Agrobacterium tumefaciens is commonly used to genetically modify dicot plants through its ability to transfer DNA to plant cells. It causes crown gall disease by transferring oncogenic T-DNA from its tumor-inducing plasmid into wounded plant cells. The T-DNA encodes genes that cause tumor formation and the production of opines, which the bacteria can use as nutrients. The binary vector system was developed to overcome challenges with manipulating large Ti plasmids, allowing foreign genes to be stably introduced between the T-DNA borders and transferred to plant cells. Agrobacterium-mediated transformation is now widely used to produce transgenic plants.

1. AGROBACTERIUM TUMEFACIENS
MEDIATED GENE TRANSFER
IN PLANTS
SUSHANTA SARMA
M.Sc. BIOTECHNOLOGY

2. Agrobacterium - mediated Gene Transfer
 Most common method of engineering dicots, but also used for monocots
 Pioneered by J. Schell (Max-Planck Institute, Cologne)
 Agrobacterium-
 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. Agrobacterium tumefaciens
 the species of choice for engineering dicot plants; monocots are generally
resistant.
 some dicots more resistant than others (a genetic basis for this).
 complex bacterium – genome has been sequenced; 4 chromosomes; ~ 5500
genes.

4. Infection and tumorigenesis
 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.

5. 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. Elucidation of the TIP (tumor-inducing
principle)
 It was recognized early that virulent strains could
be cured of virulence, and that cured strains could
regain virulence when exposed to virulent strains;
suggested an extra-chromosomal element.
 Large plasmids were found in A. tumefaciens and
their presence correlated with virulence: called
tumor-inducing or Ti plasmids.

7. 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. Ti Plasmid
(14 bp repeat)
(7 bp repeat)

9. Ti plasmids and the bacterial chromosome
act in concert to transform the plant
 Agrobacterium tumefaciens chromosomal genes: chvA, chvB, pscA
required for initial binding of the bacterium to the plant cell and code
for polysaccharide on bacterial cell surface.
 Virulence region (vir) carried on pTi, but not in the transferred region
(T-DNA).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. Generation of the T-strand
overdrive
Right
Border
Left
Border
T-DNA
virD/virC
5’
VirD nicks the lower strand (T-strand) at the
right border sequence and binds to the 5’ end.

11. Generation of the T-strand
Right
border
Left
border
T-DNA
D
virD/virC
gap filled in
T-strand
virE
1. Helicases unwind the T-strand which
is then coated by the virE protein.
2. ~one T-strand produced per cell.

12. Right
border
Left
border
D
T-DNA
T-strand coated with virE
virD nicks at Left Border sequence
1. Transfer to plant cell.
2. Second strand synthesis
3. Integration into plant chromosome

13. Overview of the Infection Process

14. Important points:
 Monocots don't produce AS in response
to wounding.
 Put any DNA between the LB and RB of
T-DNA it will be transferred to plant cell.
Engineering plants with
Agrobacterium:
Two problems had to be overcome:
 Ti plasmids large, difficult to manipulate
 couldn't regenerate plants from tumors

15. Binary vector system:
 Strategy:
 Move T-DNA onto a separate, small plasmid.
 Remove aux and cyt genes.
 Insert selectable marker (kanamycin
resistance) gene in T-DNA.
 Vir genes are retained on a separate plasmid.
 Put foreign gene between T-DNA borders.
 Co-transform Agrobacterium with both
plasmids.
 Infect plant with the transformed bacteria.

16. Binary vector system:

17. Practical application of
Agrobacterium-mediated plant
transformation:
 Agrobacterium mediated transformation methods
are thought to induce less rearrangement of the
transgene.
 Lower transgene copy number that direct DNA
delivery methods.
 Successful production of transgenic plants
depends on the suitable transformation
protocols.

18. Recent research

19. Conclusion:
Agrobacteria are biological vector for introduction of
genes into plants. Agrobacterium-mediated
transformation is not restricted to eukaryotes as
Agrobacterium is also able to act on the gram positive
bacterium Streptomyces lividans. Agrobacterium can
transfer not only DNA but also proteins to the host
organisms through its type four secretion system.

20. References
 Bevan, M. (1984) “Binary Agrobacterium vectors for plant transformation”. Nucleic
Acids Res 12: 8711-8721.
 Deblaere R., Bytebier B., De Greve H., Deboeck F., Schell J., Van Montagu M. and
Leemans J. (1985) “Efficient octopine Ti plasmid-derived vectors for Agrobacterium-mediated
gene transfer to plants”. Nucleic Acid Research 13:4777-4788.
 Chilton, M.D. (1983) “A vector for introducing new genes into plants”. Scientific
American, 248, 50-9.
 Nadolska-Orczyk, A., Orczyk, W. and Przetakiewicz, A. (2000) “Agrobacterium-mediated
transformation of cereals – from technique development to its
application”. Acta Physiologiae Plantarum, 22, 77-8.
 Kakkar, A. and Verma, V.K.,(2011) Agrobacterium mediated biotransformation.
Journal of Applied Pharmaceutical Science 01 (07); 2011: 29-35.