Pre-conditioning treatments to improve plant transformation
Provided herein are methods for increasing plant cell transformation efficiency. These methods include exposing the plant cells to a liquid medium containing a surfactant. Following exposure to the surfactant-containing medium, the cells can become more amenable to transformation and may be genetically transformed using methods known in the art. Exposure of the cells to the surfactant-containing medium prior to transformation can increase plant transformation efficiency when compared to transformation efficiency of cells not exposed to the surfactant-containing medium.
1. A method of improving transformation of plant cells comprising:
i) pre-conditioning plant cells by exposure to a surfactant containing medium, wherein said surfactant is a non-ionic trisiloxane surfactant,
ii) removing said plant cells from said surfactant containing medium, and subsequently,
iii) contacting said plant cells with at least one polynucleotide sequence to introduce said at least one polynucleotide sequence into said plant cells, wherein said contacting occurs in a medium that lacks surfactant.
2. The method of claim 1 , wherein said surfactant containing medium comprises a non-ionic surfactant.
3. The method of claim 1 , wherein said surfactant containing medium comprises surfactant at a concentration of 0.001-0.1% (v/v).
4. The method of claim 1 wherein said exposure to a surfactant containing medium lasts for 5-60 minutes.
5. The method of claim 1 wherein said introducing one or more polynucleotide sequence(s) includes the use of Agrobacterium cells harboring a plant transformation construct.
6. The method of claim 5 wherein said Agrobacterium cells harboring a plant transformation construct comprise a binary vector.
7. The method of claim 5 wherein said Agrobacterium cells harboring a plant transformation construct comprise a superbinary vector.
8. The method of claim 1 wherein said improving transformation of plant cells comprises an increased percentage of plant cells exhibiting transient expression of said at least one polynucleotide sequence relative to control plant cells not exposed to said surfactant containing medium.
9. The method of claim 1 wherein said improving transformation of plant cells comprises an increased percentage of callus pieces developing stably transformed sectors.
10. The method of claim 1 wherein said improving transformation of plant cells comprises an increased number of transformed plants regenerated from transformed tissue.
11. The method of claim 1 wherein said plant cells are derived from a monocot.
12. The method of claim 11 wherein said plant cells are derived from Zea mays, Oryza sativa, Setaria viridis, Sorghum bicolor, Triticum aestivum , or Saccharum sp.
13. The method of claim 1 wherein said plant cells are derived from a dicot.
14. The method of claim 13 wherein said plant cells are derived from Pisum sativum, Lactuca sativa , or Solanum lycopersicum.
15. The method of claim 1 wherein said at least one polynucleotide sequence comprises a polynucleotide sequence that shares at least 80% sequence identity with a sequence selected from the group of sequences consisting of SEQ ID NOs:1 and 15, or that encodes a protein that shares at least 80% sequence identity with a sequence selected from the group of sequences consisting of SEQ ID NOs:2 and 16.
16. The method of claim 1 wherein said at least one polynucleotide sequence comprises a polynucleotide sequence that shares at least 80% sequence identity with a sequence selected from the group of sequences consisting of SEQ ID NOs:3, 5, and 7, or that encodes a protein that shares at least 80% sequence identity with a sequence selected from the group of sequences consisting of SEQ ID NO:4, 6, and 8.
17. The method of claim 1 wherein said introducing at least one polynucleotide sequence comprises biolistic transformation.
18. The method of claim 1 wherein said at least one polynucleotide sequence encodes at least one CRISPR nuclease.
19. The method of claim 18 wherein said pre-conditioning results in improved genome editing relative to control cells not exposed to said surfactant containing medium.