Methods for rapidly transforming monocots

Patent metadata

Field	Value
Publication number	US-9783813-B2
Application number	US-201414483748-A
Country	US
Kind code	B2
Filing date	Sep 11, 2014
Priority date	Aug 31, 2006
Publication date	Oct 10, 2017
Grant date	Oct 10, 2017

Abstract

The present invention provides methods for transforming monocot plants via a simple and rapid protocol, to obtain regenerated plants capable of being planted to soil in as little as 4-8 weeks. Associated cell culture media and growth conditions are also provided, as well as plants and plant parts obtained by the method. Further, a method for screening recalcitrant plant genotypes for transformability by the methods of the present invention is also provided. Further, a system for expanding priority development window for producing transgenic plants by the methods of the present invention is also provided.

First claim

What is claimed is:

1. A method for producing a transgenic corn plant comprising:
a) transforming an explant with at least a first selected DNA via bacterially mediated transformation of the explant;
b) culturing the explant in a first culture medium comprising an effective ratio of cytokinin and auxin to promote development of regenerable structures capable of root and/or shoot formation; and
c) culturing the explant in at least a second culture medium comprising a reduced amount of auxin relative to the first culture medium, and/or a third culture medium that lacks auxin and cytokinin, wherein the second culture medium and/or third culture medium supports the simultaneous growth of root and shoot tissues, to produce a regenerated transgenic corn plant;
wherein the ratio of cytokinin to auxin in the first culture medium is from about 0.005 to about 0.03 (w/w), wherein the first culture medium comprises from about 0.001 mg/L to about 10 mg/L of cytokinin and from about 0.1 mg/L to about 15 mg/L auxin, and wherein the regenerated transgenic corn plant is produced within about 4-8 weeks of transforming the explant.

2. The method of claim 1 , further comprising:
d) transferring the regenerated transgenic corn plant to a plant growth medium.

3. The method of claim 2 , wherein the plant growth medium is a non-sterile matrix.

4. The method of claim 3 , wherein the non-sterile matrix is comprised in a plug.

5. The method of claim 1 , wherein the regenerable structures are formed within about 6-14 days of transforming the explant.

6. The method of claim 1 , wherein step b) is completed within about 6-14 days of transforming the explant.

7. The method of claim 1 , wherein steps a) and b) are carried out without proliferating a callus for more than about 10 days to two weeks.

8. The method of claim 1 , wherein the first culture medium comprises a bactericidal compound.

9. The method of claim 1 , wherein the second and/or third culture medium comprises sucrose at a concentration higher than in the first culture medium.

10. The method of claim 1 , wherein the first culture medium comprises Lynx 1947 medium of Table 2.

11. The method of claim 1 , wherein step (c) comprises culturing the explant in an added plant growth regulator-free liquid culture medium that supports the simultaneous growth of root and shoot tissues, to produce the regenerated transgenic corn plant.

12. The method of claim 11 , wherein the third culture medium comprises Lynx 2067 medium of Table 2.

13. The method of claim 1 , wherein step (b) is carried out for a length of from about 6 days to about 12 days.

14. The method of claim 1 , wherein the cytokinin is selected from the group consisting of BAP, zeatin, kinetin, and TDZ; and the auxin is selected from the group consisting of IAA, 2,4-D, NAA, IBA, and dicamba.

15. The method of claim 1 , and wherein step (c) comprises culturing the explant in the second culture medium, wherein the second culture medium comprises an increased ratio of cytokinin to auxin relative to the first medium to promote development of root(s) and shoot(s) simultaneously.

16. The method of claim 15 , wherein the second culture medium of step (c) is Lynx 2202 medium of Table 2 and/or Lynx 2068 medium of Table 2.

17. The method of claim 1 , wherein step (c) comprises culturing the explant in a third culture medium lacking plant growth regulators.

18. The method of claim 15 , wherein the ratio of shoot forming growth regulator to auxin in the second medium is from about 0.02 to about 0.06 (w/w).

19. The method of claim 15 , wherein the second medium is Lynx 2202 medium of Table 2 or Lynx 2068 medium of Table 2.

20. The method of claim 1 , wherein fresh growth medium is not added subsequent to the start of step (c).

21. The method of claim 15 , wherein the explant is further cultured on a fourth medium between culturing on the first culture medium and the second culture medium, wherein the fourth medium comprises an effective amount of auxin and cytokinin to promote callus proliferation.

22. The method of claim 21 , wherein the fourth medium is Lynx 2063 medium of Table 2.

23. The method of claim 21 , wherein the explant is further cultured on a fifth medium between culturing on the second culture medium and the third culture medium, wherein the fifth medium comprises an amount of cytokinin effective to promote shoot growth.

24. The method of claim 23 , wherein the fifth medium is Lynx 2066 medium of Table 2.

25. The method of claim 1 , wherein the bacterially mediated transformation is carried out using a bacterium selected from the group consisting of Agrobacterium sp., Rhizobium sp., Sinorhizobium sp., Mesorhizobium sp., and Bradyrhizobium sp.

26. The method of claim 18 , wherein the shoot forming growth regulator comprises cytokinin, abscisic acid, or a combination of cytokinin and abscisic acid.

27. The method of claim 26 , wherein the second culture medium comprises less than half as much auxin as the first culture medium.

28. The method of claim 1 , wherein the first culture medium and the at least second and/or third culture medium that supports the simultaneous growth of root and shoot tissues are liquid media.

29. The method of claim 1 , wherein the first culture medium is a semi-solid medium.

30. The method of claim 1 , wherein each medium used subsequent to the first culture medium is a liquid medium.

31. The method of claim 1 , wherein steps b) and c) are carried out in a single container.

32. The method of claim 1 , wherein the explant is an immature embryo.

33. The method of claim 1 , wherein the first culture medium comprises from about 0.1 mg/L to about 10 mg/L auxin, and from about 0.005 mg/L to about 0.05 mg/L cytokinin.

34. The method of claim 17 , wherein the third culture medium lacks silver nitrate.

Assignees

• Monsanto Technology Llc

Inventors

• Rout Jyoti R
• Lowe Brenda
• Purcell John
• Spelletich Anne
• Spencer Michael
• Way Melissa

Classifications

• C12N15/8209

  Selection, visualisation of transformants, reporter constructs, e.g. antibiotic resistance markers · CPC title

• C12N15/8201 Primary

  Methods for introducing genetic material into plant cells, e.g. DNA, RNA, stable or transient incorporation, tissue culture methods adapted for transformation · CPC title

• C12N15/8265

  Transgene containment, e.g. gene dispersal · CPC title

• C12N15/8205 Primary

  Agrobacterium mediated transformation · CPC title

• C12Q2600/13

  Plant traits · CPC title