Method for selection of transformed cells
The invention provides methods for the selection of transgenic cells. The invention relates to the unexpected finding that cells expressing a gene conferring tolerance to auxin-like herbicides such as dicamba may be directly selected from non-transgenic cells using auxin-like herbicides as a selective agent. In this manner, plants exhibiting tolerance to auxin-like herbicides can be directly produced without the need for separate selectable markers.
1. A method for selecting a transformed soybean plant cell comprising the steps of:
a) contacting a population of plant cells comprising a transgenic soybean plant cell transformed with a polynucleotide encoding dicamba monooxygenase with medium comprising auxin-like herbicide in an amount that inhibits the growth of cells from the population lacking the polynucleotide, wherein the polynucleotide comprises a nucleic acid sequence selected from: (1) a nucleic acid sequence encoding the polypeptide of SEQ ID NO: 6, (2) a nucleic acid sequence comprising the sequence of SEQ ID NO: 5, and (3) a nucleic acid sequence encoding a polypeptide with at least 90% sequence identity to the polypeptide of SEQ ID NO:6, wherein the polypeptide has dicamba monooxygenase activity; and
b) selecting the transformed soybean plant cell from the population of plant cells based on tolerance exhibited by the transformed cell to the auxin-like herbicide
further wherein:
said polynucleotide encoding dicamba monooxygenase is operatively linked to a chloroplast transit peptide coding sequence.
2. The method of claim 1 , comprising culturing the population of plant cells on a medium lacking the auxin-like herbicide prior to step a) and/or between step a) and step b).
3. The method of claim 2 , wherein the medium lacking the auxin-like herbicide contains a cytokinin.
4. The method of claim 2 , wherein the medium lacking the auxin-like herbicide contains 6-benzyl amino purine (BAP).
5. The method of claim 4 , wherein the 6-benzyl amino purine is in a concentration of about 10 mg/l of medium or less.
6. The method of claim 1 , wherein the polynucleotide encoding dicamba monooxygenase is not genetically linked to a selectable or screenable marker gene other than dicamba monooxygenase.
7. The method of claim 1 , further comprising the step of:
c) regenerating a fertile transgenic soybean plant from the transformed soybean plant cell.
8. The method of claim 1 , wherein the medium contains at least two auxin-like herbicides.
9. The method of claim 8 , wherein the medium contains dicamba and 2,4-dichlorophenoxyacetic acid.
10. The method of claim 1 , wherein the population of cells comprises a cotyledon explant.
11. The method of claim 10 , wherein the transformed plant cell is prepared by Agrobacterium -mediated transformation.
12. The method of claim 1 , wherein said auxin-like herbicide comprises 2,4-D, MCPA or 2,4-DB.
13. The method of claim 1 , wherein said auxin-like herbicide comprises dicamba in a concentration of from about 0.001 mg/L to about 0.02 mg/L.