somatic Hybridization and it's significance

Simatic Hybridization, types, steps, technique, method , advantages of somatic hybrid, disadvantage of somatic hybrid,

1. SOMATIC HYBRIDIZATION AND IT’S SIGNIFICANCE

2. INTRODUCTION Somatic Hybridization: The technique of hybrid production through the fusion of isolated somatic protoplasts under in vitro conditions and subsequent development of their product (heterokaryon) to a hybrid plant. Somatic Hybrids: Hybrids obtained by somatic hybridization. In somatic hybridization the nucleus and cytoplasm of both parents are fused in the hybrid cell.

3. Types of Somatic Hybridization: Based on taxonomic relationship of species involved in the hybridization, somatic hybrids are of three types, viz.: i. Interspecific Hybrids: ii. Intergeneric Hybrids: iii. Intertribal Hybrids: Based on combination of chromosomes and cytoplasm from parental species, somatic hybrids are of three types, viz.: (i) Symmetrical hybrids, (ii) Asymmetrical hybrids, and (iii) Cybrids.

4. The term protoplast- Hanstein(1880). The first isolation of protoplasts was achieved by Klercker (1892) - mechanical method. In 1960, Cocking used an enzymatic method for the removal of cell wall. Somatic hybrids was firstly introduced by Carlson in 1972 in Nicotiana guauca. Literature....... Hanstein Klercker

5. Somatic hybridization Involves 4 major steps: 1.Isolation of Protoplasts. 2.Culture of Protoplasts. 3.Fusion of Protoplasts and 4.Subsequent regeneration of hybrid cells into whole plants.

6. Sexual hybridization: involves fusion of the nuclear genes of both the parents. Somatic hybridization: involves even cytoplasm from both the parental species in hybrid obtained by protoplast fusion. Cybridization: Hybrids containing nuclear genome of one parent but cytoplasm from both the parents.

7. MECHANICAL METHOD: A small piece of epidermis from a plant is selected. The cells are subjected to plasmolysis. This causes protoplasts to shrink away from the cell walls.  The tissue is dissected to release the protoplasts.

8. ENZYMATIC METHOD: 1.Sequential Method (Two Step): The tissue is first treated with pectinase (macerozyme) to separate cells by degrading middle lamella. These free cells are then exposed to cellulose to release protoplasts. Pectinase breaks up the cell aggregates into individual cells while cellulose removes the cell wall proper . 2. Simultaneous Method (One Step): This is the preferred method for protoplast isolation. It involves the simultaneous use of both the enzymes — macerozyme and cellulose .

9. Protocol... The cells and tissue are incubated in the enzyme mixture for few to several hours; naked protoplasts devoid of cell wall are gradually released in the enzyme mixture. Protoplast have been isolated from virtually all plant parts, but leaf mesophyll is the most proffered tissue, for this purpose. In general, fully expanded leaves are surface sterilized, their lower epidemies is peeled off with a pair of forceps and the peeled areas are cut into small pieces with the enzymes mixture; vacuum infiltration may be used to facilitate the entry of enzymes into the tissues. After the period of incubation, protoplast are washed with a suitable washing medium in order to remove the enzymes and the debris. The protoplasts may be cultured on a suitable medium in a variety of ways: (1) Bergmann`s plating technique (in agar medium), (2) in a thin layer of liquid medium, and (3) in small micro drops of 50-100 ul. Protoplast readily regenerate cell wall (within 2-4days)and undergo mitosis to form macroscopic colonies, which can be induced to regenerate whole plants .

10. PROTOPLAST CULTURE Nutritional Media: In general, the nutritional requirements of protoplasts are similar to those of cultured plant cells (callus and suspension cultures). MS and B5 media with suitable modifications are used. The medium should be devoid of ammonium, and the quantities of iron and zinc should be less. The concentration of calcium should be 2-4 times higher than used for cell cultures. This is needed for membrane stability. High auxin/kinetin ratio is suitable to induce cell divisions while high kinetin/auxin ratio is required for regeneration. Glucose is the preferred carbon source by protoplasts although a combination of sugars (glucose and sucrose) can be used. The vitamins used for protoplast cultures are the same as used in standard tissue culture media.

11. As the isolated protoplasts are devoid of cell wall, in vitro fusion becomes relatively easy. There are no barriers of inability for the protoplast fusion. Somatic cell fusion appears to be the only means through which two different parental genomes can be recombined among plants that cannot reproduce sexually. Protoplast fusion involves the mixing of protoplast of two different genomes. This can achieved by spontaneous, mechanical, or induced fusion methods. A. Fusion of protoplasts

12. 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 protoplast may fuse to form homokaryons. These fused cells may sometimes contain high number of nuclei(2-40). Spontaneously fused protoplast, however, cannot regenerate into whole plants, except undergoing a few cell divisions.

13. Mechanical fusion The protoplast can be fused by pushing them together mechanically. Protoplast of lilium and trillium can be fused in enzyme solution by gentle trapping in a depression slide. Mechanical fusion may damage protoplasts by causing injuries.

14. Mechanism of fusion The fusion of protoplast involves three phases 1.Agglutination 2.Plasma membrane fusion and 3.Formation of heterokaryons. 1.Agglutination(adhesion): when two protoplast are in close contact with each other, adhesion occurs.Agglunation can be induced by fusogens eg.PEG,high pH and high Ca2+. 2. Plasma membrane fusion : Protoplast membrane get fused at localized sites at the points of adhesion. This leads to the formation of cytoplasmic bridges between protoplasts. The plasma membrane fusion can be increased by high pH and high Ca2+,high temperature and PEC,as explained below. High pH and high Ca2+ions neutralized the surface charges on the protoplasts. This allows closer contact and membrane fusion between agglutinated protoplast.

15. Cont... High temperature helps in the intermingling of lipid molecules of agglutinated protoplast membrane so that membrane fusion occurs. PEG causes rapid agglutination and formation and formation of clumps of protoplast. This results in the formation membrane fusion occurs. 3.Formation of heterokaryons The fused protoplast get rounded as a result of cytoplasmic bridges leading to the formation of spherical homokaryon or heterokaryon.

16. Induced fusion Freshly isolated protoplasts can be fused by induction. There are several fusion-inducing agents which are collectively referred to as fusogens eg. NaNO3,high pH,ca2+,polyethylene glycol, polyvinyl alcohol…. A) Sodium nitrate Treatment – Transfer protoplasts to 0.25M NaNO 3 and then centrifuge. – Low rate of heterokaryons. – Not used much today. • This method was successfully utilized for fusion of protoplasts from root tips of oat and maize seedlings but is not preferred due to low frequency of fusion, particularly when highly vacuolated mesophyll protoplasts are used.

17. Click to add Title b ) High pH/Ca++ Induced Fusion Treatment – Solution of 0.4M mannitol containing 0.05 M CaCl 2 with pH at 10.5, 37 C – Aggregates within 10 minutes – Successful method. This method involves centrifugation of the protoplasts in a fusion inducing solution (0.05M CaCl 2 2H 2 O in O.4M mannitol at pH 10) for 30 minutes , after which the tubes are placed in a water bath (37°C) for 40-50 min. • This leads to fusion of 20-50% of the protoplasts. • The method has been found superior to other methods in some cases, but high pH may be toxic in other cases.

18. Reproducible high production of heterokaryons Add 0.6 ml of PEG solution to pellet of protoplasts in a tube. Incubate at room temperature for 40 minutes with occasional rocking. After fusion, add 0.5 - 1.0 ml culture medium and centrifuge. Re-suspend protoplasts in culture medium Disturbs the charge on the membrane Protoplast fusion has been successfully achieved in several crops, using polyethylene glycol as a fusogen . The technique gives high frequency of fusion with reproducible results and involves low cytotoxicity. The technique can be used for fusion of protoplasts from unrelated plant taxa (e.g. soybean ­tobacco, soybean-maize & soybean-barley), a nd also between animal and plant cells.) C) PEG (polyethylene glycol) - 6000-8000 MW

19. The protoplasm are introduced into a small fusion chamber containing parallel wires or plates which serve as electrodes. A low-voltage and rapidly oscillating AC field is applied, which causes protoplasts to become aligned into chains of cells between electrodes. This creates complete cell-to-cell contact within a few minutes. Once alignment is complete, the fusion is induced by application of a brief spell of high-voltage DC pulses (o.125kVcm-1). A high voltage DC pulse induces a reversible breakdown of the plasma membrane at the site of cell contact, leading to fusion and consequent membrane reorganization. The entire process can be completed within 15 min. ELECTROFUSION

20. B. Selection of hybrid cells: About 20-25% of the protoplast are actually involved in the fusion. After the fusion process, the protoplast population consists of a heterogenous mixture of un-fused chloroplast,homokaryyons and heterokaryons. 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 method 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 difference in the expression of characters. There are two approaches in this direction – Drug sensitivity and auxotrophic mutant selection method.

21. Visual method Visual selection of hybrid cells, although tedious is very efficient. In some of the somatic hybridization experiments, chloroplast deficient protoplast of one parent are fused with green protoplast of another parent. This facilitates the visual identification of heterokaryons under a light microscope. The heterokaryons are bigger and green in colour while the parental protoplast 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.

22. C. 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 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. Isoenzyme analysis of hybrid plants –The multiple forms of an enzyme catalysing the same reaction are referred to as isoenzymes. Electrophoretic pattern of isoenzymes have been widely used to verify hybrids. Symmetric and asymmetric hybrids- If the chromosome number in the hybrid is the sum of the chromosome of the two parental protoplast, the hybrid is said to be symmetric. Asymmetric hybrids have abnormal or wide variations in the chromosome number than the exact total of two species.

23. CULTURE OF THE HYBRID CELLS Hybrid cells are cultured on suitable medium provided with the appropriate culture conditions .

24. REGENERATION OF HYBRID PLANTS

25. SOMATIC HYBRIDIZATION IN CITRUS/ORANGE

26. Solanaceae The family Solanaceae contains the most commonly used species for somatic hybridization. The genera from this family that have been often used for somatic hybridization include Nicotiana, Datura, Petunia, Solanum, Lycopersicon , etc. Other families include Umbellifereae .

27. ADVANTAGES OF SOMATIC HYBRIDIZATION Production of novel interspecific and intergenic hybrid Pomato (Hybrid of potato and tomato) Production of fertile diploids and polypoids from sexually sterile haploids, triploids and aneuploids.

28. conclution Somatic hybridization is an important tool of plant breeding and crop improvements through the production of inter-specific and inter-generic hybrids. It is valuable for plants which is asexual and sterile.

29. Ignacimuthu S.J.,1997, Plant Biotechnology-somatic hybridization and cybridization,5 th edition, Oxford and IBH publishing Co.Pvt.Ltd., New Dehli. Pp: 224-247. REFERENCES http://www.biologydiscussion.com/somatic-hybridization/somatic-hybridization-aspects-applications-and-limitations/10686