The process of somatic fusion is also known as protoplast Fusion, is a kind of genetic modification of plants in which two plant species are merged into a hybrid plant that has the traits of both species the species, an somatic hybrid. Hybrids are created among different species that are of the exact same species (e.g. between non-flowering potato plant species and flowers-producing potato plant) and between distinct species (e.g. among wheat Triticum as well as Rye Secale to make Triticale).

Somatic fusion is a method of creating potato plants that are resistant to the potato leaf-roll disease. Through somatic fusion the plant that produces crop potatoes Solanum tuberosum whose production of which has been drastically decreased by a disease that is transmitted by the aphid vector is fused with wild, non-tuber-bearing, Solanum brevidens, invulnerable to disease. The hybrid that results has two chromosomes and, consequently, is like polyploid plants. The concept of somatic hybridization first discovered in the late 1970s by Carlson et al. in Nicotiana Glauca.

What is Protoplasts fusion?

Protoplast fusion is a natural phenomenon. In the process of fusion when two or more protoplasts come into contact to one another, either by themselves or when they are in contact with chemicals that induce fusion. Following adhesion, the membranes of protoplasts join in particular areas. Then, finally the cytoplasm of two protoplasts meld.

Methods of Protoplast Fusion

In general protoplast fusion could be divided into two groups:

1. Spontaneous fusion;
2. Induced fusion.

In the process of somatic hybridisation, spontaneous Fusion is of no significance. The techniques used to induce the fusion process can also be classified.

1. Spontaneous Fusion

Protoplasts in isolation frequently fuse in a spontaneous manner. This phenomenon is known as spontaneous fusion. A simple physical contact is enough to trigger spontaneous fusion between protoplasts that are similar to their parents. In the process of treating enzymes to isolate protoplasts, it is discovered that protoplasts of adjacent cells join by plasmodesmata, forming the multinucleate protoplast.

The studies using electron microscopy have demonstrated that when cell walls undergo enzymatic degradation the plasmodesmatal link between adjacent cells expands due to the elimination of constriction, as well as the expansion of pit fields.

The larger size of plasmodesmata allows organelles to be inserted into adjacent cells. Then, a complete coalescence of adjacent cells takes place. The spontaneous fusion process is completely intraspecific and leads to homokaryon.

The protoplasts, after they have been isolated in a free manner and isolated, don’t fuse spontaniously with one another. One exception is the protoplast of microsporocytes from some plants in the this lily family. The isolated protoplasts spontaneously fuse. This kind of spontaneous fusion was used to generate inter-generic-specific the fusion e.g. it is the spontaneous bonding of microsporocytes that is part of Lolium longiflorum, and Trillium kamtschaticum.

2. Induced Fusion

The fusion of protoplasts that are free and isolated from various sources using chemical agents that induce fusion is referred to as inducing Fusion. Normally isolated protoplasts cannot connect due to the fact that the surface of the isolated protoplast has a negative charge (-10 mV-30 mg) around the exterior of the plasma membrane . Therefore, there is a tendency for protoplasts to resent each other because of their similar charges. This type of fusion requires a producing chemical agent or system that actually decreases the electro-negativity of protoplasts that are isolated and permits them to fusion with each with each other.

In reality, induced fusion is an extremely important and useful technique since protoplasts from a variety of and sexually incompatible plants could be utilized in this method. This technique offers the possibility and capability to mix various genotypes outside of the boundaries set by the sexual processes. The primary goals of somatic hybridization is primarily built on the induced protoplast fusion.

The plant protoplasts that are not isolated are able to be made to fuse in three ways:

1. Mechanical Fusion

In this method the protoplasts isolated from the surrounding environment are brought into close physical contact under microscopes by using micromanipulator and a micropipette. This micropipette has been partially blockage within 1 mm of the tip with a sealed glass rod. This way, protoplasts remain in place and are compressed by the flow liquid. Through this method, the occasional fusion of protoplasts is observed.

2. Chemo-Fusion

Many chemicals have been employed to stimulate protoplast fusion. NaN03, sodium Nitrate (NaN03) as well as Polyethylene Glycol (PEG) as well as calcium Ions (Ca2+) and Polyvinyl alcohol. are the most frequently employed protoplast fusion inducers often referred to by the name chemical fusogens. The majority of the time, chemo-fusion strategies are used in the majority of the experiments that induce fusion.

Chemical fusogens cause isolated protoplasts to bind to one another. This leads to tight agglutination, followed by the fusion of protoplast. The adhesion between isolated protoplast occurs due to the decrease in negative charge of protoplast or the electrostatic attraction of protoplast forces induced from chemical fusogens.

Chemo-fusion Procedures

A variety of chemo-fusion techniques have been suggested from time to time in order to increase the frequency of fusion as well as the reproducibility of the fusion product. Every technique has its own strengths and drawbacks. A few chemo-fusion strategies are discussed in the following paragraphs:

(i) Fusion induced by Sodium or Potas­sium Nitrate

Fusion of onion sub-protoplasts plasmolysed by Sodium salts was accomplished first time by Kiister (1909). In the following years, Michel (1937) demonstrated the possibility of fusion between protoplasts by using potassium nitrate as a plasmolyticum. Power et al. (1970) published a report on sodium nitrate-induced fusion of protoplasts from cereal roots.

In this way, identical amounts of protoplast from two sources are mixed, and then centrifuged at 100g for five minutes to create a thick pellet. Then, you add to 4 ml 5.5 percent sodium nitrate in 10.2 percent sucrose solution in order to re-suspend the protoplast. The protoplasts that are suspended are placed in water baths at 35°C for 5 minutes before being centrifuged at 200g for five minutes.

The pellet is placed in the water-bath at 30°C during 30 minutes. The protoplast is fused during the incubation process. The pellet is then suspended with 0.1 percent sodium nitrate for 5-10 minutes. The protoplasts are rinsed two times with the liquid culture medium through repeated centrifugation. Then, the protoplasts get placed in semisolid medium.

Utilizing the principle above intra and interspecific fusions have been accomplished by a variety of researchers. However, sodium Nitrate is harmful to cells in fusogenic concentrations. The rate of fusion isn’t very significant in this process. However, it’s only useful for protoplasts that are derived from the meristematic cells.

(ii) Fusion induced by Calcium ions at high pH

The year 1973 was when Keller as well as Melcher from Germany created a technique to cause fusion of tobacco protoplasts at high temperatures (37degC) within media that contained a large amounts of Ca2+ ions (i.e. calcium chloride) in a highly alkaline state (pH 10.5). The protoplasts have equal density collected into a centrifuge tube. Then the protoplasts spin at 100g for 5 mins.

The suspension is placed in 0.5 milliliters of medium. 4 milliliters of 0.05M CaCl2, 2H2O, in 0.4M Mannitol at pH 10.5 is added into this protoplast suspension. The centrifuge tube with protoplasts that have high pH/Ca2+, is put in the bath of water at 30°C for 10 minutes and then spun at 50g for 3-4 mins. Then, it is kept in the tubes in the bath (37deg C) for between 40 and 50 minutes. Aproximately 20-30% protoplasts will be involved in this experiment to fusion.

(iii) Fusion induced by PEG

in 1974 Kao as well as Michayluk of Canada discovered a different fusion-inducing chemical called polyethylene glycol (PEG) that is the most effective chemical found so far. Numerous fusion experiments are carried out using the polyethylene glycol. PEG triggers protoplast aggregation and then the fusion. However, the concentration and molecular mass of PEG are crucial with regard to the process of fusion.

An solution of 37.5 percent w/v PEG that has molecular weight 1,440 (or 6,000) aggregates of mesophyll as well as proplasts of cultured cells during an incubation time of 45 minutes at the temperature of room. The protoplast is fused when slow elution occurs of PEG in the medium for liquid cultures. The protoplast of carrot can be used by 28 percent PEG 1540. This process of fusion is facilitated by Ca2+ ions at a concentration of 3.5 millimeters.

However, a higher concentration of Ca2+ ions (10 or 50 millimeters) has been deemed to be beneficial. In certain studies that have used high pH/Ca2+, the PEG methods were combined. Through this method, protoplasts’ agglutination can be achieved by with the use of sufficient amounts (0.1-5 mg) of protoplast in a centrifuge tubes or the microdensities (150 one) of protoplast on coverslip. This PEG procedure has been altered slightly to fuse plant protoplasts higher in the plant.

The modifications are given below:

• It is most effective when mixed with 10 to 15% dimethyl sulfuroxyde (DMSo).
• Concanavalin A (Con A) to PEG enhances the protoplast fusion rate.
• The sea water is utilized as a stand-alone or in conjunction with PEG to connect protoplasts.

Other chemicals have been discovered to stimulate protoplast fusion

• A 15% concentration of polyvinyl alcohol (PVP) along and 0.05 CaCl2 and 0.3 M mannitol is utilized to fuse protoplasts of plants.
• Lectins are also identified as having the ability to Agglutinate protoplasts.
• Different proteins are also utilized to agglutinate protoplasts.

3. Electro-Fusion

Recently, mild electrical stimulation is be­ing used to fuse protoplasts. This technique is known as electro-fusion of protoplasts. Two glass capillary microelectrodes are placed in contact with the protoplasts. An electrical field of low strength (10 kv m-1) gives rise to di-electrophoretic dipole generation within the protoplast sus­pension.

This leads to pearl chain arrangement of protoplasts. The number of protoplasts within the pearl chain depends upon the population density of the protoplast and the distance be­tween the electrodes. Subsequent, ap­plication of high intensity electric impulse (100 kv m-1) for some microseconds results in the electric breakdown of membrane and subsequent fusion.

Zimmermann and Scheurich (1981) im­proved the method for the large scale fusion of plant protoplast. There are indications that this electrical method may increase fusion frequency and reproducibility.

Recently, gentle electrical stimulation is being utilized to combine protoplasts. This method is called electro-fusion of protoplasts. Two glass capillary microelectrodes have been connected to protoplasts. A field of electrical force (10 kV m-1) causes di-electrophoretic dipoles within this protoplast suspension.

This results in a proplasts in a chain called pearl. There are many protoplasts in the pearl chain is dependent on the density of population in the protoplast as well as distance between electrodes. The subsequent application of a high-intensity electric impulse (100 kV 1 m-1) for a few microseconds causes the rupture of membranes and the subsequent the fusion.

Zimmermann and Scheurich (1981) developed a method for large-scale protoplast of plants fusion. There are signs that this method of electrical fusion could increase the frequency of fusion as well as reproducibility.

Mechanism of Protoplast fusion

Protoplast fusion comprises three primary phases:

Step I: Agglutination or adhesion:

• A protoplast or two are brought in close to each other.
• The adhesion can be caused through a range of treatments, e.g. concanavalin A, PEG high pH, and high Ca+ Ions.

Step II: Plasma membrane fusion at localized sites:

• Membranes of protoplasts that are stuck together by fusogen and then to fuse when they are adhesion-free, which results in the formation of cytoplasmic bridges connecting protoplasts.
• Plant protoplasts have the negative charge of 10 to -30 microvolts.
• Due to the common charge, plasma membranes in two protoplasts agglutinated do not meet enough to connect.
• The membranes be brought to each other, with the distance that is 10A and less.
• The high pH, high CaIon treatment been shown in neutralizing the usual charge on the surface to allow protoplasts that have agglutinated meet in close proximity.
• The high temperature causes membrane fusion because of the disturbance of lipid molecules within plasma membranes. Fusion occurs because of the intermixing of lipid molecules inside the protoplast membranes of agglutinated protoplasts.
• PEG Agglutinates to form protoplast clumps.
• Adhesion that is tight can be observed on a small or a large isolated location.
• Localized fusion between closely connected plasma membranes takes place in areas of tight adhesion. This result in the creation of bridges between cytoplasm and plasmosomes.
• It has also been thought that PEG which is positively polarized, can create hydrogen bonds with proteins, water carbohydrates, water, etc. that are positive in the direction of polarity.
• If the PEG chain of molecules is large enough, it functions as a molecular bridge protoplasts on the opposite sides and adhesion takes place.

Step III: Formation of heterokaryon:

• The rounding off of protoplasts fused by an expansion in cytoplasmic bridges that form homokaryon or spherical heterokaryon.

Characteristics of somatic hybridization

• Somatic cell fusion is believed to be the only method by the use of which two distinct parental genomes can be recombined between plants that can’t reproduce sexually (asexual or sexually sterile).
• Protoplasts of sexually-sterile (haploid triploid, haploid as well as aneuploid) plants can be fused to make fertile diploids as well as polyploids.
• Somatic cell fusion is a way to overcome the barriers to sexual incompatibility. In some cases , somatic hybrids of two plants are also being used in the fields of agriculture or in industry.
• Somatic cell fusion can be useful in studying the cytoplasmic gene and its functions and can be used in breeding plant experiments.

References

• Downey, R.K. (1993). [Advances in Agronomy] Volume 50 || Agronomic Improvement in Oilseed Brassicas. , (), 1–66. doi:10.1016/s0065-2113(08)60831-7 
• https://padeepz.net/protoplast-fusion-and-practical-applications-of-protoplasmic-fusion/
• https://biocyclopedia.com/index/biotechnology/plant_biotechnology/in_vitro_culture_techniques/biotech_protoplast_fusion_hybridization.php
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