Methods of Protoplast Fusion: Spontaneous and Induced Fusion

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.