Use of multiple transformation enhancer sequences to improve plant transformation efficiency
The present invention relates to methods and compositions for improving the efficiency of Agrobacterium - and Rhizobium -mediated plant cell transformation by use of additional transformation enhancer sequences, such as overdrive or TSS sequences, operably linked to a T-DNA border sequence on a recombinant construct that comprises T-DNA.
1. A method of increasing the efficiency of Rhizobia -mediated plant transformation, comprising:
transforming a plant cell with a Rhizobia bacterium, said Rhizobia bacterium comprising a plant transformation vector which comprises at least one T-DNA border sequence and a transformation enhancer sequence operably linked to said T-DNA border sequence; wherein said transformation enhancer sequence comprises two or more copies of SEQ ID NO:13 or the full length complement of SEQ ID NO:13.
2. The method of claim 1 , wherein the transformation enhancer sequence is located proximal to a T-DNA right border (RB) sequence.
3. The method of claim 2 , wherein the transformation enhancer sequence is from a nopaline or octopine Ti plasmid of Agrobacterium tumefaciens.
4. The method of claim 1 , wherein the Rhizobia -mediated transformation is Agrobacterium -mediated transformation.
5. The method of claim 1 , wherein the Rhizobia -mediated transformation is Agrobacterium -, Rhizobium -, Sinorhizobium -, Mesorhizobium -, or Bradyrhizobium -mediated transformation.
6. The method of claim 1 , wherein the plant cell is from a plant selected from the group consisting of soybean, corn, cotton, canola, rice, wheat, alfalfa, common bean, peanut, tobacco, sunflower, barley, beet, broccoli, cabbage, carrot, cauliflower, celery, Chinese cabbage, cucumber, eggplant, leek, lettuce, melon, oat, onion, pea, pepper, peanut, potato, pumpkin, radish, sorghum, spinach, squash, sugarbeet, tomato and watermelon.
7. The method of claim 1 , wherein the plant cell is a corn or soybean cell.
8. The method of claim 1 , wherein the transformation enhancer sequence comprises from 2 to about 18 copies of SEQ ID NO:13 or the full length complement of SEQ ID NO:13.
9. The method of claim 1 , further comprising the step of:
c) regenerating a transgenic plant from said plant cell.
10. A recombinant DNA construct comprising a T-DNA border sequence operably linked to a transformation enhancer sequence, wherein said transformation enhancer sequence comprises two or more copies of SEQ ID NO:13, the full length complement of SEQ ID NO:13, or combinations thereof.
11. The construct of claim 10 , wherein the enhancer sequence comprises at least about four copies of SEQ ID NO:13 or the full length complement of SEQ ID NO:13.
12. The construct of claim 10 , wherein the border sequence is a right border (RB) sequence.
13. The construct of claim 10 , wherein the border sequence is a left border (LB) sequence.
14. The construct of claim 12 , wherein the RB sequence is from a nopaline Ti plasmid.
15. The construct of claim 12 , wherein the RB sequence is from an octopine Ti plasmid.
16. A transgenic cell transformed with the construct of claim 10 .
17. The cell of claim 16 , defined as a plant or bacterial cell.
18. The cell of claim 17 , wherein the cell is an Agrobacterium cell.
19. The cell of claim 17 , wherein the cell is a Rhizobium cell.
20. The cell of claim 17 , wherein the plant cell is from a plant selected from the group consisting of soybean, corn, cotton, canola, rice, wheat, alfalfa, common bean, peanut, tobacco, sunflower, barley, beet, broccoli, cabbage, carrot, cauliflower, celery, Chinese cabbage, cucumber, eggplant, leek, lettuce, melon, oat, onion, pea, pepper, peanut, potato, pumpkin, radish, sorghum, spinach, squash, sugarbeet, tomato, and watermelon.
21. A transgenic plant transformed with the construct of claim 10 .
22. The transgenic plant of claim 21 , defined as selected from the group consisting of soybean, corn, cotton, canola, rice, wheat, alfalfa, common bean, peanut, tobacco, sunflower, barley, beet, broccoli, cabbage, carrot, cauliflower, celery, Chinese cabbage, cucumber, eggplant, leek, lettuce, melon, oat, onion, pea, pepper, peanut, potato, pumpkin, radish, sorghum, spinach, squash, sugarbeet, tomato, and watermelon.