Gene transfer to plants
The Pharma-Planta consortium uses several different technologies for getting genes into plants. To transform tobacco we use Agrobacterium tumefaciens. To transform maize we use particle bombardment. To transform plastids of tobacco, tomato and lettuce, we use particle bombardment or PEG-mediated protoplast transformation.
The figure below shows the general Agrobacterium method for DNA transfer to tobacco plants.
The figure below shows some tobacco protoplasts, cells denuded of their cell walls, which are used for plastid transformation.
Gene expression in plants
Our pharmaceutical genes are expressed under the control of promoters that function in specific plant cells and species. In tobacco, we use a ubiquitous promoter which drives expression in leaves and all other tissues. In maize, we use seed-specific promoters to make the pharmaceutical protein accumulate in the kernels. For plastid transformation, the gene inserts in line with an endogenous plastid gene and is expressed under that gene’s promoter.
The figure below shows a protein blot, which proves that the pharmaceutical protein (the HIV antigen p24) is present in the tobacco tissue.
To make sure we can track transgenic plant material, all our transgenic pharmaceutical plants are cotransformed with the visible marker DsRed. This is a fluorescent protein that makes the plant tissue glow red under an appropriate light source.
The figure below shows second generation maize cobs with a mixture of transgenic (orange) and non-transgenic (green) seeds.