The following points highlight the two methods of protoplast fusion. They are: (1) Spontaneous Fusion and (2) Induced Fusion.
Method # 1. Spontaneous Fusion:
Protoplasts during isolation often fuse spontaneously and this phenomenon is called spontaneous fusion. Simply physical contact is sufficient to bring about the spontaneous fusion among the similar parental protoplasts.
During the enzyme treatment for the isolation of protoplasts, it is found that protoplasts from adjoining cells fuse through their plasmodesmata to form a multinucleate protoplast.
Electron microscopic studies have shown that as the cell wall are enzymatically degraded, the plasmodesmatal connection between the adjacent cells enlarge due to removal of its constriction and the enlargement of pit fields.
Eventually, the greater enlargement of plasmodesmata allows the entry of organelles into neighbouring cells. Finally a complete coalescence of adjacent cell takes place. Spontaneous fusion is strictly intraspecific and gives rise to homokaryon.
The protoplasts, once they are freely isolated, do not fuse spontaneously with each other. An exception is the protoplast from microsporocytes of some plants of lily family where the freely isolated protoplast fuses spontaneously. This type of spontaneous fusion has been used to produce inter generic fusion, e.g. the spontaneous fusion of microsporocyte protoplast of Lolium longiflorum and Trillium kamtschaticum.
Spontaneous fusion of two or more adjoining somatic protoplasts is of no practical use, but this may be important in studies of the nature and function of plasmodesmata, the physiology and control of mitosis in multinucleated cells and nuclear fusion. Perhaps spontaneous fusion has some practical importance for chromosome doubling.
Method # 2. Induced Fusion:
Fusion of freely isolated protoplasts from different sources with the help of fusion inducing chemical agents is known as induced fusion. Normally, isolated protoplasts do not fuse with each other because the surface of the isolated protoplast carries negative charge (-10 mV—30 mV) around the outside of plasma membrane and thus there is a strong tendency for protoplasts to repel one another due to their same charges. So this type of fusion needs a fusion inducing chemical agent or system which actually reduces the electro-negativity of the isolated protoplasts and allows them to fuse with each other.
Actually, induced fusion is a highly important and a valuable technique because the protoplast from widely different and sexually incompatible plants can be used by this procedure. This technique has the possibility and ability to combine different genotype beyond the limits imposed by sexual process. The fundamental objectives of somatic hybridization are mainly based on induced protoplast fusion.
The isolated plant protoplasts can be induced to fuse by three ways:
Mechanical Fusion:
In this process, the isolated protoplasts are brought into intimate physical contact mechanically under microscope using micromanipulator and perfusion micropipette. This micropipette is partially blocked within 1 mm of the tip by a sealed glass rod. In this way the protoplasts are retained and compressed by the flow of liquid. By this technique occasional fusion of protoplast has been observed.
Chemo-Fusion:
Several chemicals have been used to induce protoplast fusion. Sodium nitrate (NaN03), polyethylene glycol (PEG), Calcium ions (Ca2+), Polyvinyl alcohol etc. are the most commonly used protoplast fusion inducing agents which are commonly known as chemical fusogens. Generally, chemo-fusion techniques are followed in most of induced fusion experiments.
Chemical fusogens cause the isolated protoplasts to adhere to one another and leads to tight agglutination followed by fusion of protoplast (Fig 13.1). The adhesion of isolated protoplast takes place either due to reduction of negative charges of protoplast or due to attraction of protoplast by electrostatic forces caused by chemical fusogens.
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 (Fig 13.2). 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.
Chemo-fusion Procedures:
Several chemo-fusion procedures have been proposed time to time to improve the fusion frequency and reproducibility of the fused product. Each and every method has its own merits and limitations also.
Some chemo-fusion methods are described below:
(i) Fusion induced by Sodium or Potassium Nitrate:
Fusion of isolated onion sub-protoplasts plasmolysed with Sodium salts was achieved for the first time by Kiister (1909). Subsequently, Michel (1937) demonstrated fusion between protoplasts using potassium nitrate as plasmolyticum. Power et al. (1970) reported sodium nitrate induced fusion of cereal root protoplasts.
By this method, equal densities of protoplast from two different sources are mixed and then centrifuged at 100g for 5 minutes to get a dense pellet. This is followed by addition of 4 ml of 5.5% sodium nitrate in 10.2% sucrose solution to re-suspend the protoplast pellet. The suspended protoplasts are kept in water-bath at 35°C for 5 minutes and again centrifuged at 200g for 5 minutes.
The pellet is once again kept in water-bath at 30°C for 30 minutes. Fusion of protoplast takes place at the time of incubation. The pellet is again suspended by 0.1% sodium nitrate for 5-10 minutes. The protoplasts are washed twice with liquid culture medium by repeated centrifugation. Finally, the protoplasts are plated in semisolid culture medium.
Using the above principle, intra and interspecific fusions have been achieved by several workers. However, sodium nitrate is toxic to cell at fusogenic concentration. The frequency of fusion is not very high in this method. Yet it is useful only for the protoplasts derived from meristematic cells.
(ii) Fusion induced by Calcium ions at high pH:
In 1973, Keller and Melcher from Germany, developed a method to effectively induce fusion of tobacco protoplast at high temperature (37°C) in media containing high concentration of Ca2+ ions, (i.e. calcium chloride) at a highly alkaline condition (pH 10.5). Equal densities of protoplasts are taken in a centrifuge tube and the protoplasts are spun at 100g for 5 minutes.
The pellet is suspended in 0.5 ml of medium. 4 ml of 0.05M CaCl2, 2H2O in 0.4M mannitol at pH 10.5 is mixed to the protoplast suspension. The centrifuge tube containing protoplasts at high pH/Ca2+ is placed in the water bath at 30°C for 10 minutes and is spun at 50g for 3-4 minutes. This is followed by keeping the tubes in water bath (37° C) for 40-50 minutes. About 20-30% protoplasts are involved in this fusion experiment.
(iii) Fusion induced by PEG:
In 1974, Kao and Michayluk from Canada discovered another fusion inducing chemical polyethylene glycol (PEG) which is the most effective agent discovered so far. Many fusion experiments are performed by a polyethylene glycol. PEG induces protoplast aggregation and subsequent fusion. But the concentration and molecular weight of PEG are important with respect to fusion.
A solution of 37.5% w/v PEG of molecular weight 1,540 or 6,000 aggregates mesophyll and cultured cell protoplasts during a 45 minutes incubation period at room temperature. Fusion of protoplast takes place during slow elution of PEG with liquid culture medium. Carrot protoplast can be used by 28% PEG 1540 and the fusion can be promoted by Ca2+ ion at the concentration of 3.5 mM.
But higher concentration of Ca2+ ion (10 or 50 mM) has been considered beneficial. In some studies, high pH/Ca2+ and PEG method have been combined. By this method, the agglutination of protoplasts can be brought about using sufficient quantities (0.1-5 ml) of protoplast in centrifuge tube or micro-densities (150 n 1) of protoplast on a coverslip. The PEG method has been modified slightly to fuse higher plant protoplast.
The modifications are given below:
(a) PEG is more effective when it is mixed with 10-15% dimethyl sulfoxide (DMSo).
(b) Addition of concanavalin A (Con A) to PEG increases protoplast fusion frequency.
(c) Sea water has been used alone or in combination with PEG to fuse protoplasts.
(iv) Fusion induced by Other Chemicals:
Some other chemicals have also been observed to promote protoplast fusion:
(a) 15% solution of Polyvinyl alcohol (PVP) in combination with 0.05 CaCl2 and 0.3 M mannitol are used to fuse plant protoplasts.
(b) Lectins are also known to agglutinate protoplasts.
(c) Various proteins are also used for agglutination of protoplast.