2.2.  Choice of plasmolyticum

The two most commonly used compounds are the sugar alcohols - mannitol and sorbitol. Of these, mannitol is the most preferred since it is not metabolized by the plant cells. Once the protoplast divides and regenerates the cell wall, no more osmoticum is required. It is, therefore, should be removed gradually from the medium otherwise cell division stops. To slowly remove the osmoticum from the medium, the protoplast can be isolated in a high osmoticum mixture consisting of both mannitol and sucrose, the sucrose will be metabolized by the dividing protoplasts and thus, will reduce the osmolarity of the medium. Normally, mannitol is used at concentration range of 11-13%.

A solution into which the osmoticum is often, but not always, added is called CPW salts mix or CPW for short. This has been observed much more beneficial than using distilled water as a solvent in obtaining high yields of viable protoplasts:

 

Table 12.1: Salt mix of protoplast washing media solution (Cocking, Peberdy and White – CPW)

Although CPW is most widely used solution into which osmoticum or enzymes are added, some times culture medium used to grow cells or plants can also be utilized for protoplast isolation at one tenth concentration. Low concentration of culture medium is much more advantageous when compared with CPW.