2.3.  Protoplast purification

Enzyme treatment results in suspension of protoplast, undigested tissues and cellular debris. This suspension is passed through a metal sieve or a nylon mesh (50-100 µm) in order to remove undigested cellular clumps. The filtered protoplast-enzyme solution is mixed with a suitable volume of osmoticum, solution is centrifuged to pellet the protoplasts, pellet of protoplast is resuspended in osmoticum of similar concentration as used in enzyme mixture. The protoplast band is sucked in Pasteur pipette and is put into other centrifuge and finally suspended in culture medium at particular density; this is explained by the Figure 12. 2.

 

 

 

Figure 12.2: schematic diagram of protoplast purification