.5-2%(w/v) Macerozyme RlO (Yakult Pharmaceutical Industry Co. Ltd.) and 0.5% driselase (w/v) sigma aldrich . I have also used cell cultures which were 1,2,3,4,5 day old to find out the best possible time for harvesting my cells. I have even studied the growth kinetics of callus and enzymatically treated the callus at early log phase but all this is not working. Although the cells look healthy and rich in cytoplasm and shows plasmolysis but cell wall is not being removed. My plant is a dicot. I am using the protocol of Power and chapman (1985)
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