An agrobacterium based vector to introduce plant genomic fragments., very much used to produce genetic modified plants Pla

2. Introduction
• Ti plasmid is of natural occurrence in in a soil
bacteria – Agrobacterium tumefaciens infecting
dicot plant plant cell and induced tumour
formation ( Crown Gall)
• Entry of this bacteria is facilitaed by secretion of
some phenolic compound from wounded tissues
like Acetosyringone,hydroxyacetosyringone
T DNA is introduced in to plant cell , get integrated in the
plant cell chromosomes and induces un controlled growth

3. Analogous to pUC19 with enormous size of 200kb as compared to pUC19 of
size 4.9 kb
Size -200 kb , independent , extra chromosomal, circular DNA
T DNA -12-24 Kb
Ti plasmid DNA has three regions
T DNA – contains gene for biosynthesis of auxin, opine and cytokiine ( CYT)
and opine (ocs)and flanked by left & rt boarder.
T DNA boarders - a 24 kb on eithe side of Lt & Rt of T DNA
Rt is more critical for Transfer
2. Virulence Regions- gene for promoting transfer of T DNA and located
outside T DNA, is vir ( Virulene regions)- ~ 40 kb at least 8~11 vir genes
Nine vir gene, -vir A, G, B1, C1, D1, D2 D4 E1 & E2
Opine catabolism regions – encode a protein helping uptake and metabolism
of opine to provide C & N
Ori region – replication
After infection T DNA is excised and get integrated with chromosomal DNA by
homologous recombination.

4. Classification -On the basis of Opines
• Nopaline plasmids : carry gene for synthesizing
nopaline in the plant and for utilization
(catabolism) in the bacteria.
• Octopine plasmids : carry genes to synthesize
octopine in the plant and catabolism in the
bacteria.
• Agropine plasmids : carry genes for agropine
synthesis and catabolism.

5. Genetic Map
30 kb
a o7 kb
a 14 kb
Two component of Ti plasmid are responsible for transformation
1.T DNA
Vir region
T Dna integrates at random site or Multiple sits

6. Structure of T DNA
30 Kb

7. GENE WITHIN T DNA
Gene Product Function
ocs Octopine synthetase Opine synthesis
nos Nopaline synthetase Opine synthesis
Tms 1 Tryptophan 2 mono
oxygenase
Auxin
Tms 2 Indole acetamide hydrolase auxine
tmr Isopentyl transferase cytokinin
ags Agropine synthetase cytokinin
Opine – condensation product of either aa & keto acids or
aa and a sugar which act as carbon & nitrogen source

8. Nopaline
Plasmid
Lt & Rt has imperfect terminal repeats and rt removal blocks transfer of T DNA , vir
gene for enzymes needed in excision , integration and transfer during
transformation. Located either cis or in trans positiion T DNA express in very low
levelsin bacteria grwing in soil but in plant increase.within 10-15 hrs
acetosyringosine act as inducer of vir gene

9. The process of transfer and integration is mediated by
Signal transduction – The release chemical acts as signal
molecules to attract Agrobacterium
Attachment –Cellulosic fibres of bacteria helps in
attachment. For this chv ( Chromosomal virulence gene of
bacterial cell to plant cell
Production of virulence protein –a series of event follows
the attachment resultin production of virulence protein
At first vir A expresses which in turn activate vir G and the
DI, D2, E2 B etc are expressed
Some sugars like glucose , galactose , xylulose act as
stimulator.

10. Transfer
Lt & Rt boarder of T DNA are recognised by vir D /vir D2 protein for the
production of ssT DNA ,its production and exportto plant cell The SST
DNA gets attached to vir D2
SS T DNA –vir D2 complex in association with vir G is exported
from bacterial cell . Vir B forms transport apparuts
Transfer of T DNA and integration
TDNA – vir D@ complex cross plasma membrane . Complex interact with
vor E2 protein whch prevent T DNA degradation from nuclease
Interaction of vir D2 and E2 and other proteins inflence transport and
integration.
The T DNA –vir D2 vir E2 complex enters through nuclear pore complex

11. Function of vir genes
• virA - transports acetosyringone into bacterium,
activates virG post-translationally (by
phosphorylation)
• virG - promotes transcription of other vir genes
• virD2- endonuclease/integrase that cuts T-DNA at
the borders but only on one strand.
• virE2 - can form channels in membranes
• virE1 - chaperone for virE2
• virD2 & virE2 also have NLSs, gets

12. Vir gene
Protein products needed for T DNA transfer by
acting as certain receptor for the ligands
secreted by wounded sites in plant . It is neve
trasfeerd in to plant
Vir A-H ( eight types ) and has separate operon
Excision , transfer and translocation in the nucleus

13. Signal transduction
• Acetosyringone interacts with Vir A and auto
Phosphorylte at Histindine
• Transfer of P to aspartate of vir G which act as TF for other V
genes
• Activated vir G binds to enhancer (12 bp) present within promotor
of vir A,B,C D E operons
• Expression generates SS T DNA
• Vir D1 2 act as endonuclease and cut at 5’sit of T boarder while
cutting it remains attach to prevent exonucleotic activity
• Excised ss T DNA is transferred and with the help of vir E and D get
translocated in to the nucleus as they have NLS
• SST DNA converted into Ds T DNA
• By recombination it gets integrated into the plant genome for
transformation

14. Transfer Mechanism

15. Overview of Infection Process

16. Neopaline Ti plasmid is important
• synthesis enzymes for production of Auxin
(IAA) & Cytokine Isopentyl adenosine. For
growth production .
• Antibiotic marker for selection Kanamycin,and
the realted aminoglycoside G418
• Hygromycin and to the drug
• methotrexate

17. Ti plasmid as vector
This ability wass consideed to craete vector to transfer the desired gene for
for transformation of plant cell
Limitation mto be used as vector
Ti Plasmid are large so to be used as vector it
has reduce its size
Absence of RE so , it has to be incorporated
Phytoharmones and auxin synthesising gen
have to remove.
Opine production lowers the transformed yield
so has to be tremoved

18. Ti p as vector
• T DNA is transfer so it is replaced by desired gene to
be consided as vector in transformin plant cell.
• F If it is disarmed i.e removed Rt & lt donot cause
tumour so transformed can not be identifed
• For this a marker kanamycin R e.coli transposan Tn5
from is added with disarmed T I plasmid / This forms
neomycin phosphotransferase type II ( NPTII which
detoxify the kanamycin by phosphorylation
• Since it is bacterial specofic therefore can not express
in plant so plant specific protein is ,termination and
poly adenyaltionsequece. Such marker I s calleed
chimeric selectable marker

19. • 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

20. 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.

21. Practical application
• 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.

22. Process of Transformation
1
• Explants ( cotyledon, Hypocotyls , microspore
2
• Co-cultivation to allow infection with agrobacterium containg Ti vector
• Careendiline to kill bacteria
3
• Transformed or nontransformed
• Selective medium to kill non transformed 9 addition of kanamycin
4 •Transformed shoots
5 •Rooted shoots
6 •Adult plant

23. 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.