Protoplast Fusion

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

Spontaneous Fusion or Natural Fusion of Protoplasts

The process of cell fusion occurs naturally as in the process of egg fertilization. Some of the neighbouring protoplasts may combine to produce homokaryocytes when the cell walls are being broken down by enzymes (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 joining of cell-to-cell connections known as plasmodesmata. 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 of Protoplasts

The protoplasts can be mechanically pressed together to fuse. For example, by gently trapping the protoplasts of Lilium and Trillium in an enzyme solution, they can be united. Although protoplasts may get injured as a result of mechanical fusing.

Induced Fusion of Protoplasts

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 polyethylene glycol (PEG) treatment approach is often utilized in protoplast fusion:

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

Electro-Fusion of Protoplasts

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 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 major drawback of this method is its need for expensive and specialized equipment.

Fusion Mechanism of Protoplasts

Three processes are involved in the fusing of protoplasts:

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.

Somatic hybridization is a technique of fusing protoplasts from different plant species to create hybrid plants. It is different from conventional ways involving sexual hybridization because it does not need sexual reproduction. Instead, it combines traits from different plants without being limited by species barriers. In this article, we will cover somatic hybridization notes, steps, and its applications.