Protoplast fusion and isolation

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Protoplast is defined to as the first organized body of a species. Frequently, protoplast is that unit of biology which is composed of a nucleus of the cell and the surrounding materials i.e. protoplasmic materials. Protoplast is used to study biology of the membrane, including up taking macromolecules.
Protoplasts are also used for transformational purposes of DNA. Plant cells, protoplasts may be made to regenerate into the plant, first by growing a group of plant cells that develops a callus and then by shoot regeneration's from the callus using PTC (Plant Tissue Culture) technique. This requires growth of protoplasts into callus and the proper balance of plant growth regulators in culture medium.
Isolation of Protoplast :-
Release of protoplast from plant tissue can be infer from one of two methods. The 1st method includes physical isolating the naked cells through dissection of the cell walls. The most common method deals with treating the plant tissue with enzymes such that it digest the cell wall material. Mostly used are combination of three enzymes:
• 0.5% Macroenzyme, Pectinase, to dissolve the tissue.
• 2 % Cellulase R10 and
• 0.1% Driselase, to dissolve the cell walls.

For the successful regeneration the starting plant material being used as a source of protoplasts proves to be very important. Plants able to be regenerated from protoplasts then only young shoots from tissue culture are used as the starting material. By centrifugation the enzymes are removed, alginate is used to embed the protoplast.
Agrose plated protoplast in liquid or in a medium solidifies did not develop. The medium containing macronutrient and micronutrients and high concentrations of different sugars is used to stabilize the naked protoplasts until the cell wall is reformed. Cell walls when formed, the cells began shows division after 8 to 10 days of growth in the dark phase .
The medium also contained two plant growth regulators:-
• 1 mg/liter naphthaleneacetic acid (an auxin);
• 1 mg/liter benzyladenine (a cytokinin).
The osmotic strength and the concentration of growth regulators was reduced after 14 days growth on the initial culture medium,. The osmotic strength had been reduced 10 days later. Application of agrose solidifies about 4 weeks of culture, then the small clumps of unorganized cells, or calli, could be removed and plated on a medium. These calli are made to grow in the dark until or unless they reached a definite size of 3 to 4 mm in diameter, and then, they are transferred to a new medium containing 2 mg/liter benzyladenine such that to induce plant formation.
The cultures were moved to the light and placed on a shoot elongation medium as soon as the young plants were visible under a stereomicroscope. The number of plants per callus, ranges between five and fifty. The green house is used for the plants rooted behaviour with few losses.
Fusion of Protoplasts –
Fusion of protoplasts is facilitated by mixing of two whole new genomes and could be done in between interspecific, intergeneric, or even interkingdom levels, which are not possible by conditional techniques due to incompatibility. Easy tools for undergoing fusion in vitro due to the fact that isolated protoplasts are devoid of walls.
Mixing the two genomes opens the door for gene transfer and clarifies the study of gene expression, stability of traits, and at micro levelthe genetic changes of the cell.

Protoplast fusion can be :-
1. Spontaneous
2. Induced.
Spontaneous Fusion:
In this the callus or cells which are grown in suspension are made to subject of the enzymatic degradations of the cell wall, then there is expansion of plasmodesmata instead of the break down, this facilitates the spontaneous fusion of the protoplasts to form the homokaryons. These plurinucleate cell contains 2-40 nuclie. Such type of protoplast fusion is usually seen when protoplasts are isolated from tissue cultures grown in vitro. This spontaneous fusion products don't regenerate into whole plant except undergoing a few divisional phases.

Fusion of protoplast
Induced Fusion: In this the protoplasts are made to fuse with .the help of an external agent such as mechanical force or fusing chemicals or electric charges. Induced fusion is reproducible and efficient. The fusion involving the mechanical means requires the protoplast of two different species, they are made to pass through micropipette, the tip of micropipette is blocked partially. This is the golden period at which protoplast comes to intimate contact of each other, thus retained and remain compressed by the flow of the liquid. The fusion doesn't depend upon the presence of inducing agent. In this method the protoplast are likely to be facing the injury.


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