somatic hybrid.pptx

Somatic hybridization

1. Presented by : Dr. Nidhi Mahendru Assistant Professor Dept of Biotechnology GNKC,YNR

2. Somatic Hybridization Somatic hybridization broadly involves in vitro fusion of isolated protoplasts to form a hybrid cell and its subsequent development to form a hybrid plant. Protoplasts provide a novel opportunity to create cells with new genetic constitution. Protoplast fusion is a wonderful approach to overcome sexual incompatibility between different species of plants.

3. Som a tic h ybrid i z a t i o n i n v ol v es the following aspects: Fusion of protoplasts Selection of hybrid cells Identification of hybrid plants.

4. A. Fusion of Protoplasts: Isolated protoplasts are devoid of cell walls, therefore, 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. t w o di f f e r e n t P r o t opla s t fus i o n of genomes can be ach i e v ed by spontaneous, mechanical, or ind u c ed fusion methods.

5. Spontaneous fusion: Enzymatic degradation of cell walls leads to fusion of the adjoining protoplasts to form homokaryons. These fused cells may sometimes contain high number of nuclei (2-40). This is mainly because subsequent dissolution of e x p a nsio n a n d of p las mode rm al connections between cells. The frequency of homokaryon formation is likely 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.

6. 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.

7. Induced fusion: Freshly isolated protoplasts can be fused by induction. There are several fusion- inducing agents which are collectively referred to as fusogens e.g. NaN0 3 , high pH/Ca 2+ , polyethylene glycol, polyvinyl alcohol, lysozyme, concavalin A, dextran, dextran sulfate, fatty acids and esters, electro fusion

8. Fusion of protoplasts

9. The fusion of protoplasts involves three phases namely agglutination, plasma membrane fusion and formation of heterokaryons. 1. Agglutination (adhesion): When two protoplasts are in close contact with each other, adhesion occurs. Agglutination can be induced by fusogens e.g. PEG, high pH and high Ca 2+ .

10. 2. 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. The plasma membrane fusion can be increased by high pH and high Ca 2+ , high temperature and PEC

11. 3. Formation of heterokaryons: The fused protoplasts get rounded as a result of cytoplasmic bridges leading to the formation of spherical homokaryon or heterokaryon. B. Selection of Hybrid Cells: About 20-25% of the protoplasts are actually involved in the fusion. After the fusion process, the protoplast population consists of a heterogenous mixture of un-fused chloroplasts, homokaryons and heterokaryons.

12. Fusion products of protoplasts

13. It is, therefore, necessary to select the hybrid cells (heterokaryons). The commonly used methods employed for the sel e ction of visual h yb r id cells a r e and cytometric bioc h e mi c al, methods.

14. C . Ide n tifi c a tion o f Hyb r id (C e ll s ) 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. Some of the commonly used approaches for the identification of hybrid plants are:

15. Molecular techniques: Recent developments in molecular biology have improved the understanding of genetic constitution of somatic plant hybrids. Differences in the restriction patterns of chloroplast and mitochondrial DNAs. Molecul a r mar k e r s suc h as RFL P , AFL P , RAPD and microsatellites. PCR technology.

16. Chromosome number in somatic hybrids: The chromosome number in the somatic hybrids is generally more than the total number of both of the parental protoplasts. Wide variations are reported which may be due to the following reasons: Fusion of more than two protoplasts. Irregularities in mitotic cell divisions.

17. In fusogen or electro-induced fusions, about one third of the fusions occur between more than two protoplasts. Differences in the status of protoplasts (actively dividing or quiescent) from the two species of plants result in formation of asymmetric hybrids. Asymmetric hybrids may be due to unequal replication of DNA in the fusing protoplasts. Protoplast isolation and culture may also lead to somaclonal variations, and thus variations in chromosome number.

18. Symmetric hybrids: If the chromosome number in the hybrid is the sum of the chromosomes of the two parental protoplasts, the hybrid is said to be symmetric. Symmetric hybrids between incompatible species are usually sterile. This may be due to production of 3n hybrids by fusing 2n of one species with n of another species.

19. Asymmetric hybrids Asymmetric hybrids have abnormal or wide variations in the chromosome number than the exact total of two species. These hybrids are usually formatted with full somatic complement of one parental species while all or nearly all of the chromosomes of other parental species are lost during mitotic divisions.

20. Cybrids: The cytoplasmic hybrids where the nucleus is derived from only one parent and the cytoplasm is derived from both the parents are referred to as cybrids. The phenomenon of formation of cybrids is regarded as cybridization. are produced Normally, cybrids p r o t opl a s ts f r o m t w o p h y t o g en e t i c ally w hen di s tin c t species are fused. Gen e t i c all y , cybrids a r e h y bri d s onl y f or cytoplasmic traits.

21. Hybrids and Somatic Incompatibility: M a n y a times, p r o d u c t ion o f ful l - pl e d g e d h y brid s t h r ou g h fusio n o f p r o t opla s ts of di s t a n t l y r el a t e d highe r pla n t speci e s is rather difficult due to instability of the two dissimila r g e nome s in a c om m on cytoplasm. This phenomenon is referred to as somatic incompatibility.

22. Hybrids formed despite somatic incompatibility may exhibit structural and developmental abnormalities. Several generations may be required to eliminate the undes i r able t o this g e nes . Due limitation in somatic h yb r idi z a tion, cybridization involving protoplast fusion for partial genome transfer is gaining importance in recent years.

23. Methodology of Cybridization : A diagrammatic representation of the formation of hybrids and cybrids is given

24. Hybridization and Cybridization A s the f orm a t i o n o f h e t e r o k ar y o n o ccu r s du r ing h y bri d i z a tion, t h e nucle i c an be one stimulated to segregate p r o t op l a s t c o n tr i bu t es to so that the cytoplasm while the other contributes nucleus alone (or both nucleus and cytoplasm). In this way cybridization can be achieved.

25. Genetic recombination in Asexual or Sterile Plants: There are many plants that cannot reproduce sexually. Somatic hybridization is a novel approach through which two parental genomes of a sexual or sterile plants can be brought together. Thus, by fusing parental protoplasts, fertile diploids and polyploidy can be produced.

26. Overcoming incomp a tibility: B a rrie r s of S e x ual c r os s ing b e t w e e n Sexual species genus (i n t e r s pecifi c ) (inter-generic) two different and two different is impos s ible by conventional breeding methods. Somatic hybridization overcomes the sexual incompatibility barriers.

27. Fusio n b e t w e e n protoplasts ( Solanum tuberosum ) ( L y c opersi c o n escul e n tu m ) of potato and t om a t o ha s c r e a t e d pomato (Solanopersicon, a new genus). 2. Interspecific o f f our different specie s o f rice fusion ( O r y z a brachyantha , O . elchnge r i , O . o f ficinali s a n d O . perrieri ) could be done to improve the crop .

28. Applications of Cybrids: Cybridization is a wonderful technique wherein the desired cytoplasm can be transferred in a single step. Cybrids are important for the transfer of cytoplasmic male sterility (CMS), antibiotic and herbicide resistance in agriculturally useful plants. Some of the genetic traits in certain plants are cytoplasmically controlled. This includes some types of male sterility, resistance to certain antibiotics and herbicides.

29. Cybrids of Brassica raphanus that contain nucleus of B. napus , chloroplasts of atrazinc resistant B. campestris and male sterility from Raphanus sativas have been developed.

30. Applications of Somatic hybridization This technique has opened new possibilities for the in vitro genetic manipulation of plants to improve the crops. 1. Disease resistance: Several interspecific and inter-generic hybrids with disease resistance have been created. Many disease resistance genes (e.g., tobacco mosaic virus, potato virus X, club rot disease) could be successfully transferred from one species to another. For example, resistance has been introduced in tomato against diseases such as TMV, spotted wilt virus and insect pests.

31. Environmental tolerance: The genes responsible for the tolerance of cold, frost and salt could be successfully introduced through somatic hybridization, e.g., introduction of cold tolerance gene in tomato. Quality characters: Somatic hybrids for the production of high nicotine content, and low erucic acid have been developed.

32. 4 . Cytoplasmic male sterility: A modification of hybridization in the form of cybridization has made it possible to transfer cytoplasmic male sterility.

33. Somatic hybridization has helped to study the cytoplasmic genes and their functions helpful in plant breeding programmes. Protoplast fusion will help in the combination of mitochondria and chloroplasts to result in a uniqu e nuclea r - c y t o p la s m i c g en e t i c combination. Somatic hybridization can be done in plants that are still in juvenile phase. Protoplast transformation (with traits like nitrogen fixation by incorporating exogenous DNA) followed by somatic hybridization will yield innovative plants.

34. Limitations of Somatic Hybridization: The r e a r e s e v e r al p r oblem s a n d limi t a t i ons associated with somatic hybridization. 1. Somatic, hybridization does not always produce plants that give fertile and visible seeds. 2. Regenerated plants obtained from somatic hybridization are often variable due to somaclona l v a ri a t i ons , c h r omos o mal elimination, organelle segregation etc.

35. 3. P r o t opl a s t culture is frequently associated with genetic instability. Protoplast fusion between different species/genus is easy, but the production of viable somatic hybrids is not possible in all instances. Some of the somatic hybrids, particularly when produced by the fusion of taxonomically different partners, are unbalanced and not viable.

36. 6. There are limitations in the selection methods of hybrids, as many of them are not efficient. 7 . The r e is expression no cer t ai n ty a s r e g a r d s the o f a n y specifi c ch a r ac t e r in somatic hybridization. 8. Somatic hybridization between two diploids results in the formation of an amphidiploid which is not favourable. For this reason, haploid protoplasts are recommended in somatic hybridization.