Agrobacterium - mediated Gene Transfer

1. Co-integration And Binary vector
Strategy

2. Agrobacterium - mediated Gene
Transfer
Most common method of engineering dicots
Soil borne, gram negative, rod shaped, motile
found in rhizosphere
Causative agents of “Crown gall” disease of
dicoltyledones
Have ability transfer bacterial genes to plant
genome
Attracted to wound site via chemotaxis in
response to chemicals (sugar and Phenolic
molecules: acetosyringone) released from
damaged plant cells
Contains Ti plasmid which can transfer its T-
DNA region into genome of host plants

3. Infection and tumor genesis
Infection occurs at wound sites.
Involves recognition and chemotaxis of the
bacterium toward wounded cells.
galls are “real tumors”, can be removed and
will grow indefinitely without hormones.
genetic information must be transferred to
plant cells.

4. Tumor characteristics
1. Synthesize a unique amino acid, called “opine”
octopine and nopaline - derived from arginine
agropine - derived from glutamate
2. Opine depends on the strain of A. tumefaciens.
3. Opines are catabolized by the bacteria, which
can use only the specific opine that it causes
the plant to produce.

6. Ti-plasmid features
 Two strains of Ti-plasmid:
 -Octopine strains- contains two T-DNA region: TL
(14 kb) and TR ( 7 kb)
 -Nopaline strains- contain one T-DNA region(20 kb)
 Size is about 200 kb
 Has a central role in Crown-gall formation
 Contains one or more T-DNA region that is
integrated into the genome of host plants
 Contain a vir region ~ 40 kb at least 8~11 vir genes
 Has origin of replication
 Contains a region enabling conjugative transfer
 Has genes for the catabolism of opines

8. Limitations
• T-DNA size is too large.
• Presence of oncogenes.
• Lack of MCS & selectable marker gene.

9. The Binary Vector Strategy
• The T-DNA does not need to be physically associated
with the vir genes in order to become integrated into
the plant genome.
• Systems in which T-DNA and vir genes are located
on separate replicons were termed T-DNA binary
systems.

11. Co-integration Strategy
• In this strategy, the gene of interest to be
introduced into the Ti plasmid vector is first sub-
cloned in a conventional E.coli plasmid vector
such as pBR322 for easy manipulation, producing
a so called intermediate vector.
• The insertion of gene of interest into a Ti plasmid
results from the recombination of intermediate
vector and a Ti plasmid.
• The recombination takes place through a
homologous region present in both plasmids.

14. Examples of these vectors include:
• SEV series: the right border of the T-DNA together with the
phytohormone genes coding for cytokinin and auxin are
removed and replaced by a bacterial kanamycin resistance
gene while the left border and a small part of the left segment
(TL) of the original T-DNA (referred to as Left Inside
Homology (LIH)) are left intact.
• pGV series: the phytohormone genes are excised and
substituted by part of pBR322 vector sequence. The left and
right border sequences as well as the nopaline synthase gene of
the Ti plasmid are conserved.

15. Disadvantages:
• Long region of homologies required between the Ti
plasmid and the E. coli plasmids (pBR322 based
intermediate vectors) making them difficult to
engineer and use
• Relatively inefficient gene transfer compared to the
binary vectors

16. THANK YOU