Cybrids

Protoplast fusion and cybrids in plants

1. CYBRIDS IN PLANT IMPROVEMENT PROGRAMME R.R.KIRUTHIKA BTH-12-009

2. CONTENTS What is cybrid ? Advantages of cybrids Need for cybrids Protoplast isolation Techniques involved Cybrids in plant breeding

3. CYBRID Cybrid – cytoplasmic hybrid . Cybrids are cells or plants containing nucleus of one species but cytoplasm from both the parental species. Cybridization – production of somatic cybrid . The process of protoplast fusion resulting in the development of cybrid is known as cybridization .

4. HISTORY In 1892 Klercker was the first to isolate protoplast from Stratiotes aloides using mechanical method. Cocking in 1960 was the first to report the isolation of protoplast from tomato root tips using concentrated solutions of cellulase from the fungus Myrothecium verrucosa . Enzymes for protoplast isolation was first employed by Takebe and his co workers in 1968. Kao and Michayluk in 1974 first proposed PEG for fusion of protoplast. Gleba 1979 fused tobacco protoplast which produced a cybrid .

5. Melchers and Labib in 1974 fused protoplast of two haploid light sensitive lines of Nicotiana tabacum . Kao and Wetter in 1976 isolated cell cybrids of Glycine max and Nicotiana glauca . Pental and Cocking proposed that triploids could be produced by fusing protoplast isolated from microspores at the tetrad stage (n) of a species with protoplast isolated from the somatic cells of other species . Pirrie and Power synthesized triploids by fusing microspore protoplast of Nicotiana glutinosa with somatic cell protoplast of Nicotiana tabacum .

6. NEED FOR CYBRIDS P roduction of full hybrids through protoplasts fusion of distantly related plants – unrealistic - wide spread instability of the two disimilar genomes in common cytoplasm. Undesirable –exhibit structural and developmental abnormalities. Partial genome transfer – fusion of normal protoplasts of the recipient with enucleated protoplasts of the donar - cybridization .

7. ADVANTAGES Two different parental genomes that cannot reproduce sexually (asexual or sterile) are recombined . Overcomes sexual incompatibility barriers. Used in study of cytoplasmic genes & their activities- plant breeding experiments . To transfer cytoplasmic male sterility ( tomato,tobacco ). To transfer antibiotic resistance character(tobacco) To transfer herbicide resistance (brassica) Used in mitochondrial research .

8. PROPERTIES OF PROTOPLAST Lack cell wall and have plasma membrane Semi permeable and selectively permeable Fusion property Regenerate the cell wall (totipotent) Phagocytises and phenocytises Spherical in shape Sensitive to osmotic presser

9. CYBRID PRODUCTION Cybrids are produced during fusion of protoplast from two phylogenetically distant species. Regeneration from phylogenetically distant species will have plastomes from both parental species but the functional genome of only one species through chromosomal elimination . The extranuclear genes which control agronomically important characters are of considerable interest .

10. PROTOPLAST FUSION-STEPS INVOLVED Isolation of protoplasts Culture of protoplasts Fusion of protoplasts Identification and selection of hybrid cells and their subsequent regeneration of protoplast into whole plants.

11. ISOLATION OF PROTOPLAST Mechanical method (non enzymatic) Cells are plasmolysed in iso -osmotic solution-tissues cut into thin strips –protoplast realeased from cut ends. Sequential enzymatic method (two step) Lower epidermis is peeled-leaf cut into pieces-macerated with macerozyme –release of mesophyll cell- cellulase to digest cell wall. Mixed enzymatic (simultaneous) Mesophyll tissue subjected simultaneously to macerozyme and cellulase treatment-macerates the tissue by breaking the middle lamella and release protoplast by digesting cell wall.

12. Enzymes used Cellulases cellulysin , driselase , meicelase p-1 Hemicellulases helicase , hemicellulase , rhozyme HP 150 Pectinases macerase,pectinol,pectolyse y-23 , zymolyase , macerozyme R-10

13. FACTORS AFFECTING ISOLATION Source material Pre-enzyme treatment Enzyme treatment Osmoticum PROTOPLAST VIABILITY AND DENSITY Flourescein diacetate method Evan’s blue staining Phenolsafranine staining

14. CULTURE OF PROTOPLAST B 5 and MS media Reduction in ammonia Two – four times increased calcium concentrations – membrane stability and integrity. Osmotica – sorbitol, mannitol,glucose and sucrose METHODS Multiple drop array method D roplet culture Plating method Micro culture chamber Feeder layer technique

15. REGENERATION OF PROTOPLAST Protoplast in culture show rapid cytoplasmic streaming ,increasing in size and chloroplast aggregate conspicuously around the nucleus. Cell wall formation begins within few hours after isolation and may be completed in two to several days. Characteristic spherical shape is lost once the wall formation is complete. The freshly formed cell wall is composed of loosely arranged microfibrils – requires readily metabolised carbon sourse (sucrose) in nutrient medium.

16. Cell increase in size and first division occurs between 2-7 days. Direct relationship between wall formation and cell division. After 1-3 weeks ,macroscopic colonies –transferred to osmotic free medium to develop callus. Embryogenesis or organogenesis . Embryogenesis- cells from protoplast culture differentiate into embroids then into whole plants. Organogenesis – callus transferred to a cytokinin free medium for shoot formation and medium containing auxin for root formation.

17. FUSION OF PROTOPLAST Facilitates mixing of two whole genomes and could be exploited in crosses at interspecic , intergeneric or interkingdom level. SPONTANEOUS FUSION P lasmodesmata of adjoining cells expand to form homokaryons . Rare and non reproducible. INDUCED FUSION External agent is used to fuse. Reproducible and sufficient. Mechanical fusion - micropipette Chemical fusion - fusogen (PEG,NaNO3,lysozyme) Electrical fusion - electric charge

18. OPTIMUM CONDITIONS FOR ELECTRIC FUSION

19. SELECTION OF SOMATIC HYBRIDS AND CYBRIDS Protoplast population consists of unfused parental types , homokaryons and heterokaryons . Fusion is random and uncontrolled . BIOCHEMICAL SELECTION Differential growth characteristics and nutritional requirementsof unfused and hybrid mesophyll protoplast. Nicotiana glauca and Nicotiana langsdorfii Petunia parodii and Petunia hybrida - drug actinomycin D Nicotiana sylvestris and Nicotiana knightiana – drug resistance cell lines.

20. COMPLEMENTATION SELECTION Complementation by auxotrophic mutants. Auxotrophs are mutants requiring specific compounds for thier growth. Only hybrid lines are expected to survive in minimal medium. Chlorophyll deficient and light sensitive protoplasts of Nicotiana tobacum under high intensities of light produced green colonies. VISUAL SELECTION V isual identification of heterokaryons at the light microscope level in fusion of chlorophyll deficient of one parent and green chloroplast of one parent.

21. MORPHOLOGICAL SELECTION Selection based on abnormal morphology. In regeneration of intergeneric somatic hybrids such as pomatoes and topatoes . Intermediate morphology of callus determined the intergeneric somatic hybrids between Vicia faba and Petunia hybrida . FLOW CYTOMETRY AND SORTING SELECTION Flow cytometry and flurescent activated cell sorting while maintaing viability. Electronic sorting of heterokaryons formed with flurescent dyes ( rhodomine isothiocynate and floreceine isothiocynate )- in cell sorter machine.

22. CYBRIDIZATION TECHNIQUE Nuclear genome of one parent “ donar ” is inactivated chemically or with irradiation before protoplast fusion. Irradiation with x-rays or gamma rays ,in doses of 50 to 300 Gy , is effective in partial or complete inactivation of donar cells. Fusion of untreated “recipient” protoplast with “ donar ” and culturing result in cybrid plants possessing the nucleus of the “recipient” and the cytoplasm of both parental species . Phylogenetically distant species produce cybrids during protoplast fusion.

23. PLANT IMPROVEMENT The cybrid produced by electrofusion between Citrus unshiu and C.sinensis . Restoration of male fertile N icotiana by fusion of protoplast derived from two different cytoplasmic male sterile cybrids . Correct deficiencies of nitrate reductase and xanthium dehydrogenase activities in tobacco by fusing its normal protoplast with protoplast of Physalis and Datura inactivated by x-ray treatments. Streptomycin resistance has been transferred from N icotiana tabacum to three other species of tobacco.

24. Cytoplasmic traits as markers Chloroplast and mitochondria possess many genes- phenotypic expression. The chloroplast trait, RuBPcase activity was exploited for indentifying the somatic hybrids of Nicotiana langsdorffii and N.glauca . cytoplasmic male sterility(CMS) is strictly maternally inherited character having control from mitochondria. CMS is controlled by nuclear/mitochondrial interaction as in Nicotiana - organelle marker. CMS was used to sort out the somatic cybrids /hybrids in Nicotiana debneyi (CMS line) and N.tabacum cross.

25. CMS-ASSOCIATED PHENOTYPES IN CYBRIDS Cybrids of Nicotiana tabacum and Hyoscyamus niger . A protoplast fusion –based alloplasmic cytoplasm transfer by conventional backcrosses- useful for generating alternative CMS sources with novel nucleo cytoplasmic compositions . These alterations resulted in modified floral and vegetative phenotypes.

26. LIMITATIONS Biparental inheritance of cytoplasm during sexual reproduction occurs in only a few genera . Plant regeneration from protoplasts is often a difficult, or even impossible task . Instability of transferred genes in somatic hybrids. R ecovering controlled asymmetric hybrids due to factors like cell fusion, nuclear fusion, nuclear genes segregation and recombination.

27. SOURCES S.Ignaciumuthu.S , PLANT BIOTECHNOLOGY Chawla , H.S . Introduction to plant biotechnooogy Agricultural biotechnology

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