Agrobacterium tumefaciens is a tool for genetic engineering in plants. It is a soil bacterium that can transfer DNA fragments called T-DNA from its tumor-inducing plasmid into the genome of plant cells. The T-DNA can be modified to contain desirable genes for traits like herbicide or pest resistance. The bacterium recognizes wounded plant cells and transfers T-DNA using virulence genes. Integrated T-DNA is then expressed stably in the plant genome. Agrobacterium-mediated transformation is widely used for genetic engineering in plants due to its simplicity, efficiency and ability to transfer large DNA segments.

1. TOPIC: Agrobacterium tumefaciens as a tool for
genetic engineering in plants
By:
Sourabh Sharma
(14MBT003)
Dr. Samantha Vaishnavi
Prof. SBT, SMVDU

2.  To increase enormous potential of plants for agriculture, producing more nutritious
and higher-yielding crops that are resistant to environmental stresses (cold and
drought), insect pests and diseases one has to introduce desired/foreign
/recombinant DNA into the plant`s genome.
 It is well known that plants can propagate vegetatively and some plant cells are
totipotent, having the potentiality of regeneration and differention to give complete
plant. (Animal cells donot have this capacity)
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INTRODUCTION

3.  Microinjection
 Biolistics- Gene gun
method/ Particle
bombardment method
 Electroporation
 Silica carbon/ fibres
 Laser mediated
 PEG
 DEAE-dextran
 Calcium
phosphate
 Artificial lipids
 Dendrimers
 Agrobacterium
tumefaciens
 Agrobacterium
rhizogenes
 Viruses mediated
Physical Chemical Biological
Plant Transformation Methods

4.  Common Features of Agrobacterium tumefaciens
 Soil borne, gram negative, rod shaped, motile found in rhizosphere.
 Causative agents of “Crown gall” disease of dicotyledons.
 Have ability to 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 .

5. Tumor inducing (Ti) Plasmid
 Vir region is organised into 8 Operons.
 And has approx. 25 genes.
 Vir region mediates the transfer of T-DNA to the plant genome.
Conjugal transfer reg.
25 bp repeat
25 bp repeat

6. Ti plasmid features…..
 Two strains of Ti plasmid:
 Octopine strains- contains two T-DNA regions 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 in the
genome of host plant.
 Contains a vir region (40 kb) with atleast 8-11 kb vir
genes that transfers T-DNA to plant cell.
 Has origin of replication.
 Contains a region enabling conjugal transfer.
 Has genes for the metabolism of opines.

7. Vir genes Functions
Vir A Kinase; Encodes acetosyringone receptor protein, also activates vir G by
phosphorylation leading to constitutive expression of all genes.
Vir B Encodes membrane protein, involved in conjugal tube formation through which
the T- DNA is transport.
Vir C Encodes Helicase enzymes; unwinding of T-DNA.
Vir D Topoisomerase activity; vir D2 is an endonuclease.
Vir E Single strand binding protein (SSBP); binds to T-DNA during transfer.
Vir F Activity not known.
Vir G Master controller DNA binding protein; vir A activates vir G by phosphorylation,
vir G dimerises and activates constitutive exp. of all vir genes.
Vir H Activity not known.
Vir genes and their
Functions....

8. Overview of the Infection Process

9.  Acetosyringone and α-hydroxyacetosyringone binds with Vir A
protein (located in the inner membrane) and activates it. It start
functioning as autokinase to phosphorylate itself by ATP.
Phosphorylated Vir A protein then phosphorylates Vir G protein which
then dimerises.
 Phosphorylated Vir G protein has DNA binding function. It induces
expression of rest of Vir operons.
 Vir D1 protein has topoisomerase and endonuclease activity.
 It binds to right border sequence of T-DNA and facilitate the action of
Vir D2 protein which is also endonuclease and nicks at the right
border and remains bound to 5’end so generated.
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Mechanism of Transfer

10.  The 3’end produced at the site of nick serves as a primer for DNA synthesis in 5’--
--3’ direction as a result of which one strand of
 T-DNA is displaced from the DNA duplex.
 The T-DNA strand is again nicked at the left border to generate a single strand
copy of T-DNA.
 To this single strand copy Vir E 2 protein (single strand DNA binding proteins )
bind for its protection against exonucleases
 Vir B operon consisting of 11 genes encode membrane bound Vir B proteins.
These along with Vir D4 proteins participate in conjugal tube formation between
bacterial and plant cells for transfer of T-DNA
 Vir D2 which remains bound to 5’end of T DNA has a signal sequence which
drives it into the nucleus of plant cell.
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11. Generation of the T-strand.
overdrive
Right
BorderLeft Border
T-DNA
virD/virC
VirD nicks the lower strand (T-strand) at the right border
sequence and binds to the 5’ end.
5’

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

13. 1.Transferto plantcell.
2.Secondstrandsynthesis
3.Integrationintoplantchromosome
Right
border
Left
border
D
T-strand coated with virE
T-DNA
virD nicks at Left Border sequence
Cont…

14. INTEGRATION OF T-DNA INTO PLANT GENOME
 T-DNA enters plant cell as a single stranded structure which is immediately converted
into double stranded form.
 Vir E2 also has nuclear localization sequence and is responsible for transfer of T DNA
into plant cell nucleus
 Double stranded T-DNA integrate at random sites in the host plant genome.
 For integration 23-79 base pair deletion takes place at the integration or target site
After integration of T-DNA into plant genome ,the genes for auxins, cytokinins and
opines express themselves which result in uncontrolled growth in the form of tumor
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15. cont.
..

16. HARNESSING AGROBACTERIUM IN TRANSFERRING FOREIGN
GENE INTO PLANT…….
 Agrobacterium’s ability to introduce its DNA into plant genome with efficiency makes Ti
plasmid an attractive vector for gene transfer into plants.
 T-DNA is modified as needed, before actual transfer of desired gene. Genetically
engineered Agrobacterium were produced with disarmed T-DNA region in which
oncogenes and opine biosynthesis genes were replaced by desired (Foreign ) gene,
selectable marker gene and a unique restriction site. The natural Ti plasmids are unsuitable
to be used directly as vectors for invitro manipulation due to following reasons;
 1) Large size 2) Tumor induction (Oncogenic ) property 3)Absence of unique restriction
enzyme site.
 Disarmed T-DNA , left and right borders along with genes of Vir region are essential
elements for designing of transformation vectors. The strategies for harnessing
Agrobacterium for introduction of new/desired/foreign genes into plants are; 16

17. Prototype disarmed Ti-vectors
 T-DNA has been disarmed by making it nononcogenic by deleting all of its
oncogenes.
Zambryski et al. (1983) substituted pBR322 sequences for almost all of the T-
DNA of pTiC58, leaving only the left and right border regions and the nos gene
and resulting construct was called pGV3850.
Opine production was exploited as a screenable phenotype, and the ocs and nos
genes have been widely used as screenable markers.
To achieve high-level expression in plants, a very active promoter is required. In
dicots, promoters from the Agrobacterium nopaline synthase (nos), octopine
synthase (ocs) and mannopine synthase (mas) genes have been widely used.
 Dominant selectable markers have been inserted into the T-DNA, so that
transformed plant cells can be easily selected on the basis of drug or herbicide
resistance.

19.  Disarmedderivativesofwild-typeTiplasmidscanbeusedforplant
transformation,becausetheirlargesizemakesthemdiffculttomanipulateinvitro.
T-DNAisolatedfromaparentTiplasmidwassubclonedinaconventionalE.coli
plasmidvectorforeasymanipulation,producingaso-calledintermediatevector.
ConjugationbetweenthetwoE.colistrainstransferredthehelperplasmidtothe
carrieroftheintermediatevector,whichwasinturnmobilizedandtransferredto
therecipientAgrobacterium.
HomologousrecombinationbetweentheT-DNAsequencesoftheTiplasmidand
intermediatevectorthenresultedintheformationofalargecointegrateplasmid,
fromwhichtherecombinantT-DNAwastransferredtotheplantgenome.

21. Intermediate vectors have been widely used, the large cointegrates are not easy for
transformation. The vir-genes of the Ti plasmid function in trans and can act on any T-
DNA sequence present in the same cell.
Therefore, the vir genes and the disarmed T-DNA containing the transgene can be
supplied on separate plasmids, and this is the principle of binary vector systems
(Hoekma et al. 1983, Bevan 1984). The T-DNA can be subcloned on a small E. coli
plasmid for ease of manipulation.
This plasmid, called mini-Ti or micro-Ti, can be introduced into an Agrobacterium
strain carrying a Ti plasmid from which the T-DNA has been removed. The vir
functions are supplied in trans, causing transfer of the recombinant T-DNA to the plant
genome.
Binary Ti-Vectors

22. Binary vector Strategy of Ti -Plasmid

23.  Glufosinate herbicide
 Sethoxydim herbicide
 Bromoxynil herbicide
 Glyphosate herbicide
 Sulphonylurea herbicide
 Bt Gene
 Modified Fatty Acid
Gene
 Flower Colour
 Flower Life
 Delayed Fruit
Ripening Gene
 Virus Resistance
Improved Genetic Traits in Plants using A. tumefaciens
r
r
r
r
r

24. TRANSFORMATION TECHNIQUE USING AGROBACTERIUM
•Agrobacterium gene transfer can be achieved in the following two
ways
 Co culture with tissue explants.
 In planta transfusion.
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25. Co- culture with tissue explants
 For transformation explants like protoplast, callus , tissue slices, leaf disc,
stem or floral tissue etc. can be co-cultured with genetically engineered
Agrobacterium with recombinant vectors for about 2 days .
 The explants can then be transferred to shoot inducing (regeneration
medium ) containing kanamycin and carbenicillin.
 Kanamycin allows only transformed plant cells to divide and regenerate
shoots in about 3-4 weeks, while carbenicillin kills Agrobacterium cells.
 The shoots are separated and transferred to root inducing medium and
finally after few weeks are transferred to soil.
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27. In planta transformation
Transformation can also be achieved by imbibition of
seeds in fresh cultures of Agrobacterium. T-DNA
becomes integrated into plant genome. It appears that
Agrobacterium cells enter the seedling during
germination, are retained within the plants, when
flowers develop, the zygote or cells become
transformed.

28. Agrobacterium mediated gene transfer is most frequenly used indirect
method, it has many advantages;
Advantages of Agrobacterium mediated gene transfer
• Simple and comparatively less expensive.
• High transformation efficiency.
• Transgenic crops obtained have better fertility percentage.
• Protocols for both dicotyledons and monocotyledon are available.
• Relatively large length DNA segment can be transferred.

29. Disadvantages of Using
Agrobacterium as Vectors
• Agrobacterium can only be used to clone
genes in dicots like potato, tomato,
tobacco, peas and beans etc. but not
monocots like wheat, rice , barley
etc.which are most important crop
plants.due to ; lack of production of
Acetosyringone or α-
hydroxyacetosyringone like phenolics that
the bacteria appear to respond to simple
molecules, such as sugars and amino acids.
• Time consuming.

30. 2005/12/16