Agrobacterium mediated gene transformation

simple and rapid explanation to process of agrobacterium mediated gene transfer

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 References 3

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 I nd irect method D irect method Agrobacterium mediated gene transfer Physical Chemical DNA imbibitions electroporation by cell, tissue, etc particle bombardment calcium phosphate 5

6. Cont n …. 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 Basis of pathogenicity. Agrobacterium tumefaciens - crown gall. Agrobacterium rhyzogen- hairy root Agrobacterium radiobacter- Avirulent Crown gall disease Hairy root disease 8

9. Cont n 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 region 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 D 1 essential for cleavage of super coiled stranded substrate vir E responsible for gene transfer protein vir C helps in DNA transfer vir B 11 ATPase activity- provides energy for DNA movement 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 chv D, ilv, mia A,and att, have virulence property. 14

15. Agrobacterium tumefaciens T-DNA transfer process Steps of gene transfer in plant:- Bacterial colonization Induction of virulence system Generation of T-DNA transfer complex T-DNA transfer 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. Cont n …… 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

20. Cont n Screening Culturing Shoot initiation Rooting Whole plant 20

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