Somatic Hybridization in Plant Biotechnology

Somatic hybridization is the process of creating hybrid plants by fusing isolated somatic (plant body/vegetal cells other than the reproductive cells) protoplasts in a lab setting. The resulting heterokaryon is then developed into a hybrid plant. A new hybrid cell with traits from both parent plants can be created by fusing the protoplasts of two separate plant cells using the somatic hybridization process.

The improvement of domesticated plants has long been accomplished by sexual hybridization between closely related species. Unfortunately, the majority of the time sexual hybridization is restricted to cultivars within a species or, at best, to a small number of closely related wild species. Thus, the value of sexual hybridization for agricultural enhancement is constrained by species barriers.

Somatic Cell Fusion

Somatic cell fusion is the joining of two separate cells' protoplasts to create functional cell hybrids. In the hybrid cell, the cytoplasm and nuclei of both parents are combined. Sometimes a fused hybrid, also known as a cybrid or cytoplasmic hybrid, contains the nuclear genome of just one parent but the cytoplasmic genes (plastome) of both parents. These hybrids produced by the fusion of somatic cells are an excellent approach to getting around any species' barriers to sexual hybridization.

Somatic Hybridization Technique

Protoplast fusion, choosing hybrid cells, and identifying hybrid plants are the three components of somatic hybridization.

Fusion of Protoplasts

Plant cells without a cell wall are known as protoplasts; nevertheless, they do have a plasma membrane. Both spontaneous and induced fusion techniques can be used to accomplish protoplast fusion, which includes combining protoplasts with two distinct genomes.

When protoplasts are isolated from callus cultures, the spontaneous fusing of the protoplasts is seen. However, aside from going through a few divisions, spontaneous fusion products do not regenerate into complete plants and must be triggered by a variety of methods.

Methods of Induced Fusion: To fuse plant protoplasts from various origins, an appropriate substance (fusogen) is introduced. The various fusogens used include lysozyme, high pH/Ca⁺⁺, NaNO₃, artificial seawater, polyethylene glycol (PEG), electrofusion, and others.

Fusion Mechanism

The three primary stages of protoplast fusion are as follows:

• Agglutination or Adhesion: Two or more protoplasts are brought together. Numerous treatments, such as PEG, high pH, and high Ca⁺⁺ ions, might cause adhesion.

• Plasma membranes of protoplasts that have been agglutinated by fusogen fuse at the point of adhesion.

• Plasma membrane fusion at limited sites cytoplasmic bridges is created between the protoplasts as a result.

• Rounding off of the fused protoplasts as a result of the expansion of cytoplasmic bridges results in the formation of spherical heterokaryon or homokaryon.

Selection of Hybrid Cells

Despite efforts to improve protoplast fusion efficiency, typically only 1 - 10% of the protoplasts in a treated population have undergone fusion. In order to choose the hybrid cells from this heterogeneous combination, procedures are developed. A few of them include:

• Complementary Auxotroph: Auxotrophs are mutants that are unable to thrive in a minimal environment. When two protoplasts harbouring distinct recessive/deficient markers fuse together, complementation results in a fusion product with functional restoration. Since the parental cells cannot grow in the minimum media, the hybrids can be chosen because they can grow there, while the parental cells cannot.

• Use of Metabolic Inhibitors: In this technique, parental cells are exposed to an irreversible biochemical inhibitor, such as iodoacetate or diethylpyrocarbonate and only hybrid cells are able to divide as a result.

• Use of Visual Characteristics: The most efficient but the most tedious method to select products of protoplast fusion is to visually identify hybrid cells and mechanically isolate individual cells. This includes the use of morphologically distinct cells, fluorescent labelling, etc.

Identification of Hybrid Plants

Hybrids that have been developed must be verified as products of somatic fusion of two different protoplasts. Some ways of identification are listed below:

• Morphology: When plant regeneration is achieved through protoplast fusion, the end products exhibit a variety of morphological characteristics. On them, hybrid verification can rely. Most of the time, the morphological traits of somatic or sexual hybrids fall somewhere in the middle between the two parents.

• Isoenzyme Analysis: Isoenzymes are the different molecular forms of the enzyme that catalyse the same reaction. Isoenzyme electrophoretic banding patterns have been widely employed to confirm hybridity. Somatic hybrids may exhibit isoenzyme bands of specific enzymes belonging to either one of the parents, or both parents, at once.

• Chromosomal Constitution: A quick and accurate way to confirm hybrid cells is by counting the number of chromosomes in the cells. Additionally, it reveals the ploidy condition of the cells.

• Molecular Techniques: Species-specific restriction fragments of nuclear DNA coding for ribosomal RNA have been shown to verify somatic hybrids. PCR technology has been utilised for hybrid identification.

Advantages

Beyond the boundaries of sexual crossability, it gives us the chance to create hybrids between taxonomically dissimilar plant species. Additionally, it produces cells with novel genomic, nuclear, and cytoplasmic constitutions that would not otherwise be possible.

Limitations

Only a few somatic hybrid plants have been used as commercial varieties, including Brassica spp and the potato. This is due to the fact that similar to sexual breeding, the more genetically distant the parents being crossed, the more chromosomally unstable and sterile are their offspring, often producing material that is unsuitable for breeding.

Applications

• Creation of brand new interspecific and intergeneric fusions between plants that are difficult or impossible to hybridise by traditional methods. It removes obstacles caused by sexual incompatibility.

• Disease Resistance: Somatic hybridization has allowed the spread of disease resistance genes from one plant to numerous others. Tomatoes now have the ability to resist a number of illnesses, including TMV, the spotted wilt virus, insect pests, and cold tolerance.

• Resistance to Abiotic Stress: Research on somatic hybridization for resistance to abiotic stress has been focused on the families Fabaceae, Brassicaceae, Poaceae, and Solanaceae and relates to cold and frost resistance.

• Cytoplasmically encoded features such as some forms of male sterility and specific antibiotic and herbicide resistance traits are among the traits that are useful for agriculture. Resistance to antibiotics, herbicides as well as CMS has been introduced in so many cultivated species.

Summary

In somatic hybridization, the cytoplasm and nuclei of both parents are combined to form a hybrid cell which has the traits of both the fused parent cells. Somatic cells can be generated by somatic hybridization which consists of 3 stages: protoplast fusion, selection of hybrid cells, and identification of hybrid plants. Hybrid plants produced by somatic hybridization enable the construction of hybrids between taxonomically distant plant species beyond the limits of sexual crossability. These hybrid plants have better qualities/traits including disease and abiotic stress resistance. Despite the benefits, only a few commercial varieties are available as the progeny obtained are sterile and chromosomally unstable.