Plant protoplasts represent the finest single cell system and offer exciting possibilities in the fields of somatic cell genetics and crop improvement. Isolated protoplasts serve as an excellent starting material for cell cloning and development of mutant lines in vitro than single whole cells. They also provide experimental material for many other fundamental and applied studies. Freshly isolated protoplasts have been employed in studies related to cell wall synthesis, membrane properties and virus infection. However, the feature of isolated protoplasts that has brought them into the limelight is the ability of these naked cells to fuse with each other irrespective of their origin.
Protoplast fusion has opened up a novel approach to raising new hybrids. This technique of hybrid production through the fusion of body cells, bypassing sex altogether, is called somatic hybridization. Unlike sexual reproduction in which organelle genomes are generally contributed by the maternal parent, somatic hybridization also combines cytoplasmic organelles from both the parents. In somatic hybrids recombination of mitochondrial genomes occurs frequently. Chloroplast genome recombination is rare but segregation of chloroplast of the two sources in hybrids causes selective elimination of chloroplasts of one or the other parent, forming novel nuclear-cytoplasmic combinations.
Fusion products with the nucleus of one parent and extra-nuclear genome/s of the other parent are referred to as hybrid and the process to obtain cells or plants with such genetic combination/s is called hybridization. Somatic cell fusion, thus offers new ground to achieve novel genetic changes in plants.
The technique of somatic hybridisation involves the following steps.
(i) Isolation of protoplasts:
Plant cell consists of cell wall which has to be degraded if the protoplasts of the cell have to be obtained to be manipulated as required. For this purpose, the plant cell is treated with enzyme-like pectinase, macerozyme, and cellulase etc., which hydrolysis the plant cell wall. The conditions are altered so that the successful release of protoplast is aided. The osmotic pressure of the solution is controlled by the addition of calcium chloride salts into it. This improves the plasma membrane activity. Since protoplasts are present in every plant cell it can be theoretically isolated from all the parts of the plant. But most successful isolation was made possible from the leaf of the plants. The leaf is surface sterilized and lower epidermis is removed and treated with enzyme solution.
(ii) Protoplast Fusion
During enzymatic degradation of cell walls, some of the adjacent protoplasts fuse together forming homokayons (also referred to as homokaryocytes, each with two to several nuclei). This type of protoplast fusion, called ‘spontaneous fusion’, has been ascribed to the expansion and subsequent coalescence of the plasmodesmatal connections between the cells. The occurrence of multinucleate fusion bodies is more frequent when protoplasts are prepared from actively dividing cultured cells. About 50% of the protoplasts prepared from callus cells of maize endosperm and suspension cultures of maize embryos were multinucleate.
A sequential method of protoplast isolation, or exposing the cells to strong plamolyticum solution before treating them with mixed enzyme solution would sever the plamodematal connection and, consequently, reduce the frequency of spontaneous fusion. So far as somatic hybridization and cybridization are concerned spontaneous fusion is of no value; these require the fusion of protoplasts of different origin. To achieve induced fusion a suitable chemical agent (fusogen) or electric stimulus is generally necessary. Since 1970 a variety of fusogens have been tried to fuse palnt protoplasts of which NaNO3 , high pH and high Ca2+, and polyethylene glycol treatments have been suceessfully used to produce somatic hybrid/cybrid plants. During the last decade fusion of protoplasts by electric stimulus (electrfusion) has gained increasing popularity.
(i) NaNO3 treatment:
Equal densities of protoplasts 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 Resuspend the protoplast pellet. The suspended protoplasts are kept in water – bath at 35ºC for 5 minutes and again centrifuged at 200 g for 5 minutes. The pellet is once again kept in water bath at 30°C for 30 minutes. The fusions of protoplast take place at the time of incubation. Finally, the protoplasts are plated in semisolid culture medium. The frequency of fusion is not very high in this method. Induced fusion by NaNO3 was first demonstrated by Power et al (1970). Isolated protoplasts were cleaned by floating in sucrose osmoticum. Transfer of the protoplasts in 0.25M NaNO3 solution and subsequent centrifugation promoted the fusion process. This procedure results in a low frequency of heterokaryon formation and protoplasts are markedly altered in their uptake capabilities.
(ii) High pH and high Ca++ Treatment:
Kelier and Melchers (1973) developed a method to effectively induce fusion of tobacco protoplasts at a high temperature (37°C) in media containing high concentration of Ca++ ions at a highly alkaline condition (pH 10.5). Equal densities of protoplasts are taken in centrifuge tube and protoplasts are spun at 100 g for 5 minutes. The pellet is suspended in 0.5 ml of medium. 4 ml of 0.05 M CaCl2, 2H2O in 0.4 M mannitol at PH 10.5 is mixed to the protoplast suspension. The centrifuge tube containing protoplast at high PH or Ca++ is placed in water bath at 30°C for 10 minutes and is spun at 50 g for 3 to 4 minutes. This followed by keeping the tubes in water bath (37°C) for 40- 50 minutes. About 20-30% protoplast are involved in this fusion experiment. This method was developed by Keller and Melchers (1973) for fusing two different lines of tobacco protoplasts. Isolated protoplasts are incubated in a solution of 0.4M mannitol containing 0.05M CaCl2, with pH at 10.5 (0.05 M glycine – NaOH buffer) and temperature 370C. Aggregation of protoplasts generally takes place at once and fusion occurs within 10min. Many intraspecific and interspecific somatic hybrids have been produced using this procedure.
(iii) Polyethylene glycol treatment:
PEG has been used as a fusogen in a number of plant species because of the reproducible high frequency of heterokaryon formation. About 0.6ml of PEG solution is added in drops to a pellet of protoplasts in the tube. After having capped the tube, protoplasts in PEG are incubated at room temperature for 40min. Occasional rocking of tubes helps to bring the protoplasts in contact. This is followed by elution of PEG by the addition of 0.5-1 ml of protoplast culture medium in the tube after every 10min. Preparations are now washed free of fusogen by centrifugation and the protoplasts resuspend in the culture medium. After treatment with fusogen, protoplasts are cultured following the standard procedures. PEG either provides a bridge by which Ca++ can bind membrane surfaces together or leads to a disturbance in the surface charge during the elution process.
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 1500 to 6000 aggregates mesophyll and cultured cell protoplasts during a 45 minutes incubation at room temperature. Fusion of protoplast takes place during slow elusion of PEG with liquid culture medium. Carrot protoplast can be fused by 28% PEG 1500 and fusion can be promoted by ca++ ion at concentration of 3.5 mM. But higher concentration of Ca++ ion has been considered beneficial. In some studies, high PH/Ca++ and PEG method have been combined.
Recently, mild electrical stimulation is being used to fuse protoplasts. This technique is known as electrofusion of protoplasts. Two glass capillary microelectrode are placed in contact with the protoplasts. An electric field of low strength (10kv m-3) give rise to dielectrophoretic pole generation within the protoplast suspension. This lead to pearl chain arrangement of protoplasts.
The number of protoplasts within a pearl chain depends upon the population density of the protoplast and the distance between the electrodes. Subsequent application of high intensity electric impulse (100kv m-3) for some microseconds results in the electric breakdown of membrane and subsequent fusion.
After the protoplast fusion, heterokaryons (a cell formed by fusion of two or more protoplasts of different species and the cell contains individual nucleus from both the species) have to be selected from the protoplast population that comprises of homokaryons and heterokaryons.
One such approach used for the selection of heterokaryons involves the culturing of hybrids on such a medium that favors the growth of hybrids and restricts the growth of parent cells.
Another approach involves the selection of hybrids in the form of green callus (this approach was used for selction of Datura hybrids). Staining the protoplasts with fluorescent dyes of different colors such as red and green allows easy visual selection of hybrid varieties.
Some researchers have used selectable antibiotic or herbicide-resistant markers to identify the hybrid cell. Another powerful approach involves the culturing of the entire protoplast population on a suitable growth medium (devoid of any selection marker) under appropriate environmental condition followed by identification of hybrid calli based on protein banding patterns, chromosome constitution, etc.