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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