Efficiency agrobacterium-mediated plant transformation method
View Patent ↗The present invention relates to a rapid transformation and regeneration system for plants. In particular, the invention relates to a plant tissue preparation system. The transformation method is efficient and reliable for production of fertile plants with improved agronomic qualities.
1. A method for producing a fertile transgenic plant, comprising the steps of:
(a) inoculating a regenerable plant cell or tissue selected from the group consisting of wheat immature embryos, maize immature embryos, wheat embryogenic callus, maize embryogenic callus, soybean hypocotyl sections and soybean callus suspension cell cultures with a solution comprising Agrobacterium containing a genetic component, said genetic component comprising a nucleic acid sequence of interest encoding a selectable or screenable marker that functions in the identification of a transformed plant cell or tissue, to produce an Agrobacterium -inoculated explant;
(b) substantially removing said solution of Agrobacterium ;
(c) co-culturing said Agrobacterium -inoculated explant in a vessel with about 100 to about 300 microliters of media, said media not containing a gelling agent wherein the weight of the Agrobacterium -inoculated explant is reduced from about 20% to about 35% by the end of the co-culture period;
(d) identifying and selecting a transformed plant cell or tissue comprising said genetic component; and
(e) regenerating a transgenic plant therefrom.
2. The method of claim 1 wherein the regenerable cell or tissue is an immature embryo and is precultured prior to step (a).
3. The method of claim 1 wherein the co-culture period is from one hour to about 6 days.
4. The method of claim 1 wherein the co-culture period is from about one day to about 4 days.
5. The method of claim 1 wherein the co-culture period is from about one day to about 3 days.
6. A method for producing a fertile transgenic plant, comprising the steps of:
(a) inoculating a regenerable plant cell or tissue selected from the group consisting of wheat immature embryos, maize immature embryos, wheat embryogenic callus, maize embryogenic callus, soybean hypocotyl sections and soybean callus cell suspension cell cultures with a solution comprising Agrobacterium containing a genetic component, said genetic component comprising a nucleic acid sequence of interest encoding a selectable or screenable marker that functions in the identification of a transformed plant cell or tissue, to produce an Agrobacterium -inoculated explant;
(b) substantially removing said solution of Agrobacterium ;
(c) co-culturing said Agrobacterium -inoculated explant in a vessel with about 100 to about 300 microliters of media, said media not containing a gelling agent, wherein the weight of the Agrobacterium -inoculated explant is reduced from about 20% to about 35% by the end of the co-culture period and wherein the manner for controlling said reduction in the weight of the Agrobacterium -inoculated explant comprises limitation or removal of water from the vessel containing said explant;
(d) identifying and selecting a transformed plant cell or tissue comprising said genetic component; and
(e) regenerating a transgenic plant therefrom.
7. The method of claim 1 wherein the vessel contains filter paper.
8. The method of claim 6 wherein the vessel contains filter paper.