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Agrobacterium mediated gene transformation
- 1. Presented by – Aware Swapnil Registration No- BTN-2012/07 Valuable guidance by- Prof S. A. Belge. Agrobacterium mediated gene transformation 1
- 2. WEL -COME 2
- 3. Contents Introduction to genetic transformation. Agrobacterium general introduction. Classification of Agrobacterium. History of Agrobacterium mediated gene transformation. T-DNA. Virulence genes. Chromosomal genes Agrobacterium tumefaciens T-DNA transfer process. Methods of Agrobacterium tumefaciens References3
- 4. Introduction to genetic transformation. The process of transferring foreign gene. It’s a natural process seen in bacteria. In plants it’s manually carried out. Two methods of genetic transformation in plants- 1)Direct method. 2)Indirect method. Direct method e.g.- Electroporation, microprojectile bombardment. Indirect e.g.- Agrobacterium mediated gene transformation 4
- 5. Methods of gene transformation Indirect method Direct method Agrobacterium mediated gene transfer Physical Chemical DNA imbibitions electroporation by cell, tissue, etc particle bombardment calcium phosphate 5
- 6. Contn …. macro Injection PEG mediated micro injection polication DMSO liposome mediated Sonication DNA transfer via pollen 6
- 7. Agrobacterium general introduction. Rod shape gram negative bacterium Found in soil Causes diseases like crown gall and hair roots in dicot plants The bacteria transfers a tumor-inducing plasmid The plasmid incorporates with host genome and transcribes 7
- 8. Classification of Agrobacterium. Two basis of classification 1. Basis of pathogenicity. Agrobacterium tumefaciens- crown gall. Agrobacterium rhyzogen- hairy root Agrobacterium radiobacter- Avirulent Crown gall disease Hairy root disease 8
- 9. Contn 2. On growth pattern Biotype I Biotype II Biotype III 9
- 10. History of Agrobacterium mediated gene transformation. Smith & Townsend (1907)- said bacteria caused crown gall disease Brown & Stonier (1958)-proposed that not whole bacteria but some part of it causes disease Zenen et.al (1974)- noted virulent strain- Agrobacterium tumefaciens Chilton et.al (1977)-reported Ti & Ri plasmid transfer to plant causing disease 10
- 11. T-DNA. The transfer DNA (T- DNA) is the transferred DNA The T-DNA initiates at right border and terminates at left border Its about 24000 base pairs Contains genes for enzymes synthesizing opines and phytohormones It also contains a vir 11
- 12. Virulence genes. Virulence region consists of 24 genes in total Virulence genes are located in 8 operons from vir A- vir H vir A vir F and vir G are monocistronic operons , where as vir B,C,D,E,H are polycistronic vir A senses Acitosyringone vir G transcriptional activator of vir box vir B conjugational pores between plant cell and bacteria vir D1 essential for cleavage of super coiled stranded substrate vir E responsible for gene transfer protein vir C helps in DNA transfer vir B11 ATPase activity- provides energy for DNA 12
- 13. Location of virulence genes. 13
- 14. Chromosomal genes Chv A& B major role in exopolysaccharide production psc A major role in T-DNA transport chv E glucose and galactose transport chvD, ilv, miaA,and att, have virulence property. 14
- 15. Agrobacterium tumefaciens T-DNA transfer process Steps of gene transfer in plant:- 1. Bacterial colonization 2. Induction of virulence system 3. Generation of T-DNA transfer complex 4. T-DNA transfer 5. Integration of T-DNA into plant. 15
- 16. Methods of Agrobacterium tumefaciens genes transfer. Infection through wound Leaf disk method Co-cultivation 16
- 17. Infection through wounds Preparation of vector Explants selection Wounding explants Infection of Agrobacterium Incubation Culturing 17
- 18. Contn …… Callus generation Shoot formation Rooting Regeneration of plant 18
- 19. Leaf disk method Preparation of vector selection of explants Cutting leaf disk Infection process Washing using antibiotics like cefotoxime and carbenicillin Selection of transferred and non-transferred tissues 19
- 21. Co cultivation Isolation of protoplast Incubation with Agrobacterium Culturing cells Regeneration of plant 21
- 22. Lets recall 22
- 23. References Introduction to plant tissue culture.(second edition) by M.K.Razdan Agrobacterium mediated gene transformation. Google scholars article. Gene transfer- Wikipedia the free encyclopedia. Plant biotechnology by H.S.chawala. 23
- 24. Thank you 24