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

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

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

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

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.

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.

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

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.

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

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)

Transformed shoots are grown in tissue culture (under selection)

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.

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