ii. Enzymatic method

In 1960, E.C. Cocking demonstrated the possibility of enzymatic isolation of a large number of protoplasts from cells of higher plants. This method involves leaf sterilization followed by peeling of the lower epidermis to release cells which are plasmolyzed and added to enzyme mixture followed by harvest of protoplast as shown in (Figure 12.1). Either of the procedures for enzymatic isolation can be used: sequential enzymatic hydrolysis or mixed enzymatic hydrolysis.

In the sequential isolation, firstly, cells are separated by the use of a maceration enzyme – a pectin hydrolyzing enzyme such as, macerozyme or Pectolyase. Once the cells are separated, they are washed in CPW solution free of enzymes but containing plasmolyticum by gentle centrifugation (100g). The pellet is retained and resuspended in the second enzyme like, cellulases and hemicellulases, used to hydrolyse the remaining cell was component. Once the protoplasts are released they are washed with CPW to remove the debris.

In the mixed enzymatic approach, Plant tissues are plasmolyzed in the presence of a mixture of pectinases and cellulases, thus, inducing simultaneous separation of cells and degradation of their walls to release the protoplasts directly in a single step.

 

 

Figure 12.1: Steps involved in protoplast isolation, fusion and regeneration