This document summarizes Agrobacterium-mediated gene transfer. It begins with an introduction to gene transformation and describes Agrobacterium as a soil bacterium that causes crown gall disease in plants by transferring genes. It then discusses the classification of Agrobacterium, the history of using Agrobacterium for gene transfer, and components involved like the T-DNA, virulence genes, and chromosomal genes. The document outlines the process of T-DNA transfer from Agrobacterium to plant cells and concludes with some common methods for Agrobacterium-mediated gene transfer.
2. CONTENTS
Introduction to gene 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.
Method of agrobacterium tumefaciens .
References.
3. GENE TRANSFER INTRODUCTION
Gene transfer is to transfer a gene from one
DNA molecule to another DNA molecule .
The direct desirable gene transfer from one
organism to another and the subsequent
stable integration & expression of foreign gene
into the genome is referred as genetic
transformation .
Transient transformation occur when DNA is
not integrated into host genome
4. Agrobacterium mediated gene transfer
Agrobacterium:
Soil borne ,gram negative ,rod shape motile found in
rizosphere .
Causative agents of “crown gall "disease of
dicotyledons .
Have ability transfer bacterial genes to plant genome .
Attracted to wound site via chemotaxis in response
to chemical (sugar and phenolic molecules
:acetosyringone )released from damaged plant cells .
Contain Ti plasmid which can transfer its T-DNA
region into genome of host plants
8. 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 cause
disease .
Zenen et.al (1974 ) –noted virulent stain
Agrobacterium tunefaciens .
Chilton et.al ( 1977 ) –reported Ti & Ri
plasmid transfer to plant causing disease .
9.
10. 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
opiens and phytohormones .
It also contains a vir .
13. VIRULENCE GENES
Virulence region consists of 24 genes in total
Virulence genes are located in 8 operons from virA-virH
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
17. AGROBACTERIUM TUNEFACIENS T-DNA
TRANSFER PROCESS
Steps of gene transfer in plants
I. Bacterial clonization
II. Induction of virulence system
III. Generation of T-DNA transfer
complex
IV. T-DNA transfer
V. Integration of T-DNA into plants
18. METHODS OF AGROBACTERIUM TUMEFACIENS
GENE TRANSFER
Infection through wound
Leaf disk method
Co –cultivation