1. RB LB D1 D1
VirD1 nicks RB / LB 5’ 3’ RB LB D2
Vir D2 binds to the 5’ end of the nicks

2. Single strandedT-DNA is displaced, and replacement synthesis
reforms the ds T-DNA RB LB 5’ 3’ 5’ 3’
Displaced ss T-DNA strand

3. Single stranded T-DNA exits the
bacterium via a pore composed
of the products of other vir
operon genes. 5’ 3’

4. Plant cytoplasmic
proteins complex with
VirE2 proteins to target
the T-DNA into the
plant nucleus.

5. Once integrated into the nucleus, the auxin and cytokinin genes present on the T-DNA are expressed, and this expression
leads to cell proliferation and the formation of the callus.
Parasitic relationship – plant is “forced”
to synthesise opines for the bacterium.

6. Requirements for a gene transfer vector:
1) It must be non-oncogenic,
2) Vector must be relatively small,
3) Way of introducing foreign DNA into the vector,
4) Selection system.

7. Plant Transformation Vector Bin (Binary) 19:
Within the borders:
Onc+ genes deleted
RE sequences
LacZ selection
Nos-Npt11 gene
RB/LB unaltered

8. pAL 4404 carries the Vir operon
T-DNA and Vir are
present on different
replicons – Binary
System
pAL4404 is completely
non-oncogenic – only
carries the Vir genes.

9. To introduce the gene to be transferred into the Bin 19 system:
Gene to be transferred is inserted into the RE polylinker. – E. coli
LacZ system allows identification of recombinants (Blue>White)
KanR allows selection of transformed tissue
This plasmid is now transferred to Agrobacterium LBA4404

10. Stages in plant transformation using disarmed Agrobacterium vectors:
1) Leaf discs are cut from the plant to be transformed (wound)
2) They are floated on an Agrobacterium suspension
3) Placed on selective plates containing kanamycin (Bin19 kanR)
4) 2-4 weeks, transformants regenerate from leaf disc edges.
5) Control, non transformed discs bleach and die (killed by kanamycin)

11. Transformed shoots are grown in tissue culture (under selection)

12. Transgenic plants are grown to maturity in a transgenic containment
facility to minimise spread of pollen and seed to the environment
Transgenic plants are normal in every way - flower, set seed.
Technical limitation:
Integration of transgene is random, so a number of transformants,
all expressing the transgene to different levels, are produced.
Transgenics have to be selected to identify high expressors.

13. Transgenic tomatoes
with altered softening
characteristics.
Developed in the U.K.
By Prof. Don Grierson
Nottingham University
Not for sale in the U.K!