In Agrobacterium-mediated transformation, the tumor inducing T-DNA segment of the plasmid DNA harboured by the Agrobacterium, is replaced by gene of interest. The Agrobacterium then has to be mobilised into the plant in order to transform the plant by incorporating the gene of interest into the genomic DNA of infected cells. The transformed cells are then regenerated into whole plants with genomic DNA of the whole plant containing the gene of interest.
Transferring the gene into the Agrobacterium
The gene of interest firstly has to be incorporated into the plasmid DNA in the Agrobacterium. Different techniques are used routinely in the lab to do this. They are heat shock, freeze and thaw, electroporation and tri parental mating. For the first three, competent cells have to be made that will be used for transformation. For heat shock and freeze and thaw methods, chemical competent cells are used and for electroporation the cells have to be electro-competent. In tri-parental mating, a helper plasmid contained in Escherichia coli strain is used to transfer the gene from an E.coli strain containing the gene of interest, to the Agrobacterium strain that is going to be used for plant transformation.
1. Transformed Agrobacteria colony is picked and grown in 2ml of YEP (10 g/l peptone, 10 g/l yeast extract powder, 5 g/l sodium chloride) liquid media containing antibiotics used for selection for 48 hours in the dark in a shaking incubator at 28°C.
2. Then 100µl of these is grown in a bigger tube or Erlenmeyer flask (must be sterile) containing 100ml of YEP liquid still with antibiotics. Acetosyringone is added at a concentration of 250µm. Shake at 28°C in the dark until an OD of 1-2 is reached.
3. Then harvest the cells, about 25ml of Agrobacterium liquid culture can do, to harvest the cells at 7000x g at 25°C for 15 minutes.
4. Discard the supernatant and re-suspended the pellet in 15 ml liquid MS media and centrifuge for 10 min still at 7000x g at 25°C.
5. Discarded the supernatant and re-suspended the pellet in 10 ml liquid YEP media.
6. Incubate plant explants, explants can be leaves, seeds, seedlings, nodal sections, stem pieces depending on which of this is ideal for the particular plant, in the suspension for 30minutes to an hour at room temperature (25°C).
7. Remove the explants and blot them dry on sterile tissue paper and place in the dark for 3 days on MS media containing 250 µM acetosyringone.
8. After 3 days rinse the explants in ½ MS media containing 250 µg/ml cefotaxime to wash off the bacteria.
9. Place the explants on ½ MS media containing appropriate antibiotics for plant selection. MS can be supplemented with hormones such as 2,4D to produce callus or with cytokinin (BA is mainly used) and auxin (IAA and NAA are mainly used) at the appropriate concentrations for the particular plant.
The explants that grow in the plant selection media are regarded as putative transformants. To ascertain whether they have been transformed, molecular techniques such as polymerase chain reaction (PCR) are used to analyse or check whether the gene of interest is present in the plants genomic DNA. To do this plant's genomic DNA is extracted and then the primers that are complementary to the gene sequence are used to amplify it after which the PCR product is electrophoresed on agarose gel to visualise and ascertain that the gene is present. This will then mean a successful transformation of the plant by the Agrobacteria after which they will now be referred to as being transgenic.
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