Modifying the Plant

Fig. 1. Tobacco leaf, cut into small discs and soaked in Agrobacterium, being cultured.

In the research on PAL-840, the gene construct was inserted into both Nicotiana benthamiana (a tobacco commonly used as a model species in plant biotechnology) and into cassava itself. To produce transgenic plants, young leaves are cut into small disks and treated with plant hormones. The Agrobacterium is applied (Figure 1), so that the chimeric gene construct will be inserted into the plant's genome. The leaf discs are then transferred to a culture medium containing antibiotics to kill the Agrobacterium (whose job is now complete) and to select for the successful genetic transformation events. Once again kanomycin is used, this time to kill all the plant cells which have not received the transgenes. Only those containing the kanomycin resistant gene (and the gene of interest; in this case the GUS marker gene under control of the PAL-840 promoter) can survive and grow. Plant growth hormones then stimulate regeneration of whole shoots from the transgenic cells.

The antibiotic-resistance gene plays a crucial role in the production of genetically engineered plants, but after this stage it has no agronomic value. Indeed, presence of antibiotic genes and other "selectable marker genes" in transgenic crops is controversial from an environmental point of view, and biotechnologists are under increasing pressure to develop alternative strategies for recovering transformed cells in culture.

Several weeks later, thin sections of the regenerated plants can be examined under a microscope to determine the action of the promoter in the genetically modified plants (Figure 2).


Fig. 2. Expression of GUS as controlled by PAL-840 promoter. From left to right: petiole, young stem, semi-woody stem. Upon treatment with an appropriate chemical, the protein produced by the GUS gene produces a blue colour, enabling the localised action of the PAL-840 promoter to be visualized.