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ABSTRACT

The technique of fusion of isolated protoplasts from somatic cells and regeneration of hybrid plants from the fusion products is called somatic hybridization which completely bypasses the sex and allows combining the genomes of two desirable parents, irrespective of their taxonomic relationship. The hybrid cells that are formed from the fusion of two unrelated protoplasts combines a set of three genomes from the parents, viz. nuclear genome, mitochondrial genome, and plastid genome. Applications of somatic hybridization in crop improvement are constantly evolving. However, it must be appreciated that genomic incompatibility following protoplast fusion continues to be a serious drawback in somatic hybridization.

Plant somatic hybridization through protoplast fusion is an important tool in plant improvement, which allows researchers to combine somatic cells (whole or partial) from different cultivars, species or genera resulting in novel genetic combinations including symmetric hybrids, asymmetric hybrids or cybrids. Somatic hybridization has a characteristic potential to combine both nuclear and cytoplasmic genes simultaneously unlike sexual hybridization or genetic engineering techniques. This technique can facilitate breeding and gene transfer, bypassing problems which are associated with conventional sexual crossing, including sexual incompatibility, polyembryony, male or female sterility. Cocking (1960) published his pioneer work on plant protoplast isolation. However, the first somatic hybrid production was reported by Carlson et al. (1972) in the genus tobacco through the technique of cell fusion. This has now been extended to a large number of genera to produce symmetric somatic hybrids (with complete nuclear genomes of both the parents), asymmetric hybrids (nuclear genome from the donor parent into the genome of the recipient parent), and cybrids (nuclear genome of a parent with mitochondrial genome of the other parent). Since, then hundreds of reports have been published during the past three decades which extend the procedures to additional plant genera and evaluate the utilization potential of somatic hybrids in many crops species like rice, rapeseed, tomato, potato and citrus. The essential steps followed in the technique of somatic hybridization are:

(a) Isolation of protoplasts,
(b) fusion of protoplasts,
(c) culture of protoplasts to raise entire plant,
(d) selection of hybrid cells, and
(e) hybrid verification.

 Protoplast Isolation

Protoplast isolation has been reported from mesophyll cells of in vivo and in vitro grown plants, aseptic seedlings, embryogenic and non-embryogenic suspension cultures, cotyledons, hypocotyls, and male and female gametes. The young leaves from in vitro grown aseptic shoot cultures are the widely used tissue to isolate protoplasts. The mesophyll cells in the leaves are loosely arranged hence, the enzymes have an easy access to the cell wall. Isolation of protoplasts requires at least two enzymes like a pectinase enzyme to dissolve the middle lamella that binds the adjacent cells together and a cellulose enzyme to digest the cell walls and release the protoplasts. Some tissues may require hemicellulase in addition to cellulose and pectinase to release protoplasts. The activity of these enzymes is dependent on pH and temperature. The pH of the enzyme mixture is adjusted at 4.7–6.0. The maximum activity of the enzymes is at 40 to 50°C but the tissues are generally, incubated in enzyme solution at 25 to 30°C as the higher temperatures may be injurious to the cells. The isolated protoplasts are osmotically fragile and a variety of solutes, ionic and nonionic are used as osmoticum for isolation and culture of protoplasts. The most commonly used osmotic stabilizer is mannitol, a metabolically inert sugar, in the amount of 450–800 mM. The protoplast preparation is contaminated with undigested cells and tissues and debris of overdigested broken cells, which must be removed before their culture and manipulation and the most commonly used purification method is filtration followed by centrifugation.

 Protoplast Fusion

Freshly isolated protoplasts fuse when brought into intimate contact and held together for a few minutes. The most widely used techniques to fuse plant protoplasts are chemical fusion by PEG (polyethylene glycol) and electric stimulation. PEG has been widely accepted as a fusogen of plant protoplasts as it induces high frequency heterokaryon formation with low toxicity to plant cells. A combination of the original PEG method and high Ca²⁺ high pH method is widely used. The fused protoplasts with two nuclei of different parents are called heterokaryons and those with nuclei of the same parent are called homokaryons. The fusion of nuclei in the heterokaryons during culture results into a hybrid cell. The major disadvantages of the chemical fusion method are: (1) the fusogen is toxic to some cell systems (2) it produces random and multiple cell aggregates, and (3) fusogen must be removed before culture. Additionally, electrofusion is rapid (completes within 15 min), synchronous, convenient method and the treatments are relatively nontoxic to protoplasts, allowing fusion of defined protoplasts. Electrical fusion is more reproducible and often gives greater fusion frequency than the chemical fusion. For electrofusion the protoplasts should be of good quality as the poor quality protoplasts burst during electrofusion and release salts changing the conductivity of the fusion mixture. For efficient electrofusion, the osmolarity of the fusion mixture should be proper, the strength, duration, and number of DC pulses should be optimized.

 Protoplast Culture

The density of purified protoplasts is determined using hemocytometer and adjusted to the desired level before culture. A variety of methods have been used to culture freshly isolated protoplasts and after fusion treatment, which are similar to the methods used for cell culture Under optimal culture conditions protoplasts synthesize a wall within 24 hour and lose their characteristic spherical shape. Generally, a proper somatic cell wall is necessary for the cell to divide by normal mitosis. In the absence of a proper wall nuclear divisions may occur but these are not followed by cytokinesis. The time taken for the first cell division in protoplast cultures varies with the species, genotype, protoplast source, procedure of protoplast isolation, protoplast viability, medium composition and culture conditions.

Selection of Somatic Hybrids

After chemical fusion, the fusion mixture is generally a heterogeneous mixture of homokaryons, heterokaryons, parental types, and a variety of nuclear cytoplasmic combinations. The frequency of the desirable fusion products is always much lower than the parental types. Several strategies have been followed to select or enrich the population of hybrid cells. Of these, biochemical mutants and antibiotic and herbicide resistance are used very frequently. So far the biochemical mutants are chlorophyll or nitrate reductase deficient and albino mutants have been widely used.

 Characterization of Somatic Hybrids

Even after a passage through an effective selection method, it is desirable to confirm the hybridity of the plants regenerated from the fusion products because of the chances of escapes through the selection pressure. Morphological, cytological, and molecular methods have been applied for the purpose. Use of molecular techniques such as isozyme analysis and DNA analysis by RFLP, RAPD, SSR, AFLP, and 5S rDNA spacer sequence have been used to characterize the somatic hybrids (Liu et al. 2005).

 Plant Regeneration

Plant regeneration from freshly isolated protoplasts or after their fusion occurs via organogenesis or embryogenesis. The first report of plant regeneration from isolated protoplasts of Nicotiana tabacum was published by Takebe et al. (1971).

General applications of somatic hybridization

The most common target using somatic hybridization is the generation of symmetric hybrids that contain the complete nuclear genomes of both parents. Somatic hybrid recovery following protoplast fusion is often facilitated by hybrid vigour. In a few cases, a new somatic hybrid may have direct utility as an improved cultivar. The most important application of somatic hybridization is the building of a germplasm as a source of superior breeding parents for various types of conventional crosses in cases of many different crop species. It has expanded opportunities for wide hybridization especially the production of intergeneric combinations that maximize genetic diversity. Many somatic hybrids have been produced to access genes that confer disease resistance. Somatic cybridization is the process of combining the nuclear genome of one parent with the mitochondrial or chloroplast genome of the other parent. Cybrids can be produced by the donor-recipient method or by cytoplast–protoplast fusion but can also occur spontaneously from intraspecific, interspecific or intergeneric symmetric hybridization. A primary target of somatic cybridization experiments is to transfer the cytoplasmic male sterility (CMS) to facilitate conventional breeding. In addition to somatic cybridization, incomplete asymmetric somatic hybridization also provides opportunities for transfer of fragments of the nuclear genome, including one or more intact chromosomes from one parent which is the donor into the intact genome of a second parent that is the recipient.

 Somatic Hybridization and Genetic engineering

In vitro technology has contributed significantly to enlarge the scope of wide hybridization for genetic up gradation of crop plants by providing methods to overcome pre-zygotic and post zygotic barriers of sexual incompatibility. Genetic engineering is a precise, aim-oriented sophisticated technology for genetic manipulation of crop plants. However, its applications are limited due to lack of target genes, difficulty in transferring polygene traits (e.g. yield, stress resistance). Moreover, presence of the selection or reporter marker genes in the transgenic plants poses negative impact on public acceptability of the transgenic products. From bio-safety viewpoint, somatic hybridization has an edge over genetic engineering because in the latter some of the genes are alien to plants, derived from other kingdoms.

 Conclusion

Somatic hybridization is a promising technique for the introgression of alien genes, including polygenic traits into crop plants and the technique has advanced from the academic stage to field applications. A unique advantage of somatic hybridization is in creating new combinations of nuclear and/or cytoplasmic organelles, leading to more variation enrichment of current gene pool. The hybrid cells may give rise to hybrid plants with full nuclear genomes of both the parents (Symmetrical hybrids). However, very often interactions between the genomes of the two parents result in various combinations of nuclear and cytoplasmic genomes. The nuclear genome of one of the parents may be partially or completely eliminated during successive cell cycles, before regeneration of plants, leading to asymmetric hybrid or cybrid formation, respectively. Asymmetric somatic hybridization to transfer part of the gene of the donor parent and cybridization to transfer cytoplasmic traits has considerably enhanced the importance of somatic hybridization. It has also been well documented that protoplast fusion can be used to create useful bridging material for breeding programs.

  References

 

Carlson, P.S., Smith, H., Dearing, R.D. (1972). Proceedings of the National Academy of Sciences. 69:2292–2294.

Cocking, E. C. (1960). A method for the isolation of plant protoplasts and vacuoles. Nature 187:962–963

Takebe, I., Labib, G., Melchers, G. (1971). Regeneration of whole plants from isolated mesophyll protoplasts of tobacco. Naturwissenschaften 58:318–320.

Liu, J., Xu, X., Deng, X. (2005). Intergeneric somatic hybridization and its application to crop genetic improvement. Plant Cell Tissue and Organ Culture 82:19–44.

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