Agrobacterium-mediated transformation assisted by vacuum infiltration
Vacuum infiltration had already been used by plant physiologists before researchers interested in improving transformation efficiency started using it. Plant physiologists use the method to allow the penetration of pathogenic bacteria into the inter cell spaces and in that way study the interaction between plants and pathogenic bacteria.
Physically, vacuum generates a negative atmospheric pressure that causes the air spaces between the cells in the plant tissue to decrease. The longer the duration and the lower the pressure of the vacuum, the less air space there is within the plant tissue. An increase in the pressure allows the infiltration medium, including the infective transformation vector, to relocate into the plant tissue. For plant transformation, vacuum is applied to a plant part in the presence of Agrobacterium for a certain time period. The length of time that a plant part or tissue is exposed to vacuum is critical as prolonged exposure causes hyperhydricity.
Vacuum infiltration-facilitated transformation can be performed in planta, in which the plant part to be transformed, e.g., flower, is not excised from the plant, thus eliminating the need for in vitro regeneration of plants. It also offers several other advantages such as the generation of many independently transformed plants from a single plant, a reduction in somaclonal variation because there are no tissue culture steps, and the possibility of high throughput testing because the process is fast. The method is also potentially useful for transformation of plants recalcitrant to plant tissue culture and regeneration.
The use of Agrobacterium-mediated transformation assisted by vacuum infiltration was first reported in 1993 for transforming Arabidopsis and since then many improvements have been made. Others plants such as soybeans, duckweed, wheat, petunia, and rice have also been transformed by this method.
Several organizations have patents and patent applications directed to Agrobacterium-mediated transformation assisted by vacuum infiltration. The selected disclosures describe either transformation of any plant or transformation of monocotyledonous plants. They also discriminate between any plant part or a selected plant part or tissue, e.g., flower.
Transformation of any plant
- The Samuel Roberts Foundation Noble Foundation has patent applications filed in Australia and Europe on methods for direct plant transformation of any plant with Agrobacterium using vacuum infiltration. Some limitations of the filed claims lie in the plant part or plant phase to be transformed: seedlings and plants in flowering stage. The methods generally entail suspending Agrobacterium cells containing a vector with a gene of interest in vacuum infiltration medium, and the plant portions to be transformed are immersed in the suspension and subjected to vacuum infiltration. Infiltrated plants subsequently produce transformed seeds from which transformed plants are obtained.
Transformation of monocot plants
- Paradigm Genetics has a PCT application disclosing the use of vacuum infiltration to transform monocots with Agrobacterium. In addition, the disclosure describes the transformation of rice by applying a vacuum to a rice panicle immersed in an Agrobacterium suspension. This disclosure is discussed in detail under the section Monocots – General transformation methods. The claims as filed are limited to the use of a monocot flower as tissue to be transformed with Agrobacterium and subjected to vacuum. The apparatus to perform the vacuum infiltration is also part of the filed claims.