Protoplast culture Protoplast is a cell without

Protoplast culture

Protoplast is a cell without cell wall. In plants, the cells are invariably surrounded by thick cell wall made of cellulose and pectin. During the process of tissue culture and using it for crop improvement, cell wall acts as a barrier to induce mutations or some times to develop the callus.

Methods of protoplast isolation: l mechanical; l enzymatic.

Mechanical method of protoplasts isolation Cut plasmolyzed tissue and subsequent deplasmolysis results in expansion and release of the protoplasts from the cut ends of the cell. In practice this technique is difficult and the yield of viable protoplasts is meager. One advantage, however, is that the deleterious effects of the wall-degrading enzymes on the metabolism of the protoplasts are eliminated.

Enzymatic method of protoplasts isolation: Cellulase enzymes digest the cellulose in plant cell walls while pectinase enzymes break down the pectin holding cells together. Once the cell wall has been removed the resulting protoplast is spherical in shape. Digestion is usually carried out after incubation in an osmoticum (a solution of higher concentration than the cell contents which causes the cells to plasmolyse). This makes the cell walls easier to digest.

Enzymatic method of protoplasts isolation: Osmotics are: l High concentrations of sugars; l Sorbitol; l Mannitol.

Protoplasts can be isolated from a range of plant tissues: l Leaves; l Stems; l Roots; l Flowers; l Pollen;

There are few methods of protoplasts cultivation: l l l Hanging microdrops; Nurse culture; Suspension of protoplasts; Plating method; Cultivation on a “nutrient layer”; Cultivation of immobilized protoplasts.

Protoplasts fusion and somatic hybridization With the development of techniques for enzymatic isolation of protoplasts and subsequent regeneration, a new tool of genetic manipulation of plants has now become available. Moreover, the fusion of protoplasts of genetically different lines or species has also been possible. For example, some plants that show physical or chemical incompatibility in normal sexual crosses, may be produced by the fusion of protoplasts obtained from two cultures of different species. Somatic hybridization of crop plants represents a new challenge to plant breeding and crop improvement.

Protoplast fusion can be broadly classified into two categories: l l Spontaneous fusion Induced fusion: Fusion of freely isolated protoplasts from different sources with the help of fusion inducing chemicals agents is known as induced fusion. Normally isolated protoplast do not fuse with each other because the surface of isolated protoplast carries negative charges (-10 m. V to -30 m. V ) around the outside of the plasma membrane. So this type of fusion needs a fusion inducing chemicals which actually reduce the electronegativity of the isolated protoplast and allow them to fuse with each others.

The isolated protoplast can be induced to fuse by three ways: 1. Mechanical fusion: In this process the isolated protoplast are brought into intimate physical contact mechanically under microscope using micromanipulator or perfusion micropipette.

2. Chemofusion: Several chemicals has been used to induce protoplast fusion such as sodium nitrate , polyethylene glycol (PEG), Calcium ions (Ca++ ). Chemical fusogens cause the isolated protoplast to adhere each other and leads to tight agglutination followed by fusion of protoplast

3. Electrofusion: Subsequent application of high strength of electric fields (100 kvm-1) for some microseconds results in electric breakdown of membrane and subsequent fusion