09a Somatic Cell Hybridization.pdf

Somatic Cell Hybridization Development of hybrid plants through the fusion of somatic protoplasts of two different plant species/varieties is called somatic hybridization Techniques of Somatic Hybridization 1) Isolation of protoplast 2) Fusion of the protoplasts of desired spec...

Somatic Cell Hybridization Development of hybrid plants through the fusion of somatic protoplasts of two different plant species/varieties is called somatic hybridization Techniques of Somatic Hybridization 1) Isolation of protoplast 2) Fusion of the protoplasts of desired species/varieties 3) Identification and Selection of somatic hybrid cells 4) Culture of the hybrid cells 5) Regeneration of hybrid plants The process by which protoplasts of two different plant species fuse together to form hybrids is known as somatic hybridization and the hybrids so produced is known as somatic hybrids. The technique of somatic hybridization involves the following steps. i) Isolation of protoplasts: Plant cell consist of cell wall which has to be degraded if the protoplasts of the cell has 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 successful release of protoplast is aided. The osmotic pressure of the solution is controlled by 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 plant. But most successful isolation was made possible from leaf of the plants. The leaf is surface sterilized and lower epidermis is removed, and treated with enzyme solution. ii) Fusion of different protoplasts: Different protoplasts isolated are treated with different mechanisms so that they fuse together. The mechanisms followed; iii) High calcium high pH treatment: Here, the different protoplasts in one solution are together treated with conditions like high calcium and high pH so that they fuse together. In some cases such extreme conditions has proved to be toxic to certain protoplasts. iv) Polyethylene glycol (PEG) treatment: This has proved to be one of the most effective methods for protoplast fusion. The cells are treated with a concentration of around 30% poly ethylene glycol which binds to plasma membrane. This is treated with calcium solution which being cationic binds to PEG. During washing, the PEG pulls out the plasmalemma leading to fusion of protoplasts in close proximity. This leads to fusion of protoplasts randomly and so is a non-selective fusion process. v) Electro fusion technique: This process involves passing low voltage electric pulses in a solution of protoplasts to be fused so that they line up for  fusion. The protoplast can be fused by subjecting it to brief exposure to high voltage electric current which leads to alteration of membrane so that the adjacent protoplast fuse. The protoplasts so lined up can be moved by the use of micromanipulator so that required protoplast can be fused. This is carried out in an electroporator. Selection of hybrid cells after a successful protoplasmic fusion experiment, a variety of structure are available like unfused protoplast and protoplast of same species fused together and protoplasts of different species fused together or hybrid cells. In order to separate the hybrid cells from other residue several techniques are followed. Mechanical isolation of fused protoplasts is done or taking advantage of natural properties exhibited by host cells so that the cells showing absence in that property indicate the hybrid cells. Another important method is by culturing all the residues and formation of calli which is then studied to identify the hybrids. The hybrids formed are of different types like: Symmetric hybrids: These contain the somatic chromosome of both the parental species. These are very significant as they show all the properties exhibited by parent species. Asymmetric hybrids: These are those hybrids which preserve the genetic material of one parent organism. The chromosome content of other parent species is lost. Cybrids: These consist of nucleus of one species and cytoplasm from both the species. They are produced by fusion of one species with another having enucleate protoplast or having inactivated nucleus or loss of chromosomes of one parent by repeated mitotic division. It can even be induced by inactivating nucleus of a protoplast prior to fusion. The cybrids produce many advantages like transfer of plasma gene of one species into the nuclear background of another species, formation of recombinants between mitochondrial or chloroplast genomes. Advantages of Somatic Hybridization 1) Production of novel interspecific and intergenic hybrid – Pomato (Hybrid of potato and tomato). 2) Production of fertile diploids and polypoids from sexually sterile haploids, triploids and aneuploids. 3) Transfer gene for disease resistance, abiotic stress resistance, herbicide resistance and many other quality characters. 4) Production of heterozygous lines in the single species which cannot be propagated by vegetative means. 5) Production of unique hybrids of nucleus and cytoplasm. Problem and Limitation of Somatic Hybridization 1) Application of protoplast technology requires efficient plant regeneration system. 2) The lack of an efficient selection method for fused product is sometimes a major problem. 3) The end-product after somatic hybridization is often unbalanced. 4) Regeneration products after somatic hybridization are often variable. 5) It is never certain that a particular characteristic will be expressed. 6) Genetic stability is still a matter of research. 7) Sexual reproduction of somatic hybrids is still under research. Practical application of somatic hybridization 1. Means of Genetic Recombination in Asexual or Sterile Plants Somatic cell fusion appears to be the only approach through which two different parental genomes can be recombined among plants that cannot reproduce sexually. Similarly, protoplast of sexually sterile plants can be fused to produce fertile diploids and polyploids. There are several reports describing the amphidiploid and hexamploid plants produced from fusion of haploid protoplasts of tobacco. Protoplasts isolated from dihaploid potato clones have been fused with isolated protoplasts of Solanum brevidens to produce hybrids of practical breeding value. Haploid protoplasts from an anther- derived callus of rice cultivars upon fusion also produce fertile diploid and triploid hybrids. 2. Overcoming Barriers of Sexual Incompatibility In plant breeding programmes, sexual crossing at Interspecific and Intergeneric levels often fails to produce hybrids due to incompatibility barriers, which can be overcome by somatic cell fusion. Schieder (1978) obtained amphidiploid Datura innoxia (+) D. discolor and D. innoxia (+) D. stramonium, by fusing their diploid mesophyll protoplast. These hybrids cannot be produced conventionally and they are industrially important because show heterosis and higher (20-25%) scopolamine content than in the parental forms. Nicotiana repanda, N.nesophila and N.stockoni are resistant to number of disease but are not sexually crossable with N. tabacum (Tobacco). However, fertile hybrids have been reported in combination N. tabacum (+) N nesophila and N. tabacum (+) N. Stockoni by protoplast fusion. Somatic hybridization of dihaploid and tetraploid potato protoplast with isolated protoplasts of Solanum brevidens, S. phureja and S. penelliii resulted in the synthesis of fertile, partially amphidiploid plants possessing important agricultural traits, e.g., resistance to potato leaf virus V and Erwinia soft rot. Similarly, somatic hybrids between Brassica napus and B. nigra have been produced which is resistant to Phoma Lingam. 3. Cytoplasm Transfer Power et.al. (1975) fused mesophyll protoplasts of Petunia with cultured cell protoplast of the crown gall of Parthenocissus and selected a line which contained the chromosomes of only Parthenocissus but exhibited some of the cytoplasmic properties of Petunia for some time. This was followed by direct application of cybridisation in agricultural biotechnology by transfer of cytoplasmic male sterility from Nicotiana techne to N. tabacum, N. tabacum to N.sylvestris and Petunia hybrida to P. axillaries. Besides cytoplasmic male sterility, the genophore of the cytoplasm codes for number of practially important traits, such as the rate of photosynthesis, low or high temperature tolerance, and resistance to disease or herbicides. In genus Brassica, two desirable traits coded by cytoplasmic genes have been genetically manipulated through interspecific cybridisation between different species of Brassica. These traits include cytoplasmic male sterility (cms) and resistance herbicide. Similarly, cytoplasmic genes coding for Atrazine and cms have been transferred into cabbage, rice and potato. Thus somatic hybridization techniques help in forming wide variety of recombinants among the plasma gene of different species and plasmagenes and chloroplast genes. It also helps to form hybrid cells exhibiting chloroplast genome of one species and mitochondrial genome of another species which is not possible by ordinary means of hybridization of two plant species. These different levels of fusion and recombination helps in production of new species which has all the qualities of parent organisms or even better.