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The somatic hybridization involves three aspects. The conventional method to improve the characteristics of cultivated plants, for years, has been sexual hybridization. The major limitation of sexual hybridization is that it can be performed within a plant species or very closely related species. This restricts the improvements that can be done in plants.

Somatic hybridization broadly involves in vitro fusion of isolated protoplasts to form a hybrid cell and its subsequent development to form a hybrid plant. Plant protoplasts are of immense utility in somatic plant cell genetic manipulations and improvement of crops. Thus, protoplasts provide a novel opportunity to create cells with new genetic constitution. And protoplast fusion is a wonderful approach to overcome sexual incompatibility between different species of plants.

More details on the applications of somatic hybridization are given later. Somatic hubridization involves the following aspects: A. Fusion of protoplasts B. Selection of hybrid cells C. Identification of hybrid plants. Fusion of Protoplasts: As the isolated protoplasts are devoid of cell walls, there in vitro fusion becomes relatively easy.

There are no barriers of incompatibility at interspecific, inter-generic or even at inter-kingdom levels for the protoplast fusion. Protoplast fusion that involves mixing of protoplasts of two different genomes can be achieved by spontaneous, mechanical, or induced fusion methods.

Spontaneous fusion: Cell fusion is a natural process as is observed in case of egg fertilization. During the course of enzymatic degradation of cell walls, some of the adjoining protoplasts may fuse to form homokaryocytes homokaryons.

These fused cells may sometimes contain high number of nuclei This is mainly because of expansion and subsequent coalescence of plasmodermal connections between cells. The frequency of homokaryon formation was found to be high in protoplasts isolated from dividing cultured cells. Spontaneously fused protoplasts, however, cannot regenerate into whole plants, except undergoing a few cell divisions.

Mechanical fusion: The protoplasts can be pushed together mechanically to fuse. Protoplasts of Lilium and Trillium in enzyme solutions can be fused by gentle trapping in a depression slide. Mechanical fusion may damage protoplasts by causing injuries. There are several fusion-inducing agents which are collectively referred to as fusogens e. Some of the fusogens and their use in induced fusion are described. A diagrammatic representation of protoplast fusion is depicted in Fig.

NaNO3 treatment results in a low frequency of heterokaryon formation, particularly when mesophyll protoplasts are fused. The method consists of incubating protoplasts in a solution of 0. The protoplasts form aggregates, and fusion usually occurs within 10 minutes.

PEG enhances fusion of protoplasts in several species. This tube is shaken and then allowed to settle. The settled protoplasts are washed several times with culture medium.

PEG treatment method is widely used protoplast fusion as it has several advantages: i. It results in a reproducible high-frequency of heterokaryon formation. Low toxicity to cells. Reduced formation of bi-nucleate heterokaryons. PEG-induced fusion is non-specific and therefore can be used for a wide range of plants. Electro-fusion: In this method, electrical field is used for protoplast fusion. When the protoplasts are placed in a culture vessel fitted with micro- electrodes and an electrical shock is applied, protoplasts are induced to fuse.

Electro-fusion technique is simple, quick and efficient and hence preferred by many workers. Further, the cells formed due to electro-fusion do not show cytotoxic responses as is the case with the use of fusogens including PEG. The major limitation of this method is the requirement of specialized and costly equipment. Mechanism of fusion: The fusion of protoplasts involves three phases agglutination, plasma membrane fusion and formation of heterokaryons.

Agglutination adhesion : When two protoplasts are in close contact with each other, adhesion occurs. Agglutination can be induced by fusogens e. Plasma membrane fusion: Protoplast membranes get fused at localized sites at the points of adhesion. This leads to the formation of cytoplasmic bridges between protoplasts. This allows closer contact and membrane fusion between agglutinated protoplasts.

This results in the formation of tight adhesions of membranes and consequently their fusion. Formation of heterokaryons: The fused protoplasts get rounded as a result of cytoplasmic bridges leading to the formation of spherical homokaryon or heterokaryon. After the fusion process, the protoplast population consists of a heterogenous mixture of un-fused chloroplasts, homokaryons and heterokaryons Fig. It is therefore necessary to select the hybrid cells heterokaryons.

The commonly used methods employed for the selection of hybrid cells are biochemical, visual and cytometric methods. Biochemical methods: The biochemical methods for selection of hybrid cells are based on the use of biochemical compounds in the medium selection medium. These compounds help to sort out the hybrid and parental cells based on their differences in the expression of characters.

Drug sensitivity and auxotrophic mutant selection methods are described below: 1. Drug sensitivity: This method is useful for the selection hybrids of two plant species, if one of them is sensitive to a drug. Protoplasts of Petunia hybride species A can form macroscopic callus on MS medium, but are sensitive to inhibited by actinomycin D. Petunia parodii protoplasts species B form small colonies, but are resistant to actinomycin D. When these two species are fused, the fused protoplasts derive both the characters — formation of macroscopic colonies and resistance to actinomycin D on MS medium.

This helps in the selection of hybrids Fig. The parental protoplasts of both the species fail to grow. Protoplasts of P. Drug sensitivity technique was originally developed by Power et al for the selection of hybrids of Petunia sp. A similar procedure is in use for the selection of other somatic hybrids e. Nitrate reductase deficient mutants of tobacco N. The parental protoplasts of such species cannot grow with nitrate as the sole source of nitrogen while the hybrids can grow. Two species of nitrate reductase deficiency— one due to lack of apoenzyme nia-type mutant and the other due to lack of molybdenum cofactor cnx- type mutant are known.

The parental protoplasts cannot grow on nitrate medium while the hybrid protoplasts can grow Fig. The selection of auxotrophic mutants is possible only if the hybrid cells can grow on a minimal medium. Visual methods: Visual selection of hybrid cells, although tedious is very efficient.

In some of the somatic hybridization experiments, chloroplast deficient albino or non-green protoplasts of one parent are fused with green protoplasts of another parent.

This facilitates the visual identification of haterokaryons under light microscope. The heterokaryons are bigger and green in colour while the parental protoplasts are either small or colourless.

Further identification of these heterokaryons has to be carried out to develop the specific hybrid plant. There are two approaches in this direction — growth on selection medium, and mechanical isolation.

Visual selection coupled with differential media growth: There exist certain natural differences in the sensitivity of protoplasts to the nutrients of a given medium. Thus, some media can selectively support the development of hybrids but not the parental protoplasts. A diagrammatic representation of visual selection coupled with the growth of heterokaryons on a selection medium is given in Fig. Mechanical isolation: The visually identified heterokaryons under the microscope can be isolated by mechanical means.

This involves the use of a special pipette namely Drummond pipette. The so isolated heterokaryons can be cloned to finally produce somatic hybrid plants. The major limitation of this method is that each type of hybrid cell requires a special culture medium for its growth. This can be overcome by employing micro drop culture of single cells using feeder layers. Cytometric methods: Some workers use flow cytometry and fluorescent-activated cell sorting techniques for the analysis of plant protoplasts while their viability is maintained.

The same techniques can also be applied for sorting and selection of heterokaryons. The hybrid cells derived from such selections have proved useful for the development of certain somatic hybrid plants.

Identification of Hybrid Cells Plants: The development of hybrid cells followed by the generation of hybrid plants requires a clear proof of genetic contribution from both the parental protoplasts.

The hybridity must be established only from euploid and not from aneuploid hybrids. Some of the commonly used approaches for the identification of hybrid plants are briefly described.

Morphology of hybrid plants: Morphological features of hybrid plants which usually are intermediate between two parents can be identified. For this purpose, the vegetative and floral characters are considered.

These include leaf shape, leaf area, root morphology, flower shape, its structure, size and colour, and seed capsule morphology. The somatic hybrids such as pomatoes and topatoes which are the fused products of potato and tomato show abnormal morphology, and thus can be identified. Although the genetic basis of the morphological characters has not been clearly known, intermediate morphological features suggest that the traits are under the control of multiple genes.

It is preferable to support hybrid morphological characters with evidence of genetic data. Isoenzyme analysis of hybrid plants: The multiple forms of an enzyme catalysing the same reaction are referred to as isoenzymes.


Protoplast Fusion and Somatic Hybridization: Importance and Limitation

Tygozragore Spontaneously fused protoplasts, however, cannot regenerate into whole plants, except undergoing a few cell divisions. The somatic hybridization involves three aspects. To be addressed then is the extent to which sexual incompatibility limits the production of other useful hybrids. Figure 1 shows a general model of the different steps of protoplast regeneration, compiling achievenents most promising research areas for further research in the near future.



The somatic hybridization involves three aspects. The conventional method to improve the characteristics of cultivated plants, for years, has been sexual hybridization. The major limitation of sexual hybridization is that it can be performed within a plant species or very closely related species. This restricts the improvements that can be done in plants.


Somatic Hybridization: Aspects, Applications and Limitations

Mikahn Achievements and limitations of protoplast research pdf Spontaneously fused protoplasts, however, cannot regenerate into whole plants, except undergoing a few cell divisions. Normally, cybrids are produced when protoplasts from two phytogenetically distinct species are fused. As the formation of heterokaryon occurs during hybridization, the nuclei can be stimulated to segregate so that one protoplast contributes to the cytoplasm while the other contributes nucleus alone or both nucleus and cytoplasm. A modification of hybridization in the form of cybridization has made it possible to transfer cytoplasmic male sterility. Differences in the status of protoplasts actively dividing or quiescent from the two species of plants result in formation of asymmetric hybrids.


Research Limitations

Plant protoplasts are of immense utility in somatic achievemrnts cell genetic manipulations and improvement of crops. Therefore, it is necessary to use the same enzyme from each plant parents and somatic hybridsfrom a specific tissue with the same age. Achievements and limitations of protoplast research pdf Expectations of success with an untested pair of species could be expected to be low. The most important objective in Solanum tuberosum somatic breeding is the introduction of resistance against the PvY virus, Colorado beetle and late blight Phytophtora.

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