VECTOR MEDIATED GENE
TRANSFER
SAVITHA
SIXTH SEM
[Link] BOTANY
AGROBACTERIUM MEDIATED GENE TRANSFER
Agrobacterium infection has been extensively
utilized for transfer of foreign dna to a number
of species.
They are gram negative bacteria
They infect both dicot and monocot plants and
causes tumorous growth in the infected
portion.
The genus agro bacterium includes four
species
[Link],[Link],[Link],[Link]
izogenes.
[Link] contains Ti plasmid it is used
as vectors for gene transfer.
SALIENT FEATURES OF Ti
PLASMID
1. The Ti plasmid is a large double
stranded circular DNA .It is found
in almost all strains of
Agrobacterium.
2. The size of Ti plasmid ranges from
150-230 kilo bases.
3. It undergoes denaturation when it is
exposed to a temperature above
370C.
4. Its molecular weight ranges from
It has a region for oncogenecity
which is depends upon the
production of some unusual
aminoacids and which causes
tumorous growth.
It has a sequence for the conjective
transfer of this plasmid from one
organism to another.
TYPES OF TI PLASMID
Octopine type plasmid-This type of Ti
plasmid encode for the synthesis of an
opine,namely octopine.
Nopaline type plasmid-This type of Ti
plasmid encode for the synthesis of of
nopaline is a kind of opine.
Agropine type plasmid-The structure of
this plasmid is similar to the structure of
other type of [Link] it contain
thegene for agropine synthesis in T-DNA.
STRUCTURE OF T-DNA
FRAGMENT
o T-DNA IS A PART OF Ti plasmid
o It has a single repeated sequence at both ends.
o These sequences act as cis-acting sites during
the transfer of Ti plasmid toplant genome.
o The removel of left borderd sequence does cot
cause any considerable changein tumour
induction in the infected tissue,
o But the removel of right borderd sequence from
the TDNA fai to inducetumor in infected tissue.
o The T-DNA capable of inducing tumour formation
on the infected tissues.
Ti plasmid is an idea
vector
Ti plasmids replicates autonomously
without any influence of
chromosomal DNA.
It has a sequence for thetransfer of
this plasmid from one organism to
another organism.
It has a definite tumorogenic
property which is usedas agenetic
markerfor identifying transformed
plant cells.
Steps for gene transfer
The desired foreign gene is isolated
from desired strain of plant.
An intermediate vector plasmid is
constructed by using naturally
occuring Co1 E1 plasmid.
The Ti plasmid is isolated from
Agrobacterium tumefaciens with the
help of enzymes.
The T-DNA is then inserted in a PBR322
plasmid. It is carried out using usual gene
clone procedure.
The T-DNA is then cut with a restriction
enzyme and the prepared dna fragment is
inserted into the T-DNA as a result
chimeric DNA is formed.
The transformed cells so obtained are
then mixed with a culture of
[Link] and incubated for a few
hours.
In A. tumefaciens the chimeric
DNAundergoes homologous genetic
recombination with the Ti plasmid of
the cell.
The transformed [Link] so
formed contain some gene which
give resistance todrugs,then they are
selected forculturing.
The selected bacteria are then
allowed to infect the cultured plant
cells whose genome are to
beimproved.
Ri plasmid
[Link] contain another
plasmid, namely Ri [Link]
hairy root diseasesome plants.
The molecular basis of this disease
led to the utilization of the bacterium
for developing gene transfer system.
STRUCTURE OF Ri PLASMID
It has two T- DNA fragments namely Tr DNA
and Tl DNA separated by 15KB segments.
Tr DNA is similar to T-DNA ,it has the gene for
auxin and agropine synthesis.
Tl DNAhas 4 genes
Rol A responsible for hairy root formation.
Rol B induces root initiation and callus
formation.
Rol C promotes root growth.
Rol D suppress callus growth.
USES
Ri plasmid can be used as substitutes for
Ti plasmid to transfer foreign genea to
plants.
Ri plasmid exchange DNA segment
between plants and bacteria.
They could be used to induce rooting in
clones where rooting from callus or explant
is difficult.
They could be also used to increase
nodulation frequency of plantlets during
micropropagation
