1. Introduction

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 described as living matter enclosed by a plant cell membrane. Protoplast isolation for the first time was carried out by Klercker in 1892 using mechanical method on the plasmolysed 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 (Bhojwani et al . 1977). 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:

• i.  regenerate a cell wall

ii.  dedifferentiate to form callus

iii.  divide mitotically and proliferate clonally

iv  redifferentiate into shoots, roots or embryos and produce a complete plantlet.

However, to fully explore the potentials for protoplast-technology, efficient and reproducible methods for protoplast isolation and purification must first be established. Since leaf tissue is a readily accessible source of genetically uniform cells, it is often desirable to use mesophyll protoplasts in somatic hybridization studies, but, leaf tissues, in general, do not yield large number of protoplasts owing to the difficulty in removing the lower epidermis (Chaturvedi 2003). An alternative, therefore, is the cultured cell material where protoplasts can show greater potential to divide (Bhojwani and Razdan 1996).

 

2.  Protoplast isolation

Protoplast isolation may be carried out by Mechanical disruption method or enzymatic method. Out of these two methods, 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 swell to force their way out of the cut surface. The released protoplasts then have to be separated from damaged ones and cell debris.

Disadvantages

• •  Lower protoplast yield.

•  Labour intensive method.

•  Protoplast obtained has low viability.

•  Method is applicable only to vacuolated cells.