1. Ti plasmid derivatives
D.INDRAJA

2. • A tumour inducing (Ti) plasmid is a plasmid found in pathogenic species
of Agrobacterium, including A. tumefaciens, A. rhizogenes, A. rubi and A.
vitis.
• Agrobacterium tumefaciens is a gram-negative soil bacterium which
naturally transforms plant cells, resulting in crown gall (cancer) tumours.
• Tumour formation is the result of the transfer, integration and expression of
genes on a specific segment of A. tumefaciens plasmid DNA called the T-
DNA (transferred DNA).
• The T-DNA resides on a large plasmid called the Ti (tumour inducing)
plasmid found in A. tumefaciens.

3. • Almost 100 years ago (1907), Smith & Townsend postulated that a
bacterium was the causative agent of Crown gall tumours.
• A tumefaciens infects wounded or damaged plant tissues, it induces the
formation of a plant tumour called crown gall.
• The entry of the bacterium into the plant tissues is facilitated by the release
of certain phenolic compounds (acetosyringone,hydroxyacetosyringone) by
the wounded sites. Crown gall formation occurs when the bacterium release
its Ti plasmid into the plant cell cytoplasm.
• A fragment of Ti plasmid, referred to as T-DNA, is actually transferred from
the bacterium into the host where it gets integrated into the plant cell
chromosome.
• The T-DNA carries genes that code for proteins involved in the biosynthesis
of growth hormones (auxin and cytokinin) and novel plant metabolites
namely opines — amino acid derivatives and agropines — sugar derivatives.
• The growth hormones cause plant cells to proliferate and form the gall. As
the bacteria multiply and continue infection, grown gall develops which is a
visible mass of the accumulated bacteria and plant material.

4. •The size of Ti plasmid range from150 to 230 kbp.
•They are large molecule exist as independent replicating circular DNA
molecules within the Agrobacterium cells.
•The T-DNA is variable in length in the range of 12 to 24 kb.
•Several dicotyledonous plants (dicots) are affected by crown gall disease e.g.
grapes, roses, stone-fruit trees.

5. Ti plasmid as a vector
• Agrobacterium plasmids are disarmed by deleting naturally occurring T-
DNA encoded oncogenes and replacing them with foreign genes of interest.
• These vectors are mainly composed of the following components:
– The right border sequence of T-DNA which is absolutely required for T-
DNA integration into plant cell DNA.
– A multiple cloning site (poly-linker DNA) that promotes the insertion of
cloned gene into the region between T-DNA borders.
– An origin of DNA replication that allows the plasmids to multiply in E.
coli.
– A selectable marker gene (e.g. neomycin phosphor transferase) for
appropriate selection of the transformed cells.
• Two types of Ti plasmid-derived vectors are used for genetic transformation
of plants—
– co- integrate vectors
– binary vectors.

6. COINTEGRATE VECTORS
• Co integrate vector is produced by integrating the modified E.coli plasmid
into a disarmed pTi.
• Co integration is achieved within Agrobacterium by homologous
recombination.
• The E.coli plasmid pGV1103 and the disarmed pTi must have some
sequences common in both for recombination to occur.
• pTi is disarmed by replacing its oncogenes with sequences from E.coli
plasmid.
• The pGV1103 is modified to produce an intermediate vector. Origin of
replication in E.coli.
• Selectable markers e.g. neo gene for selection of transformed plant cells.
• Kanamycin resistance for selection of Cointegrate vector in Agrobacterium

8. BINARY VECTORS
• The binary vector system consists of an Agrobacterium strain along with a
disarmed Ti plasmid and vir helper plasmid (the entire T-DNA region
including borders deleted while vir gene is retained).
• It may be noted that both of them are not physically linked (or integrated).
• A binary vector with T-DNA can replicate in E. coli and Agrobacterium.
The binary vector has the following components:
• Left and right borders that delimit the T-DNA region.
• A plant transformation marker (PTM) e.g. npt II that confers kanamycin
resistance in plant transformed cells.
• A multiple cloning site (MCS) for introducing target/foreign genes.
• A bacterial resistance marker e.g. tetracycline resistance gene for selecting
binary vector colonies in E. coli and Agrobacterium.
• oriT sequence for conjugal mobilization of the binary vector from E. coli to
Agrobacterium.
• A broad host-range origin of replication such as RK2 that allows the
replication of binary vector in Agrobacterium.

9. Production and use of binary vector: The target (foreign) gene of
interest is inserted into the multiple cloning site of the binary vector.
• In this way, the- target gene is placed between the right and left border
repeats and cloned in E. coli.
• By a mating process, the binary vector is mobilised from E. coli to
Agrobacterium.
• Now, the virulence gene proteins of T-DNA facilitate the transfer of T-DNA
of the vector into plant cells.

10. Advantages of binary vectors:
• binary vector system involves only the transfer of a binary plasmid to
Agrobacterium without any integration.
• This is in contrast to co-integrate vector system wherein the intermediate
vector is transferred and integrated with disarmed Ti plasmid.
• Due to convenience, binary vectors are more frequently used than co-
integrate vectors

11. Some limitations of Ti-plasmid directly as vector:-
• Ti-plasmid are large in size (200-800 kb) smaller vectors are preferred for
recombinant experiment.
• Absence of unique restriction enzyme sites on Ti plasmids.
• The phytohormones (auxin & cytokinin) produced by the plant cells so
auxin , cytokinin must be removed.
• Opine production is transformed plant cells lowers the plant yield so opine
synthesizing genes should be removed.
• Ti plasmids cannot replicate in E. coli. Considering the above limitations, Ti
plasmid- based vectors with suitable modifications have been constructed

12. CONCLUSIONS
 Agrobacterium tumefaciens is a natural tool for plant transformation .
 Agrobacterium is capable of infecting intact plant cells tissues and
organs.
 As a result tissue culture limitations are much less of a problem.
Transformed plants can be regenerated more rapidly.
 Agrobacterium is capable of transferring large fragments of DNA
very efficiently without substantial rearrangements.
 The stability of gene transferred is excellent.