Inhibition of Agrobacterium-induced necrosis
In plant biology, necrosis means death of a plant tissue; the tissue first turns brown and subsequently dies. Disrupted plant cells of a tissue at the cut edge release colorless phenolic compounds that come into contact with each other and in the presence of oxygen suffer brown discoloration accumulating in brown spots in the cells. Oxidation extends throughout the tissue and the culture media, and, if not controlled, the tissue finally dies. Enzyme activity and polymerization of phenolic compounds are some of the causes of the oxidation-browning process.
Inoculation of a plant tissue with Agrobacterium is in itself a disruptive process and triggers a hypersensitive response in the tissue. As a result, there is a poor survival rate of the target tissue. Therefore, the design of an adequate artificial environment to minimize damage due to the interaction of Agrobacterium with the plant tissue is critical for the success of genetic transformation experiments. Anti-necrotic/anti-browning treatments, applied during the transformation process, include addition of reducing agents, heat inactivation of enzymes participating in the oxidative process, lowering pH and the addition of enzyme inhibitors.
There are two entities with patents directed to reducing browning/necrosis induced by Agrobacterium:
- Syngenta (formerly Novartis) has patents in the United States, Australia and Europe and patent applications in the United States and Europe on different methods for overcoming the necrosis induced by Agrobacterium, especially in Gramineae plants. According to the applicant, Agrobacterium induces in some plants a necrotic process analogous to apoptosis in animal cells, where cell death is characterized by DNA fragmentation and defined morphological changes. The claimed methods for controlling plant cell death include:
- heat shock treatment of the plant tissue;
- addition of chemical inhibitors, such as ethylene inhibitors; and
- expression of foreign gene products in the transformed plants.
- The University of Minnesota has PCT, United States, and Australian patent applications disclosing the use of agents to inhibit enzymatic browning of plant tissue duringAgrobacterium-mediated transformation. The agents include:
- sulfhydryl-containing agents (e.g. L-cysteine); and
- iron and copper chelators.