SEMINAR ON Somatic Hybridization SOMATIC HYBRIDIZATION Introduction Definition
SEMINAR ON Somatic Hybridization
SOMATIC HYBRIDIZATION Introduction Definition Somatic Hybridization process Isolation of protoplast Fussion of protoplast Selection of hybrid cells Regeneration of hybrid plants Types of Somatic Hybridization: Symmetric Somatic Hybridization Asymmetric Somatic Hybridization Application Limitations Reference
INTRODUCTION : - v Somatic hybridization or para sexual hybridization is a process In which hybrids are obtained through the fusion off protoplast. Belonging to two distinct plant species somatic hybrid are specifically implicated in the improvement of vegetatively propagates crop plant such as a Potato, Cassava, Banana, etc. Grater success in the production of Somatic hybrid involved effective fusion of protoplast.
DEFINITION : When the two different cells of protoplast fussed with each others & made a new species or (Hybrid cell) then it is called protoplast fusion or Somatic Hybridization. SOMATIC HYBRIDIZATION PROCESS : This process is completed in following steps : Isolation of protoplast Fusion of protoplast Selection of hybrid cells Regeneration of hybrid plants
ISOLATION OF PROTOPLAST : The plant cell is surrounded by cellulogenous cell wall and connected with adjacent call by middle lamella. Separation of plant cell and removal of cell wall either experimentally or mechanically or enzymatic process. Result in this procedure of breaked, naked plant cell (with out cell walled cell ) is called protoplast. This word is given by MANSTEEN in 1880. The protoplast can be isolated from leaves and callus.
Basically two methods are used for isolation of protoplast. i) Mechanical method or non enzymatic method. ii) Enzymatic Method i) Mechanical method or non enzymatic method This method is crude and oldest method for protoplast isolation it is only an historical method. It involves chopping and plasmolysis of tissue. This method is time consuming and laborious. The number of protoplasts through this method is not too much. In 1892 KLERCKER first used this method
II. ENZYMATIC METHOD : This method is effective in protoplast isolation of many plant part but in mesophill of leaves it is very useful & effective. The following enzyme are used in disslovation of middle lamella and cell wall eg. Pectinase, cellulase, hemicellulase, macerozyme etc. The most widely used commercial enzyme are onozuka –R – 10 macerozyme –R – 10, pectolyse –Y – 23 etc. Advantage of this method : • Large quantity of protoplast isolated. Cell are not injured as mechanical method. • Osmotic shrinkage is minimum and the deleterious effects of excessive plasmolysis are minimized. • Cooking in 1960 was the first reported the isolation of protoplasts from the fungus Myrothecium verrucosa.
PROTOPLAST FUSION : Protoplast fusion is a physiochemical phenomena. üBroadly speaking, protoplast fusion can be classified in two steps, such as : (A) Spontaneous Fusion (B) Induced Fusion Protoplast Fussion Spontaneous Fussion Induced Fussion Mechanical Fussion Chemo Fussion Electro Fussion
SPONTANEOUS FUSION : In somatic hybridization spontaneous fusion is of little significance. The protoplast 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 fuse spontaneously. This type of spontaneous fusion has been used to produce interygeneric fusion, e. g. the spontaneous fusion of microsporocyte protoplast of Lolium lengiflorum and Trilium kamtschaticum.
INDUCED FUSION : This technique has the possibility and ability to combine different genotype beyond the limits imposed by sexual process. (A) – MECHANICAL FUSION: In this process the isolated protoplasts are brought into intimate physical contact mechanically under microscope using micromanipulator and perfusion micropipette. In this way the protoplasts are retained and compressed by the flow of liquid. By this technique occasional fusion of protoplast has been observed.
(B) CHEMOFUSION : Chemofusion are the must commonly used protoplast fusion inducing agents. It agents as known as chemically fusogens. e. g. Sodium Nitrate (Na. No 3). Potesium Nitrate (Kno 3). Polyvinyl alchohl and PEG. A- In 1979, Kao and Michayluk from Canada discovered an ther fusion inducing chemical PEG. B- High concentration of Ca 2+ & High p. H. • In 1973 KELLER and MELCHER from Germany, developed this method. • Take Tobacco protoplast at high temperture (370 C). • Taken in centrifuge tube and centrifuge from 3 minutes.
Basic protocol for achieving protoplast fusions through the use of chemical fusogens
Ca. Cl 2 Conditions - H 2 O Mannitol p. H – 10. 5 • If these conditions are given to protoplast mixture. • than result is protoplast fusion. ELECTRO FUSION : v. ZIMMER MANN and SCHEURICH (1981) improved method for the large scale fusion of plant protoplast. This method may increase fusion frequency reproducibity.
REGENERATION OF HYBRID PLANTS: The use of somatic hybridization in plant breeding depend on two events 1 Regeneration of somatic hybrid plant. 2 Fertility of somatic hybrid plant. Many regeneration are achieved by the somatic hybrid but some somatic hybrid not gives regeneration this characteristic is called somatic incompatibility. The reason for somatic incompatibility are not clearly understood.
The somatic hybrid are of the following two types : A – Symmetric Somatic Hybridization B – Asymmetric Somatic Hybridization SYMMETRICAL SOMATIC HYBRIDS : Some somatic hybrid plants retain the full or nearly full somatic complements of the two parental species these are called Symmetrical hybrids. These somatic hybrids can now be used in breeding programmes for limited gene/chromosome introgression from the species contributing the haploid protoplast.
ASYMMETRICAL SOMATIC HYBRIDS : In this type of hybrid have only one parent character (dominent )& receive character not high light. e. g. Dacaus carota + Nicotiana toabaco. Normally heterokaryon protoplast are selected for somatic hybridization because : Color of both cells are different Both protoplast cells are complementary of each other. Different qualities mitochondria chromosomes number Enzyme are protoplast in heterokaryon Some important selected somatic hybrids selection of hybrid cells. a) Interspecific hybridization] b) Intraspecific hybridization
INTERSPECIFIC HYBRIDIZATION: 1. Brassica juncea +B. spinescens 2. B. napus +B. carinata 3. B. napus +B. juncea 4. Citrus siensis +C. limon 5. C. sinensis +C. paradisi 6. C. sinensis +C. unshiu 7. Helianthus annuus +H. gigateus 8. Oryza sativa +O. brachyantha 9. O. sativa +O. eichingeria INTRASPECIFIC HYBRIDIZATION: - 1) Brassica campestris + Barbarea vulgaris 2) B. juncea + Diplotaxis muralis 3) B. napus + Arabidopsis thaliana 4) B. napus + B. tournefortii a 5) B. napus + Thalaspi perfoliatum a 6) C. aurantifolia + Swinglea glutinosa 7) C. sinensis + Murraya paniculata a 8) Oryza sativa + Echinochola oryzicola 9) Solanum tuberosum + Lycopersicon pimpinellifolium
APPLICATION OF SOMATIC HYBRIDIZATION : There are many important applications of somatic hybridization Production of novel interspecific & intergeneric cross between plants that are difficult or impossible by other source. 1 Production of somatic hybrid and cybride. 2 Asymmetric hybrids production. 3 Direct DNA and macro molecule uptake. 4 Protoplast of sexual sterile plants can be fused with fertile diploid & polyploid. 5 Production of Heterzygous. 6 Somatic cell fusion is useful in the study of cytoplasmic gene & there activities
LIMITATION OF SOMATIC HYBRIDIZATION : It was one suggested that somatic hybrids would be of great value in crop improvement. In certain wide crosses elimination of chromosomes from the hybrid cell is another limitation of somatic hybridization. In protoplast fusion experiments the percentage of fusion product is very low. Lastly for hybrid identification selection and isolation at the culture level. There is no standardized method which is applicable for all materials. Regeneration capacity of hybrid cell is poor. Lack of suitable selection.
REFERENCE T. A brown Book – Biotechnology & plant Generic Resources. 2005 S. S Bojwani and M. K Razdan Book – Plant tissue culture 2005 B. D Singh Book – Biotechnology 2007
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