Soybean transformation method

Patent metadata

Field	Value
Publication number	US-10214746-B2
Application number	US-201414505060-A
Country	US
Kind code	B2
Filing date	Oct 2, 2014
Priority date	Oct 4, 2013
Publication date	Feb 26, 2019
Grant date	Feb 26, 2019

Abstract

The present disclosure relates in part to a method for identifying a soybean germline transformant from a population of soybean transformants by incorporating a selection agent within rooting medium used in tissue culture during the soybean transformation process. The soybean germline transformants are selected from a population of soybean transformants which are comprised of a combination of non-germline and germline soybean transformants. The soybean non-germline transformants are identified and eliminated early in the transformation process. The soybean germline transformants are identified and selected for culturing into mature soybean plants. The method is readily applicable for screening and obtaining a soybean germline transformant at an early stage in the soybean transformation process.

First claim

What is claimed is:

1. A method of selecting soybean germline transformants, the method comprising:
a. transforming a population of cells of a soybean plant with a transgene, wherein the population of transformed cells comprises transformed germline cells and transformed non-germline cells;
b. regenerating shoots from the population of transformed cells;
c. isolating the shoots produced by the population of transformed cells;
d. subjecting the isolated regenerated shoots to a selective rooting medium comprising glufosinate, wherein (i) the subjected isolated regenerated shoots produced by the transformed germline cells create viable roots, and (ii) the subjected isolated regenerated shoots produced by the transformed non-germline cells do not create viable roots; and
e. selecting soybean germline transformants based on the ability of the selected transformants to create viable roots in the glufosinate-containing rooting medium.

2. The method of claim 1 , wherein the transforming employs a transformation method elected from the group consisting of Agrobacterium transformation, biolistics, calcium phosphate transformation, polybrene transformation, protoplast fusion transformation, electroporation transformation, ultrasonic transformation, liposome transformation, microinjection transformation, naked DNA transformation, plasmid vector transformation, viral vector transformation, silicon carbide mediated transformation, aerosol beaming transformation, or PEG transformation.

3. The method of claim 1 , wherein the population of cells of a soybean plant comprises a soybean plant tissue.

4. The method of claim 3 , wherein the soybean plant tissue is a subepidermal or corpus (L2/L3) tissue layer or an epidermal (L1) tissue layer.

5. The method of claim 4 , wherein the L2/L3 tissue layer comprises a germline cell.

6. The method of claim 4 , wherein the L1 tissue layer comprises a non-germline cell.

7. The method of claim 4 , wherein the L2/L3 tissue layer is a meristematic soybean plant tissue, a root soybean plant tissue, or a vascular soybean plant tissue.

8. The method of claim 4 , wherein the L1 tissue layer is a dermal soybean plant tissue, a ground soybean plant tissue, or a mantle soybean plant tissue.

9. The method of claim 1 , wherein the transgene is contained within at least one gene expression cassette.

10. The method of claim 9 , wherein the gene expression cassette comprises a selectable marker gene.

11. The method of claim 10 , wherein the selectable marker gene is a phosphinothricin acetyl transferase gene.

12. The method of claim 9 , wherein the gene expression cassette comprises a trait gene.

13. The method of claim 9 , wherein the gene expression cassette comprises an RNAi gene.

14. The method of claim 1 , wherein the selective rooting medium further comprises 2,4-Dichlorophenoxyacetic acid (2,4-D).

15. The method of claim 1 , wherein the glufosinate concentration within the selective rooting medium is at least 1.0 mg/L.

16. The method of claim 1 , wherein the glufosinate concentration in the selective rooting medium is from 1.0 mg/L to 10.00 mg/L.

17. The method of claim 1 , wherein the glufosinate concentration in the selective rooting medium is 1.0 mg/L.

18. The method of claim 1 , wherein the selective rooting medium comprises a basal salt, a vitamin, a mineral, and a carbon source.

19. The method of claim 18 , wherein the basal salt is Gamborg's B-5 basal salt, Schenk & Hildebrandt basal salt, White's basal salt, Chu (N6) basal salt, DKW/Juglans basal salt, Hoagland's No. 2 basal salt, Murashige & Skoog basal salt, or a combination thereof.

20. The method of claim 19 , wherein the basal salt is Murashige & Skoog basal salt.

21. The method of claim 18 , wherein the vitamin is selected from the group consisting of Gamborg's B-5 vitamin, MEM vitamin, Murashige & Skoog vitamin, Schenk & Hildebrandt vitamin, and combinations thereof.

22. The method of claim 21 , wherein the vitamin is Gamborg's B-5 vitamin.

23. The method of claim 18 , wherein the carbon source is selected from the group consisting of glucose, dextrose, mannose, fructose, galactose, glucuronate, lactose, glycerol, and combinations thereof.

24. The method of claim 23 , wherein the carbon source is sucrose.

25. The method of claim 14 , wherein the 2,4-D concentration within the selective rooting medium is at least 2.0 mg/L.

Assignees

• Dow Agrosciences Llc

Inventors

• Chennareddy Sivarama Reddy
• Pareddy Dayakar
• Samuel Jayakumar Pon
• Sarria-Milan Rodrigo
• Cicak Toby

Classifications

• 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

• C12Y203/01183

  Phosphinothricin acetyltransferase (2.3.1.183) · CPC title

• C12N5/0025

  Culture media for plant cell or plant tissue culture · CPC title

• A01H4/001

  Culture apparatus for tissue culture · CPC title