Methods for plant transformation using spectinomycin selection

• US 9,714,428 B2
• Filed: 10/06/2014
• Issued: 07/25/2017
• Est. Priority Date: 03/09/2007
• Status: Active Grant

• Current Assignee: Monsanto Technology LLC (Bayer AG)
• Original Assignee: Monsanto Technology LLC (Bayer AG)
• Inventors: Wan, Yuechun, Ye, Xudong, Williams, Edward, Petersen, Michael W., Martinell, Brian J., Somers, David A.
• Primary Examiner(s): Fox, David T
• Application Number:
  US14/507,744
• Publication Number:
  US 20150113683A1
• Time in Patent Office: 1,023 Days
• Field of Search: None
• CPC Class Codes: A01H 4/003 Cutting apparatus specially adapted for tissue culture
• CPC Class Codes: A01H 4/008 Methods for regeneration to complete plants
• CPC Class Codes: A01H 6/202 Brassica napus [canola]
• CPC Class Codes: A01H 6/4684 Zea mays [maize]
• CPC Class Codes: A01H 6/542 Glycine max [soybean]
• CPC Class Codes: A01H 6/604 Gossypium [cotton]
• CPC Class Codes: C12N 1/205 Bacterial isolates
• CPC Class Codes: C12N 15/8201 Methods for introducing genetic material into plant cells, e.g. DNA, RNA, stable or transient incorporation, tissue culture methods adapted for transformation
• CPC Class Codes: C12N 15/8202 by biological means, e.g. cell mediated or natural vector
• CPC Class Codes: C12N 15/8205 Agrobacterium mediated transformation
• CPC Class Codes: C12N 15/8209 Selection, visualisation of transformants, reporter constructs, e.g. antibiotic resistance markers
• CPC Class Codes: C12N 15/8221 Transit peptides
• CPC Class Codes: C12N 15/8261 with agronomic (input) traits, e.g. crop yield
• CPC Class Codes: C12N 15/8265 Transgene containment, e.g. gene dispersal
• CPC Class Codes: C12N 15/8271 for stress resistance, e.g. heavy metal resistance
• CPC Class Codes: C12N 15/8274 for herbicide resistance
• CPC Class Codes: C12N 15/8275 Glyphosate
• CPC Class Codes: C12N 15/8277 Phosphinotricin
• CPC Class Codes: C12N 15/8281 for bacterial resistance
• CPC Class Codes: C12N 5/0025 Culture media for plant cell or plant tissue culture
• CPC Class Codes: C12N 5/04 : Plant cells or tissues culture media C12N5/0025
• CPC Class Codes: C12N 9/1029 : transferring groups other than amino-acyl groups (2.3.1)
• CPC Class Codes: C12R 2001/41 : Rhizobium
• CPC Class Codes: C12Y 203/01081 : Aminoglycoside N3'-acetyltransferase (2.3.1.81)
• CPC Class Codes: G01N 33/0098 : Plants or trees wood G01N33/46

Abstract

The present invention relates to methods and compositions for transforming soybean, corn, cotton, or canola explants using spectinomycin as a selective agent for transformation of the explants. The method may further comprise treatment of the explants with cytokinin during the transformation and regeneration process.

Claims

1. A nucleic acid construct comprising two sequences conferring resistance to spectinomycin or streptomycin, wherein the first sequence is operably linked to a promoter active in a plant cell, the first sequence comprises an aadA gene and encodes a polypeptide comprising aminoglycoside-3′

-adenyltransferase activity, and the first sequence is fused to a chloroplast transit peptide-encoding sequence, and the second sequence is operably linked to a promoter active in a prokaryotic cell.

2. The construct of claim 1, wherein at least one of the sequences conferring said resistance comprises SEQ ID NO:

1, or the first sequence fused to a chloroplast transit peptide-encoding sequence comprises SEQ ID NO;

2.

3. The construct of claim 1, further comprising a coding sequence that confers a trait of agronomic interest or improved end use.

4. The construct of claim 3, wherein the trait of agronomic interest or improved end use comprises a coding sequence that confers herbicide tolerance.

5. The construct of claim 3, comprising a first set of left and right T-DNA borders flanking the coding sequence that confers a trait of agronomic interest or improved end use, and wherein the construct comprises a second set of left and right T-DNA borders flanking at least one of said two sequences conferring resistance to spectinomycin or streptomycin.

6. The construct of claim 5, wherein the second set of T-DNA borders flanks both sequences conferring resistance to spectinomycin or streptomycin.

7. The construct of claim 3, comprising right and left T-DNA borders, wherein the coding sequence that confers a trait of agronomic interest or improved end use is located after the right border, and wherein at least one of said two sequences conferring resistance to spectinomycin or streptomycin is located after the left border.

8. The construct of claim 7, wherein both of said sequences conferring resistance to spectinomycin or streptomycin are located after the left border.

9. A plant comprising the nucleic acid construct of claim 1.

10. A method of producing a transgenic plant comprising:

(a) transforming at least a first seed explant with the nucleic acid construct of claim 1 to obtain transformed cells; and

(b) regenerating a transgenic plant from the transformed cells, wherein the explant is contacted, prior to, concurrently with, and/or following step (a) or step (b), with at least a first media comprising spectinomycin to select transformed cells comprising said resistance to spectinomycin or streptomycin.

11. The method of claim 10, wherein step (a) further comprises transforming the cell of the explant with at least a second nucleic acid construct comprising a coding sequence that confers a trait of agronomic interest or improved end use.

12. The method of claim 11, wherein the trait of agronomic interest or improved end use comprises herbicide tolerance.

13. The method of claim 11, wherein the first and second nucleic acid constructs are integrated at different loci within the genome of the cell.

14. The method of claim 10, further comprising, prior to step (a), the step of priming the seed, wherein the priming comprises contacting the seed with a cytokinin.

15. The method of claim 10, further comprising contacting the explant with a cytokinin prior to, concurrently with and/or following step (b).

16. The method of claim 15, wherein the cytokinin is selected from the group consisting of thidiazuron, BAP (6-Benzylaminopurine), kinetin, CPPU (N-(2-Chloro-4-pyridyl)-N′

-phenylurea), 2iP (6-(y,y-Dimethylallylamino)purine), Zeatin, Zeatin-riboside, Adenine, and TIBA (2,3,5-Triiodobenzoic acid).

17. The method of claim 10, wherein the explant is a soybean, corn, cotton, or canola explant.

18. The method of claim 17, wherein the explant is a soybean explant.

19. The method of claim 10, wherein both sequences conferring resistance to spectinomycin or streptomycin comprise an aadA gene.

20. The method of claim 19, wherein the aadA gene comprises SEQ ID NO:

1.

21. The method of claim 20, wherein the aadA gene fused to a chloroplast transit peptide-encoding sequence comprises SEQ ID NO:

2.

22. A plant produced by the method of claim 10.