This document discusses Agrobacterium-mediated gene transfer in plants. It describes how Agrobacterium tumefaciens, a soil bacterium, can transfer tumor-inducing (T-DNA) segments from its Ti plasmid into the DNA of plant cells. The T-DNA is integrated into the plant genome and can include genes added through genetic engineering. The process involves inserting a gene of interest into the T-DNA, infecting plant cells with Agrobacterium, and regenerating whole plants from transformed cells. Agrobacterium-mediated transformation allows stable integration of genes and is widely used for genetic modification of crops.
1. Agrobacterium Mediated Gene
Transfer in Plants
M.Veeralakshmi
18PY21
II M.scMicrobiology
AYYA NADAR JANAKI AMMAL
COLLEGE
Department of Microbiology.
2. 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.
3. Scientific classification
• Kingdom :Bacteria
• Phylum :Proteobacteria
• Class :Alproteobacthaperia
• Order :Rhizobiales
• Family :Rhizobiaceae
• Genus :Agrobacterium
• Species :A. tumefaciens
4. 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
6. T-DNA
• The T-DNA is present in plasmid of
agrobacterium .
• Its encodes for the tumorous growth and an
opine.
• The plasmid has aVir gene is used for mobilization
o the T-DNA into the chromosomal DNA.
• The T-DNA of 21,000 base pairs size for
tomourogenesis.
• It has many genes for tumour induction.
7.
8. Ti Plasmid
• Large size plasmid of 200 kbp.
• The Ti plasmid is lost when Agrobacterium is grown above
28⁰C(curing of plasmid). Such cured bacteria do not induce
crown galls.
• Plasmids have T-DNA, right border, left border, vir genes,
origin of replication and opine region.
• Genes code for proteins that prepare the T-DNA and the
bacterium for transfer.
• T-DNA encodes genes for opine synthesis and for tumor
production.
• occ (opine catabolism) genes carried on the pTi allow the
bacterium to utilize opines as nutrient.
10. Genes of T-DNA
• Main regions:
T-DNA region(Between right and left T-DNA
border)
Oncogene
Ori
Virulence region
Opine catabolism region
Tra gene
11. Virulence gene
• Vir A: Transmembrane protein Vir
phosphorylates . Vir G binds to promoter and
activates vir genes
• Vir D1 and D2: Border endonucleases (D2
remain attached)
• Vir E: SSB protein
• Vir B: transport process
• T DNA transported and integrated into host
Complementary strand synthesis
12. Functions of gene
• Tra gene: it is used for transfer of the plasmid
one bacterium to another bacterium.
• Occ gene: it is responsible for the catabolism
of octopine.
• Ori gene(origion of replication): it is used for
autonomous replication of the plasmid.
13.
14. Steps of biological transfer
• The method of gene transfer through Agrobactrium using Ti plasmid.
Production of transgenic plants
Isolate and clone gene of interest
Add DNA segments to initiate or enhance gene
Expression
Add selectable markers
Introduce gene construct into plant cells (transformation)
Select transformed cells or tissues
Regenerate whole plants
22. Advantages of Agrobacterium gene mediated
gene transfer
• This method is used for the genetic
manipulation of plants for changing the plant.
• It is used to release improved crop varieties.
• It is used to study the gene function, promoter
analysis and inducible genes.
23. DISADVANTAGES
• Time consuming
• Not all kind of cells can be treated by this
• method
• Sometimes leads to false positive result
