The term protoplast was introduced by Hanstein in 1880. It refers to the cellular content excluding cell wall or can also be called as a naked plant cell. It is described as living matter enclosed by a plant cell membrane. Protoplast isolation for the first time was carried out by Klercker in 1892 using the mechanical method on the plasmolyzed cells. The application of protoplast technology for the improvement of plants offers fascinating option to complement conventional breeding programs. The ability of isolated protoplasts to undergo fusion and take up macromolecules and cell organelles offers many possibilities in genetic engineering and crop improvement.

The experiments involving protoplasts consist of three stages –
i. protoplast isolation
ii. protoplast fusion (leading to gene uptake)
iii. development of regenerated fertile plants from the fusion product (Hybrid).

Depending upon the species and culture conditions, the protoplasts may have the potential to:
• regenerate a cell wall
• dedifferentiate to form callus
• divide mitotically and proliferate clonally
• redifferentiate into shoots, roots or embryos and produce a complete plantlet

Protoplast isolation
Protoplast isolation may be carried out by Mechanical disruption method or enzymatic method. Out of these two methods, an enzymatic method is preferred as it provides better protoplast yield with low tissue damage while mechanical method causes maximum tissue chopping with lower protoplast yields. Both of these methods are described below:

i. Mechanical method
Klercker in 1892 pioneered the isolation of protoplasts by mechanical methods. In this method, the cells were kept in suitable plasmolyticum, for example, CPW containing 13% w/v mannitol. Once the plasmolysis is complete, while remaining in the osmoticum, the leaf lamina would be cut with a sharp-edged knife. In this process some of the plasmolyzed cells were cut only through the cell wall, releasing intact protoplasts while some of the protoplasts may be damaged inside many cells. Protoplasts that were trapped in a cell and only the corner had been cut off could be encouraged to come out by reducing the osmolarity slightly to force the protoplasts to swell to force their way out of the cut surface. The released protoplasts then have to be separated from damaged ones and cell debris.

• Lower protoplast yield.
• Labour intensive method.
• Protoplast obtained has low viability.
• Method is applicable only to vacuolated cells.

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 the harvest of protoplast. 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 enzymes 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 hydrolyze 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.