Claims
- 1. A method for selecting a MDIBOA/DIMBOA resistant Agrobacterium strain comprising:
(a) providing an Agrobacterium cell; (b) culturing said Agrobacterium cell with a phenolic inducer of the vir pathway and under other conditions supporting Agrobacterium replication, but including MDIBOA/DIMBOA at concentrations sufficient to inhibit Agrobacterium replication; and (c) isolating an Agrobacterium cell that has replicated in the culture of step (b), thereby selecting MDIBOA/DIMBOA a resistant Agrobacterium strain.
- 2. The method of claim 1, wherein the Agrobacterium cell of step (a) further comprises an antibiotic resistance gene under the control of a promoter that is upregulated by a vir gene product, and the culture conditions of step (b) include the antibiotic, resistance to which is provided by the antibiotic resistance gene, at concentrations sufficient to inhibit Agrobacterium replication.
- 3. The method of claim 2, wherein said antibiotic resistance gene is kanr, and said antibiotic is kanamycin.
- 4. The method of claim 3, wherein kanamycin is present at about 40-50 μM.
- 5. The method of claim 2, wherein DIMBOA is present at about 50-100 μM.
- 6. The method of claim 2, wherein phenolic induction uses acetosyringone.
- 7. The method of claim 6, wherein acetosyringone is present at about 30-100 μM.
- 8. The method of claim 2, further comprising culturing in the presence of a sugar.
- 9. The method of claim 8, wherein said sugar is glucose, and the concentration is 0.1%-1%.
- 10. The method of claim 2, further comprising:
(d) making a replicate culture of the selected Agrobacterium cell; and (e) culturing the selected Agrobacterium cell in the presence of MDIBOA/DIMBOA and antibiotic, but without said phenolic inducer, wherein a cell that replicates in step (e) is identified as having a constitutively activated vir pathway, and a cell that does not replicate in step (e) is identified as having a vir-mediated MDIBOA/DIMBO resistant mutation.
- 11. The method of claim 2, wherein the Agrobacterium of step (a) is A348/pAC2.
- 12. The method of claim 1, further comprising treating a replicate culture of the Agrobacterium cell of step (a) under conditions supporting Agrobacterium replication, but excluding said phenolic inducer and including MDIBOA/DIMBOA at concentrations sufficient to inhibit Agrobacterium replication.
- 13. A method for selecting a phenol hypersensitive Agrobacterium strain comprising:
(a) providing an Agrobacterium cell that comprises an antibiotic resistance gene under the control of a promoter that is upregulated by a vir gene product; (b) culturing said Agrobacterium cell with varying levels of a phenolic inducer and under other conditions supporting Agrobacterium replication, but including antibiotic resistance to which is provided by the antibiotic resistance gene, at concentrations sufficient to inhibit Agrobacterium replication; and (c) isolating an Agrobacterium cell receiving the lowest level of phenolic induction that has replicated in the culture of step (b), thereby selecting a first phenol hypersensitive Agrobacterium strain.
- 14. The method of claim 13, further comprising repeating the steps (a)-(c) with said first phenol hypersensitive Agrobacterium cell, thereby obtaining a second phenol hypersensitive Agrobacterium cell.
- 15. The method of claim 13, wherein said antibiotic resistance gene is kanr, and said antibiotic is kanamycin.
- 16. The method of claim 15, wherein kanamycin is present at about 40-50 μM.
- 17. The method of claim 13, wherein phenolic induction uses acetosyringone.
- 18. The method of claim 17, wherein acetosyringone is present at about 10 μM.
- 19. The method of claim 13, further comprising culturing in the presence of a sugar.
- 20. The method of claim 19, wherein said sugar is arabinose, and the concentration is 0.1-1%.
- 21. A MDIBOA/DIMBOA resistant Agrobacterium strain selected according to the method comprising:
(d) providing an Agrobacterium cell; (e) culturing said Agrobacterium cell with a phenolic inducer and under other conditions supporting Agrobacterium replication, but including
MDIBOA/DIMBOA at concentrations sufficient to inhibit Agrobacterium replication; and (f) isolating an Agrobacterium cell that has replicated in the culture of step (b), thereby selecting MDIBOA/DIMBOA a resistant Agrobacterium cell.
- 22. A phenol hypersensitive Agrobacterium strain comprising selected according to the method:
(d) providing an Agrobacterium cell that comprises an antibiotic resistance gene under the control of a promoter that is upregulated by a vir gene product; (e) culturing said Agrobacterium cell with varying levels of a phenolic inducer and under other conditions supporting Agrobacterium replication, but including antibiotic resistance to which is provided by the antibiotic resistance gene, at concentrations sufficient to inhibit Agrobacterium replication; and (f) isolating an Agrobacterium cell receiving the lowest level of phenolic induction that has replicated in the culture of step (b), thereby selecting a first phenol hypersensitive Agrobacterium cell.
- 23. A method for producing a MDIBOA/DIMBOA resistant Agrobacterium comprising:
(a) providing an Agrobacterium cell; (b) culturing said Agrobacterium cell with a phenolic inducer and under other conditions supporting Agrobacterium replication, but including
MDIBOA/DIMBOA at concentrations sufficient to inhibit Agrobacterium replication; (c) isolating an Agrobacterium cell that has replicated in the culture of step (b); (d) producing a culture of the isolated Agrobacterium.
- 24. A method for producing a phenol hypersensitive Agrobacterium strain comprising:
(a) providing an Agrobacterium cell that comprises an antibiotic resistance gene under the control of a promoter that is upregulated by a vir gene product; (b) culturing said Agrobacterium cell with varying levels of a phenolic inducer and under other conditions supporting Agrobacterium replication, but including
antibiotic resistance to which is provided by the antibiotic resistance gene, at concentrations sufficient to inhibit Agrobacterium replication; (c) isolating an Agrobacterium cell receiving the lowest level of phenolic induction that has replicated in the culture of step (b); and (d) producing a culture of the isolated Agrobacterium.
- 25. A DNA library prepared from an Agrobacterium cell of claim 23.
- 26. A DNA library prepared from an Agrobacterium cell of claim 24.
- 27. A method for transducing a plant using a MDIBOA/DIMBOA resistant Agrobacterium strain comprising:
(a) providing a plant cell; (b) contacting said plant cell with said MDIBOA/DIMBOA resistant Agrobacterium; and (c) culturing said plant cell under conditions suitable for Agrobacterium-mediated transformation.
- 28. The method of claim 27, wherein said plant cell is a monocot.
- 29. The method of claim 27, wherein said plant cell is a dicot.
- 30. The method of claim 27, wherein said Agrobacterium harbors a heterologous gene.
- 31. The method of claim 30, wherein said heterologous gene affects one or more performance traits in a plant of said plant cell.
- 32. The method of claim 27, further comprising obtaining seed of said plant.
- 33. The method of claim 27, further comprising obtaining progeny of said plant.
- 34. A method for transducing a plant using a phenol hypersensitive Agrobacterium strain
(a) providing a plant cell; (b) contacting said plant cell with said phenol hypersensitive Agrobacterium; and (c) culturing said plant cell under conditions suitable for Agrobacterium-mediated transformation.
- 35. The method of claim 34, wherein said plant cell is a monocot.
- 36. The method of claim 34, wherein said plant cell is a dicot.
- 37. The method of claim 34, wherein said Agrobacterium harbors a heterologous gene.
- 38. The method of claim 37, wherein said heterologous gene affects one or more performance traits in a plant of said plant cell.
- 39. The method of claim 34, further comprising obtaining seed of said plant.
- 40. The method of claim 34, further comprising obtaining progeny of said plant.
- 41. A plasmid comprising the PN25 promoter of coliphage T5 and the virA and virG genes of transcriptional control of said promoter.
- 42. The plasmid of claim 41, wherein said plasmid backbone is derived from a broad host range vector.
- 43. The plasmid of claim 42, wherein said broad host range vector is pJB20.
- 44. The plasmid of claim 41, wherein said virA gene encodes a functional deletion mutant of virA.
- 45. The plasmid of claim 44, wherein said functional deletion mutant lacks the first 284 amino acid residues of VirA.
- 46. A host cell comprising the plasmid of claim 41.
Government Interests
[0001] The United States government may own rights in this application by virtue of funding through the National Institutes of Health and Grant No. GM47369.