The term protoplast was introduced by Hanstein in 1880. It refers to the cellular content excluding cell wall or can also be called as naked plant cell. It is the living matter enclosed by a plant cell membrane. The first isolation of protoplasts was achieved by Klercker (1892) employing a mechanical method. The application of protoplast technology for the improvement of plants offers fascinating option to compliment 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 protoplast consist of three stages :
I. Protoplast isolation
II. Protoplast fusion
III. Development of regenerated fertile plants from the fusion product
I. Protoplast Isolation
Protoplasts are naked plant cells without cell wall, but they have plasma membrane and all other cellular components. They represent the functional plant cells but for the lack of the barrier, cell wall. Protoplasts of different species can be fused to generate a hybrid and this process is referred to as somatic hybridization (or protoplast fusion).
Protoplasts are isolated by
- Mechanical Method
- Enzymatic Method
1. Mechanical Method
Klercker in 1892 pioneered the isolation of protoplast by mechanical methods. In this method a small piece of epidermis from a plant is selected. The cells are subjected to plasmolysis. This causes protoplasts to shrink away from the cell walls. The tissue is then dissected to release the protoplasts.
- It is restricted to certain tissues which have large vacuolated cells.
- Yield of protoplasts is generally very low.
- The method is tedious and laborious.
- Viability of protoplasts is low because of the presence of substances released by damaged cells.
2. Enzymatic Method
Cocking in 1960 demonstrated the possibility of enzymatic isolation of protoplasts from higher plants. Mechanical isolation method of protoplast are no more practically used. Protoplasts are routinely isolated by treating tissues with the mixture of cell wall degrading enzymes in solution, which contain osmotic stabilizer. The method involves the following steps.
- Sterilization of leaves
- Peeling off lower epidermis
- Incubation in enzyme solution
- Isolation and cleaning of the protoplast
The release of protoplasts is very much dependent on the nature and composition of enzymes used to digest the cell wall. There are three primary components of the cell wall which have been identified as cellulose, hemicelluloses and pectin substances. Cellulose and hemicelluloses are the components of the primary and secondary structure of cell wall, while pectin is a component of middle lamella that joins the cells. Pectinase mainly degrades the middle lamella, while cellulose and hemicellulose are required to digest the cellulosic and hemicellulose components of the cell wall respectively.
Different enzymes preparations are available in the market by the idea is to combine one middle lamella dissolving and one cell wall digesting enzymes in proper composition to achieve maximum protoplasts release from one gm material.
Following enzymes are used:
- Macerozymes R-10
- Cellulase- Onuzuka R-10
A combination of these enzymes in a concentration of 0.5 – 2 % is used. In many cases only macerozyme and cellulose are sufficient to obtain protoplasts in significant number.