Somatic Hybridization

Somatic Hybridization. In the past blogs, we started with an what is protoplast, and then we proceeded to protoplast isolation, and then we proceeded with Protoplast Culture: Isolation, in those blogs we studied one of the applications that said we can form hybrids from protoplast right? So in this blog, we are going through how Somatic Hybrids are made.

Somatic Hybridization

It is the fusion of isolated somatic protoplasts under in vitro conditions and subsequent development of their product (heterokaryon) to a hybrid plant known as somatic hybridization.

Somatic hybridization involves in vitro fusion of the isolated protoplasts to form a hybrid cell and its subsequent development to form a hybrid plant. (It is the invitro fusion of somatic cell protoplast of two different species to form a hybrid cell and further its development into the hybrid plant is called Somatic hybridization). In somatic hybridization, the nucleus and cytoplasm of both parents are fused to form the hybrid cell.

To make a somatic hybrid somatic hybridization involves the following stages:-

1. Fusion of protoplasts

2. Selection of hybrid cells

3. Identification of hybrid plants

In this blog, we will go through the Fusion of protoplasts

1. Fusion of protoplast= It involves the mixing of protoplasts of two different genomes. As the isolated protoplasts are devoid of cell walls, they're in vitro fusion becomes relatively easy. There are no barriers to incompatibility (at Interspecific, intergeneric, or even inter-kingdom levels) for the protoplast fusion. Protoplast fusion can be achieved by spontaneous, mechanical, or induced fusion methods as shown below.

a) Spontaneous fusion = Cell fusion is a natural process as is observed in the case of egg fertilization. During the course of enzymatic degradation of cell walls, some of the adjoining protoplasts may fuse together to form homokaryocytes (homokaryons). These fused cells may contain a high number of nuclei (2-40). This is mainly because of plasmodesma connections in between cells. As The frequency of homokaryon formation was found to be high in number it is of no value as the fusion of protoplasts of different origins is required in somatic hybridization.

b) Mechanical fusion= The protoplasts can be pushed together mechanically to fuse together. Protoplasts of Lilium and Trillium mixed in enzyme solutions can be fused by gentle trapping in a depression slide. Mechanical fusion might damage protoplasts by causing injuries.

c) Induced fusion= Protoplast of different origins are induced to fuse together, with the help of several fusion-inducing agents they are also called fusogens. The different fusogens used are NaNO3, high pH/Ca2*, Polyethylene glycol, electrofusion, etc.

1. Treatment with sodium nitrate: The isolated protoplasts are exposed to a mixture of 5.5% NaNO3, in 10% sucrose solution. The Incubation is carried out for 5 minutes at 35°C, followed by centrifugation. The protoplast pellet is kept in a water bath at 30°C for about 30 min, during which period protoplast fusion occurs. But treatment results in a low frequency of heterokaryon formation, so it is not much preferred.

2. Calcium ions and high pH: Isolated protoplasts are centrifuged for 3 minutes at 50x g in a fusion-inducing solution of 0.5 M mannitol containing 0.05 M CaCl2·2H2O at a pH of 10.5. The centrifuge tubes containing the protoplasts are then incubated in a water bath at 37°C for 40–50 minutes. After this treatment, 20%–50% of the protoplasts were involved in fusion.

3. Polyethylene glycol method (PEG): 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 a culture medium (.1 ml) are mixed with an equal volume (.1 ml) of 28-56% PEG. PEG treatment method is widely used for protoplast fusion as it has several gains:-

lt results in high-frequency of heterokaryon formation.

Low toxicity to cells.

Reduced formation of binucleate heterokaryons.

PEG-induced fusion is non-specific and so can be used for a wide range of plants.

4. Electrofusion: In this method, an electrical field is used for protoplast to fuse together . When the protoplasts are placed in a culture vessel fitted with microelectrodes and an electrical shock is applied, protoplasts are induced to fuse together. The Electrofusion technique is quick, simple, and efficient, and hence preferred by many workers. Furthermore, the cells formed due to electrofusion 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 types of equipment.

For your understanding

Mechanism of fusion of all the above-Induced fusion treatments= The fusion of protoplasts involves three phases such as agglutination, plasma membrane fusion, and formation of heterokaryons.

1) Agglutination (adhesion): When two protoplasts come in close contact with each other, adhesion occurs. Agglutination can be induced by fusogens like PEG, high pH, and high Ca2+.

2) Plasma membrane fusion at localized sites: The Membranes of protoplasts agglutinated by fusogen get fused at the point of adhesion. It results in the formation of cytoplasmic bridges between the two protoplasts.

Plant protoplasts carry a negative charge. Due to the common charge, the plasma membranes of two agglutinated protoplasts do not come close enough to fuse. Fusion requires that membranes must be first brought close together at a close distance. The high pH–high Ca++ ions treatment has been shown to neutralize the normal surface charge so that agglutinated protoplasts can come in intimate contact.

High temperatures help in promoting membrane fusion due to perturbance of lipid molecules in plasma membrane and fusion occurs due to the intermingling of lipid molecules in membranes of agglutinated protoplasts.

3) Formation of heterokaryon: Rounding off of the fused protoplasts due to the expansion of cytoplasmic bridges leading to forming spherical heterokaryon or homokaryon.

Summary

Conclusion

In this blog, we discussed one of the applications of protoplast isolation which is somatic hybridization. somatic hybridization has 3 stages in it but in this blog, we only went through the first stage which is a fusion of protoplast, which is further divided into 3 ways in which spontaneous and mechanical fusion are not mostly preferred while the induced fusion method for the fusion of protoplast with the help of various fusogens is the most successful methods for obtaining viable fused protoplast. The induced fusion method uses the method of overcoming the cell membrane barrier of a plant cell with the help of fusogens such as PEG (Polyethylene glycol) etc. We also discussed the mechanism of each and every fusogens in the induced fusion method.

Reference

Biotechnology by U Satyanarayana

H.S. Chawla

Somatic Hybridization