Somatic cell hyberdization is a type of genetic modification in plants. In this process a new hybrid plant produced after the fusion of two distinct species of plants. A new hybrid species produced consist of characteristic of both.
Hybrids have been produced either between different varieties of the same species or between two different species. The process of Somatic hybridization was first introduced by Carlson.
Somatic hybridization involves in vitro fusion of isolated protoplasts to form a hybrid cell and its subsequent development to form a hybrid plant.
The somatic hybridization involves three aspects. Thses are: (A) Fusion of Protoplasts (B) Selection of Hybrid Cells and (C) Identification of Hybrid Plants.
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.
- Fusion of protoplasts
- Selection of hybrid cells
- Identification of hybrid plants.
A. Fusion of Protoplasts:
As there is no cell walls in the isolated protoplasts, there in vitro fusion becomes relatively easy. There are no hurdle of incompatibility (at interspecific, inter-generic or even at inter-kingdom levels) for the protoplast fusion. Protoplast fusion involves mixing of protoplasts of two different genomes. The process of fusion can be achieved by spontaneous, mechanical, or induced fusion methods.
Cell fusion is a natural process as is observed in case of egg fertilization. During the time of enzymatic degradation of cell walls, it has been found the some of the adjoining protoplasts may fuse which leads to formation of homokaryocytes (homokaryons). These fused cells may sometimes contain high number of nuclei (2-40).The frequency of homokaryon formation was found to be high in protoplasts which are isolated from dividing cultured cells. Spontaneously fused protoplasts, however, cannot regenerate into whole plants, except undergoing a few cell divisions.
Mechanical fusion includes the forcing of protoplasts together to fuse. Protoplasts of Lilium and Trillium can be fused by gentle trapping in a depression slide in enzyme solutions. Mechanical fusion may cause injuries leads to damage of protoplasts.
Treatment with sodium nitrate:
Treatment of NaNO3 helps to lower the frequency of heterokaryon formation when mesophyll protoplasts are fused. In this process the isolated protoplasts are exposed to a mixture of 5.5% NaNO3 in 10% sucrose solution. The solution is then Incubated for 5 minutes at 35°C and centrifuged (200 x g for 5 min). After the centrifugation, the protoplast pellet is kept in a water bath for 30 minutes at 30°C, during which period protoplast fusion occurs.
High pH and high Ca2+ ion treatment:
This is other method of protoplast fusion in which the protoplasts in a solution of 0.4 M mannitol containing 0.05 M CaCI2 is incubated for 30-40 min.utes at 3 7°C. The pH of the solution is mantaintained at 10.5 using glycine-NaOH buffer. The protoplasts form aggregates, and fusion usually occurs within 10 minutes. By this method, 20-50% of the protoplasts are involved in fusion. This method was first used for the fusion of tobacco protoplasts, and is now in use for other plants also.
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:
- It results in a reproducible high-frequency of heterokaryon formation.
- Low toxicity to cells.
iii. Reduced formation of bi-nucleate heterokaryons.
- PEG-induced fusion is non-specific and therefore can be used for a wide range of plants.
In this method, fusion of protoplast takes place with the help of electrical field applied. In this method the protoplasts are kept in a culture vessel which is fitted with micro- electrodes and an electrical shock is applied due to which 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 includes three phases agglutination, plasma membrane fusion and formation of heterokaryons.
- 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+.
- Plasma membrane fusion:
Protoplast membranes get fused at localized sites at the points of adhesion. Due to which the cytoplasmic bridges are formed between the protoplast. The plasma membrane fusion can be increased by the high pH and high Ca2+, high temperature and PEC.
(a) The High pH and high Ca2+ ions concentration leads to neutralization of the surface charges on the protoplasts. This allows closer contact and membrane fusion between agglutinated protoplasts.
(b) High temperature leads to the intermingling of lipid molecules of agglutinated protoplast membranes so that membrane fusion occurs.
(c) PEG helps in 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:
Heterokaryon or homokaryon are produced due to the formation of cytoplasmic bridges after the fusion of protoplasts.