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
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2. WEL -COME
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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
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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
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6. Contn
….
macro Injection PEG mediated
micro injection polication DMSO
liposome mediated
Sonication
DNA transfer via pollen
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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
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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
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9. Contn
2. On growth pattern
 Biotype I
 Biotype II
 Biotype III
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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
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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
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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
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13. Location of virulence genes.
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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.
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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.
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16. Methods of Agrobacterium
tumefaciens genes transfer.
 Infection through
wound
 Leaf disk method
 Co-cultivation
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17. Infection through wounds
Preparation of vector Explants
selection
Wounding explants
Infection of Agrobacterium
Incubation
Culturing
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18. Contn
……
Callus generation
Shoot formation
Rooting
Regeneration of plant
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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
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20. Contn
Screening
Culturing
Shoot initiation
Rooting
Whole plant
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21. Co cultivation
Isolation of protoplast
Incubation with Agrobacterium
Culturing cells
Regeneration of plant
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22. Lets recall
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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.
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24. Thank you
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