Agrobacterium tumefaciens is a soil bacterium that causes crown gall disease in plants. It transfers a segment of DNA called T-DNA from its Ti plasmid into the plant genome, enabling it to modify plant cell growth. The presentation discusses the bacterial characteristics, mechanism of T-DNA transfer, use of Agrobacterium for genetic engineering of plants through insertion of foreign genes in place of tumor-causing genes on the Ti plasmid, and the regeneration of transformed plants. While it is an effective tool for plant genetic engineering, some plant species are not susceptible to Agrobacterium infection.

1. PRESENTED BY
S.KAAYATHRI DEVI
19PO03
II M.SC., BIOTECHNOLOGY
ANJAC
Agrobacterium tumefaciens
mediated gene transfer
Agrobacterium
tumefaciens
mediated gene
transfer
Presented by
S.Kaayathri Devi
19PO03
II M.Sc., Biotechnology

2. Agrobacterium tumefaciens
Introduction
 Smith and 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.l (1974) – noted virulent strain
Agrobacterium tumefaciens
 Chilton.et.al (1977)- reported Ti & Ri plsmid transfer
to plant causing disease.

3. SYSTEMIC POSITION
 Kingdom: Bacteria
 Phylum: Proteobacteria
 Class: Alpha Proteobacteria
 Order: Rhizobiales
 Family: Rhizobiaceae
 Genus: Agrobacterium

4. Agrobacterium (little genetic engineer):
 Agrobacterium tumefaciens is the causal
 agent of crown gall disease
 Agrobacterium tumefaciens- Crown gall
disease
 Agrobacterium rhizogenes - Hairy root disease
 Agrobacterium radiobacter- Avirulent strain

5.  A soil-born gram negative bacterium. It is a rod
shaped and motile and belongs to the bacterial
family of Rhizobiaceae.
 It is a phytopathogen, and it is regarded as Nature’s
most effective plant genetic engineer.
 It is the natural expert of inter-kingdom gene
transfer.

6. Infects wounded or damaged part of plant causing
plant tumor called crown gall.
 The entry of the bacterium is faciliated by the
release of phenolic compounds like Acetosyringone
and Hydroxysyringone.
Crown gall occurs when the bacterium releases its Ti-
plasmid into the plant cell cytoplasm. The T-DNA is
transferred to the host cell via wounded section.

7.  The T-DNA carries genes that code for proteins
involved in the biosynthesis of growth hormones
(auxin and cytokinin) and novel plant metabolites
namely- opines and agropines.
 Growth hormones – plant cell proliferation
 Opines & agropines – source of C and energy

8. Ti Plasmid:
 A Ti plasmid is a circular piece of DNA found
 in almost all Agrobacteria
 Three main region:
 T-DNA region(Between right and left T-DNA
border)
 Oncogene
 Opine(argenine derivative)
 Virulence region
 Opine catabolism region

10. VIRULENCE GENES(24 GENES):
 Located in 8 operons from vir A to vir H
 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 supercoiled stranded
substrate
 Vir E responsible for gene transfer protein
 Vir C helps in DNA transfer
 Vir B11 ATPase activity – provides energy for DNA

11. Agrobacterium mediated gene transfer
 1. Signal induction to Agrobacterium –
phenolic compounds and some sugars, induces
biochemical changes that help in T-DNA transfer.
 2. Attachment of Agrobacterium to plant cells –
Agrobacterium attaches to cell through polysaccarides and
cellulose fibres.
 3. Production of virulence protein – signal
induction causes virulence protein to form Vir A,which
induces Vir G, this then induces the production of rest of
the virulence proteins like Vir D1/D2, Vir E, Vir B.

12.  4. Production of single stranded T-DNA – this is
recognised by vir D and thus carried forward to the host by Vir
D2.
 5. Transfer of T-DNA out of Agrobacterium – the DNA
strand is carried out by Vir D2. Through a channel made by
Vir B.
 6. Transfer of T-DNA into plant cells and integration
integration of the DNA into the host cell is helped by VirE2,
which protects the DNA from degradation by host cell
restriction modification system, while Vir D2 helps to
navigate the DNA to the nucleus.
 This process is called ILLEGIMATE RECOMBINATION,
since it does not depend on the sequence similarity.

15.  Regeneration:
for shoot organogenesis, cytokinin (lower amounts of
auxin) are required.
 Selection: (two antibiotics are required)
1. An antibiotic to kill the agrobacterium, while not
affecting the plant’s cell growth and division.
2. A second antibiotic allows growth of transformed
shoots but inhibits growth of untransformed plant
cells.

16.  Detection of the “trait” gene:
1. PCR methods can detect the presence of the trait
DNA.
2. protein detection methods are used where a gene
product is produced that defines the trait.
3. verification of the corporation of the trait gene into
the plant’s chromosome.
 By southern hybridization
 By demonstrating transfer of the trait to the original
transformant’s progeny

18. Pro’s n Con’s
 Scientists can insert any gene they want into the
plasmid in place of the tumor causing genes and
subsequently into the plant cell genome
 By varying experimental materials, culture
conditions,
bacterial strains, etc. scientists have successfully used
A. tumefaciens Gene Transfer to produce BT Corn
 This method of gene transfer enables large DNA
strands to be transferred into the plant cell without
risk of rearrangement whereas other methods like
the Gene Gun have trouble doing this.

19.  The vast majority of approved genetically engineered
agriculture has been transformed by means of
Agrobacterium tumefaciens Mediated Gene
Transfer
 Original problems existed in that Agrobacterium
tumefaciens only affects dicotyledonous plants
 Monocotyledon plants are not very susceptible to the
bacterial infection

21. References
 https://www.slideshare.net/NISHANTHSEKAR1/ag
robacterium-mediated-gene-transfer-77002058
 https://www.slideshare.net/Rahulselvaraj/agrobacte
rium-mediated-gene-transfer-140707990
 All the images and pictures used in this presentation
are taken from Google Inc.,(via google search) and
from slideshare.