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Agriculture Optional Notes On – Somatic Hybridization – For W.B.C.S. Mains Examination.
The species barriers for plant improvement encountered in sexual hybridization can be overcome by somatic cell fusion that can form a viable hybrids. Somatic hybridization broadly involves in vitro fusion of isolated protoplasts to form a hybrid cell and its subsequent development to form a hybrid plant.Continue Reading Agriculture Optional Notes On – Somatic Hybridization – For W.B.C.S. Mains Examination.
Plant protoplasts are of immense utility in somatic plant cell genetic manipulations and improvement of crops. Thus, protoplasts provide a novel opportunity to create cells with new genetic constitution. And protoplast fusion is a wonderful approach to overcome sexual incompatibility between different species of plants.
Somatic hubridization involves the following aspects:
A. Fusion of protoplasts
B. Selection of hybrid cells
C. Identification of hybrid plants.
A. Fusion of Protoplasts:
As the isolated protoplasts are devoid of cell walls, there in vitro fusion becomes relatively easy. There are no barriers of incompatibility (at interspecific, inter-generic or even at inter-kingdom levels) for the protoplast fusion. Protoplast fusion that involves mixing of protoplasts of two different genomes can be achieved by spontaneous, mechanical, or induced fusion methods.
Spontaneous fusion:
Cell fusion is a natural process as is observed in case of egg fertilization. During the course of enzymatic degradation of cell walls, some of the adjoining protoplasts may fuse to form homokaryocytes (homokaryons). These fused cells may sometimes contain high number of nuclei (2-40).
This is mainly because of expansion and subsequent coalescence of plasmodermal connections between cells. The frequency of homokaryon formation was found to be high in protoplasts isolated from dividing cultured cells. Spontaneously fused protoplasts, however, cannot regenerate into whole plants, except undergoing a few cell divisions.
Mechanical fusion:
The protoplasts can be pushed together mechanically to fuse. Protoplasts of Lilium and Trillium in enzyme solutions can be fused by gentle trapping in a depression slide. Mechanical fusion may damage protoplasts by causing injuries.
Induced fusion:
Freshly isolated protoplasts can be fused by induction. There are several fusion-inducing agents which are collectively referred to as fusogens e.g. NaN03, high pH/Ca2+, polyethylene glycol, polyvinyl alcohol, lysozyme, concavalin A, dextran, dextran sulfate, fatty acids and esters, electro fusion. Some of the fusogens and their use in induced fusion are described.
Treatment with sodium nitrate:
The isolated protoplasts are exposed to a mixture of 5.5% NaNO3 in 10% sucrose solution. Incubation is carried out for 5 minutes at 35°C, followed by centrifugation (200 x g for 5 min). The protoplast pellet is kept in a water bath at 30°C for about 30 minutes, during which period protoplast fusion occurs. NaNO3 treatment results in a low frequency of heterokaryon formation, particularly when mesophyll protoplasts are fused.
High pH and high Ca2+ ion treatment:
This method was first used for the fusion of tobacco protoplasts, and is now in use for other plants also. The method consists of incubating protoplasts in a solution of 0.4 M mannitol containing 0.05 M CaCI2 at pH 10.5 (glycine-NaOH buffer) and temperature 3 7°C for 30-40 minutes. The protoplasts form aggregates, and fusion usually occurs within 10 minutes. By this method, 20-50% of the protoplasts are involved in fusion.
Polyethylene glycol (PEG) treatment:
This has become the method of choice, due to its high success rate, for the fusion of protoplasts from many plant species. The isolated protoplasts in culture medium (1 ml) are mixed with equal volume (1 ml) of 28-56% PEG (mol. wt. 1500-6000 Daltons) in a tube. PEG enhances fusion of protoplasts in several species. This tube is shaken and then allowed to settle. The settled protoplasts are washed several times with culture medium.
PEG treatment method is widely used protoplast fusion as it has several advantages:
i. It results in a reproducible high-frequency of heterokaryon formation.
ii. Low toxicity to cells.
iii. Reduced formation of bi-nucleate heterokaryons.
iv. PEG-induced fusion is non-specific and therefore can be used for a wide range of plants.
Electro-fusion:
In this method, electrical field is used for protoplast fusion. When the protoplasts are placed in a culture vessel fitted with micro- electrodes and an electrical shock is applied, protoplasts are induced to fuse. Electro-fusion technique is simple, quick and efficient and hence preferred by many workers.
Further, the cells formed due to electro-fusion do not show cytotoxic responses as is the case with the use of fusogens (including PEG). The major limitation of this method is the requirement of specialized and costly equipment.
Mechanism of fusion:
The fusion of protoplasts involves three phases agglutination, plasma membrane fusion and formation of heterokaryons.
1. Agglutination (adhesion):
When two protoplasts are in close contact with each other, adhesion occurs. Agglutination can be induced by fusogens e.g. PEG, high pH and high Ca2+.
2. Plasma membrane fusion:
Protoplast membranes get fused at localized sites at the points of adhesion. This leads to the formation of cytoplasmic bridges between protoplasts. The plasma membrane fusion can be increased by high pH and high Ca2+, high temperature and PEC, as explained below.
(a) High pH and high Ca2+ ions neutralize the surface charges on the protoplasts. This allows closer contact and membrane fusion between agglutinated protoplasts.
(b) High temperature helps in the intermingling of lipid molecules of agglutinated protoplast membranes so that membrane fusion occurs.
(c) PEG causes rapid agglutination and formation of clumps of protoplasts. This results in the formation of tight adhesions of membranes and consequently their fusion.
3. Formation of heterokaryons:
The fused protoplasts get rounded as a result of cytoplasmic bridges leading to the formation of spherical homokaryon or heterokaryon.
B. Selection of Hybrid Cells:
About 20-25% of the protoplasts are actually involved in the fusion. After the fusion process, the protoplast population consists of a heterogenous mixture of un-fused chloroplasts, homokaryons and heterokaryons (Fig. 44.5). It is therefore necessary to select the hybrid cells (heterokaryons). The commonly used methods employed for the selection of hybrid cells are biochemical, visual and cytometric methods.
Biochemical methods:
The biochemical methods for selection of hybrid cells are based on the use of biochemical compounds in the medium (selection medium). These compounds help to sort out the hybrid and parental cells based on their differences in the expression of characters.
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