What is Somatic Hybridization?

What is Somatic Hybridization?

Somatic hybridization is the process of creating a hybrid cell through the in vitro fusing of separate protoplasts, which can then grow into a hybrid plant. Sexual hybridization has long been the preferred strategy for enhancing the traits of domesticated plants. The primary drawback of sexual hybridization is that it can only take place between closely related plant species. This limits the modifications that may be made to plants.
By fusing somatic cells to create a viable hybrid, somatic cell fusion can get beyond the species barriers to plant improvement that are present in sexual hybridization. Somatic hybridization is the act of joining separate protoplasts together in a lab setting to produce a hybrid cell that will eventually grow into a hybrid plant.
Somatic hybridization involves three events. They are as follows:

• Protoplast fusion
• Hybrid Cell Selection
• Identifying hybrid plants

(A) Protoplast Fusion

Since isolated protoplasts lack cell walls, in vitro fusing of these structures is comparatively simple. For protoplast fusion, there are no obstacles to incompatibility (at interspecific, inter-generic, or even inter-kingdom levels). A protoplast fusion that includes combining protoplasts with two distinct genomes can be accomplished naturally, mechanically, or artificially.

• Spontaneous fusion: The process of cell fusion occurs naturally, as shown in the instance of egg fertilization. Some of the adjacent protoplasts may combine to produce homokaryocytes when the cell walls are being broken down by enzymatic means (homokaryons). There may occasionally be a large number of nuclei in these joined cells (2-40). This is mostly due to the growth and subsequent coalescence of cell-to-cell connections known as plasmodesma. It was discovered that protoplasts separated from dividing cultured cells frequently became homokaryons. However, spontaneously fused protoplasts cannot grow again into whole plants without passing through a few cell divisions.
• Mechanical fusion: The protoplasts can be mechanically pressed together to fuse. By gently trapping the protoplasts of Lilium and Trillium in an enzyme solution, they can be united. Protoplasts may sustain injuries as a result of mechanical fusing.
• Induced fusion: By induction, newly isolated protoplasts can merge. Fusogenic refers to a group of fusion-inducing substances, such as NaN0₃, high pH/Ca²⁺, polyethylene glycol, polyvinyl alcohol, lysozyme, concanavalin A, dextran, dextran sulfate, fatty acids, and esters, electrofusion, among others. There are descriptions of certain fusogenic and how they are used in induced fusion.
  Due to its numerous benefits, the PEG treatment approach is frequently utilized in protoplast fusion.

1. It causes the development of high-frequency heterokaryons, which is repeatable.
2. Low cell toxicity
3. Lessening of bi-nucleate heterokaryon production.
4. Because PEG-induced fusion is non-specific, it can be used for a variety of plants.

• Electro-fusion: This technique uses an electrical field to facilitate protoplast fusion. Protoplasts are made to fuse when they are put in a culture jar equipped with microelectrodes and given an electrical shock. The electro-fusion technique is popular among employees since it is easy, quick, and effective.  Additionally, unlike when fusogenic materials are used, cells created by electro-fusion do not exhibit cytotoxic reactions (including PEG). The need for expensive, specialized equipment is this method’s main drawback.

Fusion mechanism

Three processes are involved in the fusing of protoplasts: agglutination, the fusion of the plasma membrane, and the creation of heterokaryons.

1. Agglutination/Adhesion: When two protoplasts are brought into proximity by fusogenic substances such as polyethylene glycol (PEG) and NaNO₃, they stick together.
2. Plasma Membrane Fusion: At the location of adhesion, the protoplast’s membrane fuses, creating a cytoplasmic bridge that connects the two protoplasts. The pace of membrane fusion can be accelerated by high pH and Ca²⁺ concentration.
3. Formation of Heterokaryons: A spherical homokaryon or heterokaryon is formed when the united protoplasts circle up.

(B) Hybrid Cell Selection 

A heterokaryon is produced by the fusion of just 20–25% of the protoplasts. The combination is made up of unfused protoplasts, heterokaryons, and homokaryons. From this varied mixture, methods are developed to choose the hybrid cells. Three choices are available for selection:

1. Biochemical technique: This technique separates the fused cells from the unfused cells using biological substances. There are two approaches.

• Drug sensitivity: In this procedure, one protoplast is antibiotic-resistant, preventing the other protoplast from growing in its presence. For instance, if protoplast 1 is resistant to actinomycin D but protoplast 2 is not, the fused protoplast will acquire the traits of both following unions. Protoplast 2 will not be able to develop, fused protoplasts will, and protoplast 1 produces little colonies that can be recognized and separated when the cells are cultivated on a medium containing the antibiotic.
• Mutants with auxotrophy: Mutants known as auxotrophs are unable to develop on a minimum media. The parental cells cannot grow in the minimum media, however, the hybrids can, allowing for cell selection.

2. Visual technique: Since the hybrid cells must be chosen physically and visually using this procedure, it is quite time-consuming. Using this technique, cells that develop on various mediums are combined and then visually separated. Another approach involves manually dividing the hybrid cells using a Drummond pipette. 
3. Cytometric technique: For simple cell selection, modern techniques like flow cytometry and fluorescent cells are used.

(C) Identifying Hybrid Plants

The initial somatic fusion of two distinct protoplasts must be confirmed as the source of any produced hybrids. The following list includes several methods of identification:

• Morphology: Plant regeneration is accomplished by protoplast fusion, and the resulting organisms display a range of morphological traits. Hybrid verification can rely on them. The physical characteristics of somatic or sexual hybrids often lie halfway between the two parents.
• Isoenzyme Analysis: The many molecular forms of the enzyme that catalyze the same reaction is known as isoenzymes. It is common practice to use isoenzyme electrophoretic banding patterns to establish hybridity. Somatic hybrids may display isoenzyme bands of certain enzymes that belong to both parents simultaneously and only one of the parents individually.
• Chromosomal Constitution: Counting the number of chromosomes in the cells is a rapid and accurate approach to determine whether they are hybrid. It also demonstrates the ploidy state of the cells.
• Molecular Techniques: Somatic hybrids can be confirmed using species-specific restriction pieces of nuclear DNA that code for ribosomal RNA. Hybrid identification has been carried out using the PCR technique.

Applications of Somatic Hybridization

• The development of novel interspecific and intergeneric fusions between plants that are challenging or impossible to hybridize using conventional techniques. It eliminates challenges brought on by sexual incompatibility.
• Research on somatic hybridization for abiotic stress tolerance has been concentrated on the families Fabaceae, Brassicaceae, Poaceae, and Solanaceae and is related to cold and frost resistance.
• The qualities that are helpful for agriculture include those that are cytoplasmically encoded, such as some types of male sterility and particular antibiotic and herbicide resistance traits. So many farmed species now exhibit resistance to antibiotics, herbicides, and CMS.
• Disease Resistance: Disease-resistance genes have been able to spread from one plant to many others thanks to somatic hybridization. The spotted wilt virus, TMV, insect pests, and cold tolerance can all no longer harm tomatoes.

Advantages of Somatic Hybridization

• You can do somatic hybridization on young, immature plants.
• It is now simple to research cytoplasmic genes and how they work.
• Environmental tolerance – Somatic hybridization was used to transfer genes into, for instance, tomatoes, which provide tolerance for cold, frost, and salt.
• Selective somatic hybrids with quality traits, such as the generation of high nicotine concentration, have been created.
• In the hybrid cell, the protoplast fusion results in a distinctive nuclear-cytoplasmic combination.
• It produces novel plants with desirable traits.
• An alternate technique for creating remote hybrids with desirable qualities considerably across species or genera, which cannot be achieved by the traditional approach of sexual hybridization, is somatic cell hybridization, parasexual hybridization, or protoplast fusion.

Disadvantages of Somatic Hybridization

• The created plants aren’t always healthy and fruitful.
• The regenerated plants produced through somatic hybridization occasionally display diversity for a variety of causes, including soma clonal variations, chromosomal elimination, organelle segregation, etc.
• However, it does not necessarily result in the production of viable seeds, making the fusion of distant plant genera feasible.
• Genetic instability can occasionally result from protoplast fusion.
• The effective manifestation of a certain characteristic is not necessarily ensured by somatic hybridization.
• There are only a few selection criteria.

FAQs on Somatic Hybridization

Q1. Define somatic hybridization.

Ans. Somatic hybridization is the process of fusing different protoplasts in vitro to produce a hybrid cell, which can ultimately develop into a hybrid plant. For centuries, the primary method for improving the characteristics of domesticated plants has been sexual hybridization. 

Q2. Mention the name of stages by which a somatic cell is formed.

Ans. Protoplast fusion, hybrid cell selection, and plant identification are the three steps in the process of somatic hybridization, which produces somatic cells.

Q3. Write any two limitations for somatic hybridization.

Ans. Two limitations for somatic hybridization:

1. Somatic hybridized plants that have undergone regeneration occasionally exhibit diversity due to several factors, such as somatic clonal variants, chromosomal elimination, organelle segregation, etc.
2. Somatic hybridization does not always guarantee the efficient expression of a certain trait.

Q4. Write the methods that we used to identify the hybrid plant.

Ans. Any created hybrids must have the original somatic fusion of two different protoplasts as their source validated. The following list covers several identifying techniques:

1. Morphology
2. Isoenzyme Analysis
3. Chromosomal Constitution
4. Molecular Techniques

Q5. Write about electro-fusion.

Ans. In this method, protoplast fusion is facilitated by an electrical field. When protoplasts are placed in a culture jar with microelectrodes and given an electrical shock, the protoplasts fuse. Employees favor the electro-fusion approach because it is simple, quick, and efficient. Additionally, cells produced via electro-fusion do not display cytotoxic effects, in contrast to when fusogenic ingredients are utilized (including PEG). The biggest disadvantage of this approach is the requirement for pricey, specialized equipment.