Ti plasmid derived plant vector systems:

binary and co - integrative vectors

transformation process; regeneration of
the transformed lines

Mitesh Shrestha
 Constraints of Wild type Ti/Ri-plasmid
• Very large

• Low copy number in Agrobacterium

• Difficult to isolate and manipulate in vitro

• Do not replicate in Escherichia coli, the favored host for genetic manipulation.

• T-DNA regions from wild-type Ti-plasmids are generally large and do not contain
unique restriction endonuclease sites suitable for cloning a gene of interest.

• In addition, scientists wanted to eliminate oncogenes from T-DNA to regenerate

normal plants. 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.

• Opine synthase genes were also generally deemed superfluous in constructions

designed to deliver goi to plants.
Features of efficient vector for plant transformation

• A selectable marker gene that confers resistance to transformed plant

cells. As these marker genes are prokaryotic origin, it is necessary to
put them under the eukaryotic control (plant) of post transcriptional
regulation 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.coli.

• 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 the

region between T-DNA border sequences.
 Co-integrate vectors
• Co-integrated vectors or Hybrid Ti plasmids

• First types of modified and engineered Ti plasmids devised for

Agrobacterium -mediated transformation, but are not widely used
today.

• These vectors are constructed by homologous recombination of a

bacterial plasmid with the T-DNA region of an endogenous Ti
plasmid in Agrobacterium.

• Integration of the two plasmids requires a region of homology

present in both.
 Co-integrate vectors
Three vectors are necessary in this system:
A. Disarmed Agrobacterium Ti plasmids
In these Ti plasmids, the oncogenes located in the T-DNA region have been
replaced by exogenous DNA.
Examples of these vectors include:
• SEV series: the right border of the T-DNA together with the phytohormone
genes coding for cytokinin and auxin are removed and replaced by a
bacterial kanamycin resistance gene while the left border and a small part
of the left segment (TL) of the original T-DNA (referred to as Left Inside
Homology (LIH)) are left intact.
• pGV series: the phytohormone genes are excised and substituted by part of
pBR322 vector sequence. The left and right border sequences as well as the
nopaline synthase gene of the Ti plasmid are conserved.
Ti-plasmid derived Plant vectors: Disarmed Ti-
plasmid vectors

Ti plasmid

disarmed Ti-
plant vector
 Prototype disarmed Ti vectors
• Wild-type Ti plasmids not suitable as
vector because T-DNA contains oncogenes
that cause disorganized growth of the
recipient plant cells.
• Substitute pBR322 sequences for almost
all of the T-DNA, leaving only the left and
right border regions and the nos gene to
construct pGV3850.
• Agrobacterium carrying pGV3580
transferred to plant cells, no tumor cells
were produced, but transformed cells can
produce nopaline.
• besides ocs and nos ,drug resistance
genes and herbicide resistance genes are
widely used as selectable markers
 Co-integrate vectors
Three vectors are necessary in this system:
B. Intermediate vectors
• Small pBR322-based plasmids (E. coli vectors) containing a T-DNA region.
• Used to overcome the problems derived from the large size of disarmed Ti
plasmids and their lack of unique restriction sites.
• Intermediate vectors are replicated in E. coli and are transferred into
Agrobacterium by conjugation. They cannot replicate in A. tumefaciens and
therefore, carry DNA segments homologous to the disarmed T-DNA to
permit recombination to form a co-integrated T-DNA structure.
C. Helper vectors
These are small plasmids maintained in E. coli that contain transfer (tra) and
mobilization (mob) genes, which allow the transfer of the conjugation-deficient
intermediate vectors into Agrobacterium.
 Conjugation between the two E. coli strains transferred the helper
plasmid to the carrier of the intermediate vector, and then
transferred to A. tumefaciens.

Homologous recombination between the T-DNA in intermediate vector and Ti

plasmid to form a large co-integrate plasmid which is facilitated by native
Agrobacterium rec functions.

The recombinant T-DNA is transferred to plant genome.

Donor vector
A resulting co-integrated plasmid assembled by in vitro manipulation normally
contains:
• the vir genes,

• the left and right T-DNA borders,

• an exogenous DNA sequence between the two T-DNA borders,

• plant and bacterial (E. coli and A. tumifaciens) selectable markers,

• E. coli functional origin of replication that doesn’t operate in

Agrobacterium

Disadvantages:

• Long region of homologies required between the Ti plasmid and the E. coli
plasmids (pBR322 based intermediate vectors) making them difficult to
engineer and use

• Relatively inefficient gene transfer compared to the binary vectors

Binary vector strategy: two vector strategy

• Systems in which T-DNA and vir genes are located on separate

replicons were eventually termed T-DNA binary systems

• Consists of a pair of autonomously replicating plasmid vectors

• Based on the knowledge that vir region need not be in the same
plasmid along with T-DNA for transfer
Binary vector strategy: two vector strategy
Schematic diagram of co-integration/exchange systems and T-DNA binary vector
systems to introduce genes into plants using Agrobacterium-mediated genetic
transformation.
• Binary vector/shuttle vector:
– disarmed Ti-plasmid with gene of interest between T-DNA
borders + ori for both E. coli and Agrobacterium
– also called as mini-Ti or micro Ti plasmid
• Helper Ti-plasmid:
– with virulence region that mediates transfer of T-DNA in micro
Ti-plasmid to the plant
– Constructed by removing the T-DNA
• Two different approaches have been mediated:
– Binary vector with two origin of replication: one for E.coli and the
other for A. tumifaciens
– Binary vector with single broad host range origin of replication

• In either case no vir genes are present on binary cloning

vector

• The vir genes present in a disarmed Ti plasmid from which the

T-DNA has been removed synthesize vir proteins that
eventually mobilize the T-DNA region of the binary cloning
plasmid vector into the target plant cells
 Advantages of Binary vector

• Binary vectors don’t demand in-vivo recombination as in

co-integrate vectors
• Binary vectors are more efficient and easier to obtain
• In binary system the binary plasmids exist as separate
replicons and thus their copy number remains flexible
• The size of the binary vectors are relatively small and thus
easier to manipulate
 Genetically engineered Ti-plasmid vectors

Binary systems Co-integrated vectors

Needs 2 vectors: Needs 3 vectors

Disarmed Ti plasmid
Disarmed Ti plasmid
capable for infection
with gene of interest Form
(no vir genes) co-integrated plasmid Intermediate vector
after homologous with T-region
Helper vector recombination on T-DNA and gene of interest
for infection (transferred by conjugation)
(with vir genes) Helper vector
for transfer of
intermediate plasmid into A.tumifaciens
 Super Binary Vectors
• One of the approaches toward enhancing the frequency of transformation
by binary vectors is to employ additional virulence genes, such as virB, virE,
and virG, which exhibit certain gene dosage effects.

• In the super-binary vector system, a DNA fragment that contains virB, virC,
and virG from pTiBo542 is introduced into a small T-DNA-carrying plasmid.

• A. tumefaciens strains that carry pTiBo542 are wider in host range and
higher in transformation efficiency than strains that carry other Ti plasmids,
such as pTiA6 and pTiT37.

• Super-binary vectors are highly efficient in the transformation of various

plants
 ALTERNATIVE T-DNA BINARY SYSTEMS
• Although T-DNA binary vector systems almost always consist of T-DNA and
vir regions localized on plasmids, it is not essential that they function this
way.
• Replicons containing T-DNA or vir genes do not need to be plasmids.
Indeed, several laboratories have shown that T-DNA can be integrated into
an Agrobacterium chromosome and launched from this replicon, and
specialized vectors have been generated to facilitate integration of DNA into
a specific neutral region of the chromosome of A. tumefaciens C58.
• Although launching T-DNA from the Agrobacterium chromosome can result
in lower transformation frequencies, this process has the beneficial
consequences of reducing integrated transgene copy number and almost
completely eliminating integration of vector backbone sequences into the
plant genome
• Regeneration of transformed lines
 Assignment
• Explain the constraints of using wild type Ri
plasmid. [3]
• Elaborate about the binary vector system and their
variations. [7.5]
• Differentiate between Binary and Co – integrate
vectors. [3]
• Differentiate between Super Binary and Co –
integrate vectors. [1]
• Explain about various types of vectors used for
preparation of Co – integrate and Binary Vectors. [3]