Agrobacterium Ti Plasmid

1. PLANT GENETIC ENGINEERING
Amit Kumar Sahoo
II M.Sc. Biosciences
15151

2. Amit Kumar Sahoo
II Msc B
15151
Ti plasmid derived vector systems,
protocols for transformation
Aum Sri Sai Ram

3. INTRODUCTION
WHAT ARE PLASMIDS ?
-PLASMIDS are double stranded, closed circular DNA
molecules, which exist in the cell as extra chromosomal
units.
-Transmitted from one bacterium to another (even of
another species) via horizontal gene transfer.
- Engineered plasmids are widely used as vectors in DNA
cloning .

4. Transgenic plants – An introduction
Transformation – the process of obtaining transgenic plants.
Transgenic plant – a plant with a foreign gene (or genes) from another
plant/animal that is incorporated into its chromosome.
Marc Van Montagu and Jeff Schell, discovered the gene transfer mechanism between
Agrobacterium and plants, which resulted in the development of methods to alter the bacterium
into an efficient delivery system for genetic engineering in plants.
Most common genes (and traits) in transgenic or biotech crops
herbicide resistance
Insecticide resistance
Bt genes in field corn (maize)
virus-resistance (coat-protein) genes

5. Plant Transformation Methods
Physical Chemical Biological
Microinjection
Pressure
Biolistics - gene gun/
particle bombardment
Electroporation
Silica/carbon fibers
Lazer mediated
SAT
PEG
DEAE-dextran
Calcium phosphate
Artificial lipids
Proteins
Dendrimers
A. Tumefaciens
A. Rhizogenes
Virus-mediated

6. Agrobacterium tumefaciens “The Natural Genetic Engineer”
• A. tumefaciens is a gram-negative soil
bacterium which naturally transforms plant
cells, resulting in crown gall (cancer)
tumors.
• Infects plants through breaks or wounds.
• Tumor formation is the result of integration
of T-DNA (Transfer DNA) in plant genome.

7. -A. tumefaciens causes crown gall disease, A. rhizogenes causes hairy root disease, and A. rubi
causes cane gall disease.
-Curing a particular plasmid and replacing this plasmid with another type of tumorigenic plasmid
can alter disease symptoms.
-Perhaps a more meaningful classification system divides the genus Agrobacterium into “biovars”
based on growth and metabolic characteristics.
-As a genus, Agrobacterium can transfer DNA to a remarkably broad group of organisms including
numerous dicot and monocot angiosperm species.
-In addition, Agrobacterium can transform fungi,including yeasts,ascomycetes,and basidiomycetes.Recently,Ag
robacterium was reported to transfer DNA to human cells.
-Several virulence (vir) loci on the Ti plasmid, including virC (367, 368) and virF (220, 267), were shown to deter
mine the range of plant species that could be transformed to yield crown gall tumors.
-However, it is now clear that host range is a much more complex process, which is under the
genetic control of multiple factors within both the bacterium and the plant host.
Some Points to remember

8. Ti- Plasmid
About
• A Ti or tumour inducing plasmid(approx.size 200 kb each) is a plasmid that often, but not
always, is a part of the genetic equipment that Agrobacterium tumefaciens and Agrobacterium
rhizogenes use to transduce its genetic material to plants.
• The Ti plasmid is lost when Agrobacterium is grown above 28 °C.
• The plasmid has 196 genes that code for 195 proteins. There is one structural RNA. The
plasmid is 206,479 nucleotides long, the GC content is 56% and 81% of the material is coding
genes. There are no pseudogenes.
• The modification of this plasmid is very important in the creation of transgenic plants.
• Genes in the virulence region are grouped into the operons virABCDEFG, which code for the
enzymes responsible for mediating conjugative transfer of T-DNA to plant cells.

9. Ti plasmid and virulence genes
The Ti plasmid has three important region:-
1.T-DNA region: This region has the genes for the biosynthesis of auxin (aux),cytokinin (cyt) and opine
(ocs), and is flanked by left and right borders.
 T-DNA borders- A set of 24 kb sequences present on either side (right & left) of T-DNA are also
transferred to the plant cells.
 It is clearly established that the right border is more critical for T-DNA transfer.
2. Virulence region: The genes responsible for the transfer of T-DNA into host plant are located outside
T-DNA and the region is reffered to as vir or virulence region .
 At least nine vir-gene operons have been identified. These include vir A, vir G, vir B1, vir C1, vir D1, D2,
vir D4 and vir E1,E2.
3. Opine catabolism region: This region codes for proteins involved in the uptake and metabolisms of
opines.
 Besides the above three there is ori region that responsible for origin of DNA replication which permit the
Ti plasmid to be stably maintain in A. tumefaciens.

11. Ti Plasmid stucture

12. Infection Process

13. Cellular process of Agrobacterium–host interaction
Tzvi Tzfira and Vitaly Citovsky, 2002, Trends in Cell Biol. 12(3), 121-129

14. Ti Plasmid-Derived Vector Systems
 Using Ti plasmid as a vector it is possible to
insert a desired DNA sequence (gene) into the T
DNA region of Ti plasmid.
 There are several limitations to use Ti plasmids
directly as cloning vectors :-
 LARGE SIZE.
 TUMOR INDUCTION PROPERTY.
 ABSENCE OF UNIQUE RESTRICTION SITES.

15. • Agrobacterium plasmids are disarmed by
deleting naturally occurring T-DNA encoded
oncogenes and replacing them with foreign
genes of interest.
 The right and left border sequences of T-
DNA which is required for T-DNA integration.
 A multiple cloning site.
 An origin of replication
 A selectable marker gene

16. Though Ti plasmids are effective natural vectors they had
certain limitations.
• The phytohormone produced by transformed cells
growing in culture prevents their regeneration into
mature plants. Hence auxins and cytokinin genes
must be removed from the Ti –plasmid derived
cloning vector.
• The opine synthesis gene must be removed as it m
ay divert plant resources into opine production in
transgenic plant.
• Generally, Ti- plasmids are large in size (200-800kb)
For effective cloning, large segments of DNA that
are not essential for cloning has to be removed.
• As Ti plasmid does not replicate in E.coli Ti-plasmid
based vectors require an ori that can be used in
E.coli.

17. To overcome these constraints, Ti plasmid based vectors
were organized with the following components:
• A selectable marker gene that confers resistance to transformed pla
nt
cells. As these marker genes are prokaryotic origin, it is necessary to p
ut
them under the eukaryotic control (plant) of post transcriptional regul
ation signals, including promoter and a termination- poly adenylation
sequence,
to ensure that it is efficiently expressed in transformed plant cells.
• An origin of replication that allows the plasmid to replicate in E.col
i.
• The right border sequence of the T-DNA which is necessary for T-
DNA
integration into plant cell DNA.
• A polylinker (MCS) to facilitate the insertion of cloned gene into th
e
region between T-DNA border sequences.

18. Binary vector
t-DNA
VIR genes
Plasmid DNA
Bacterial
ChromosomeBacterial ORI
Ampicillin
resistance
Construction of vector
with disired genes

19. Making of Co-Integrate Vectors
-In this strategy, both the T-DNA with our gene of interest and vir region are
present in the same vector used for transformation.
-At first; an intermediate vector is made using E.coli plasmid + vir region + T-DNA
borders + origin of replication+pBR 322 sequences.
-Second vector is a disarmed pTi vector = gene of interest+ some markers+pBR322
sequences.
-Both intermediate vector and disarmed pTi has some sequences in common
(pBR322 sequences).
-Therefore by homologous recombination, co-integration of two plasmids will take
place within Agrobacterium.
Now we have a co-integrate vector that has both T-DNA with our gene of interest
with in the T-DNA borders and vir region. This complete vector is used for
transformation eg:pGV2260.

21. Binary vector strategy: Two vector strategy
Here two vectors are used. This vector was devised based on the knowledge that vir
region need not be in the same plasmid along with T-DNA for T DNA transfer.
Binary vector consists of a pair of plasmids
1) A disarmed Ti plsmid: This plasmid has T-DNA with gene of interest + ori for both
E.coli and Agrobacterium. Also called as mini-Ti or micro Ti plasmid eg: Bin 19
2) Helper Ti plasmid has virulence region that mediates transfer of T-DNA in micro Ti
plasmid to the plant.

22. In general, the transformation procedure is as follows:
the recombinant small replicon is transferred via bacterial conjugation or direct
transfer to A. tumefaciens harboring a helper Ti plasmid, the plant cells are
co-cultivated with the Agrobacterium, to allow transfer of recombinant T-DNA in
to the plant genome, and transformed plant cells are selected under appropriate
conditions.
1.binary vector system involves only the transfer of a binary plasmid to
Agrobacterium without any integration.
2.This is in contrast to co-integrate vector system wherein the intermediate
vector is transferred and integrated with disarmed Ti plasmid.
3.Due to convenience, binary vectors are more frequently used than co-integrate
vectors.
Compared with co-integrated vectors, binary vectors present some
advantages:
-No recombination process takes place between the molecules
involved.
-Instead of a very large, recombinant, disarmed Ti plasmid, small vector
s are used, which increases transfer efficiency from E. coli to
Agrobacterium.
This vector system is most widely used nowadays. Different types of
binary vectors have been devised to suit different needs in a plant
transformation process.

23. Agrobacterium mediated transformation
The important requirements for Agrobacterium-
mediated gene transfer in higher plants are as
follows:-
 The plant explants must produce
acetosyringone for activation of Vir genes.
 The induced Agrobacterium should have
access to cells that are competent for
transformation.
 Explants include cotyledon, leaf, thin tissue
layer, peduncle, hypocotyls, stem,
microspores

24. Process of Transformation

25. Transformation Protocols
Transformation was performed using minor
modifications of published protocols, using
a.Leaf disk
b.Scutellum-derived callus, or
c.Floral dip methods, respectively.

26. An Example
Example-scutellum-derived callus method
Transformation Protocol for Rice – Abbreviated
Seed plating on 2N6 – dark
↓ 4 weeks
Subculture onto 2N6 – dark
↓ 4 – 10 days
Co cultivation onto 2N6-AS – dark
↓ 3-7 days
Selection on 2N6-TCH – dark
↓ 4 weeks, subculture onto 2N6-TCH every 2 weeks
Transfer proliferating calli onto 2N6-TCH-dark
↓ 2 weeks
Regeneration onto RGH6-dark
↓ 7 days
Transfer to light
↓ 4-6 weeks
Plantlets onto ½ MSH - light
↓
Transfer plants to the glasshouse

27. Seed Material: Oryza sativa L. ssp japonica cvs. Millin or Nipponbare.
Steps-
1. Callus Induction
2. Callus Subculture
3. Bacteria Preparation
4. Transformation
5. Callus washing
6. Selection
7. Regeneration
8. Plantlet Formation
-To ensure that the gene transfer did not result from contamination with
Agrobacterium cells, controls including species specific PCR, selective plating,
and use of a tagged binary vector were implemented.
-Thus, diverse plant associated bacteria, when harbouring a disarmed Ti plasmid
and binary vector(or presumably a cointegrate or whole Ti plasmid), are readily
able to transfer TDNA to plants. The Ti plasmid is self transmissable, perhaps in
dicating the existence of a ubiquitous natural mechanism effecting horizontal
gene transfer from bacteria to plants.

28. Procedure for plant transformation
IMAGE: Mol bio of the cell by Albert (pg no:599)

29. Regeneration, Selection And Detection...
Regeneration: for shoot organogenesis, cytokinin (lower amounts
of auxin) are required
Selection: two antibiotics are required
• An antibiotic to kill the Agrobacterium, while not affecting the plant's
cell growth and division
• a second antibiotic allows growth of transformed shoots (w/selectable
marker) but inhibits growth of untransformed plant cells.
Detection of the "trait" gene
PCR methods can detect the presence of the "trait" DNA
protein detection methods are used where a gene product is produced that
defines the trait verification of the incorporation of the trait gene into the
plant's chromosome:
• by Southern hybridization
• by demonstrating transfer of the trait to the original transformant's
progeny.

30. Ø Scientists can insert any gene they want into the plasmid in place of the tumor
causing genes and subsequently into the plant cell genome.
Ø By varying experimental materials, culture conditions, bacterial strains, etc.
scientists have successfully used A. tumefaciens Gene Transfer to produce BT Corn.
Ø 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 .
Ø The vast majority of approved genetically engineered agriculture has been
transformed by means of Agrobacterium tumefaciens Mediated Gene Transfer.
Ø Original problems existed in that Agrobacterium tumefaciens only affects
dicotyledonous plants.
Ø Monocotyledon plants are not very susceptible to the bacterial infection.
Benefits and Problems with Agrobacteria

31. References:
1.www.asgct.org › General Public › Educational Resources
2.www.link.springer.com
3.www.bios.net
4.en.wikipedia.org

32. Thank You