Agrobacterium mediated gene transformation
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Transcript of Agrobacterium mediated gene transformation
Presented by – Aware Swapnil
Registration No- BTN-2012/07
Valuable guidance by- Prof S. A. Belge.
Agrobacterium mediated gene
transformation
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WEL -COME
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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
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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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
Contn….
macro Injection PEG mediated
micro injection polication DMSO
liposome mediated
Sonication
DNA transfer via pollen
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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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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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Contn
2. On growth pattern
Biotype I
Biotype II
Biotype III
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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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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 virregion
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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 movement
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Location of virulence genes.
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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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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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Methods of Agrobacterium
tumefaciens genes transfer.
Infection through
wound
Leaf disk method
Co-cultivation
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Infection through wounds
Preparation of vector Explants
selection
Wounding explants
Infection of Agrobacterium
Incubation
Culturing
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Contn ……
Callus generation
Shoot formation
Rooting
Regeneration of plant
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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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Contn
Screening
Culturing
Shoot initiation
Rooting
Whole plant
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Co cultivation
Isolation of protoplast
Incubation with Agrobacterium
Culturing cells
Regeneration of plant
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Lets recall
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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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Thank you
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