Methods for plant transformation using spectinomycin selection

• US 8,030,544 B2
• Filed: 03/10/2008
• Issued: 10/04/2011
• Est. Priority Date: 03/09/2007
• Status: Active Grant

• Current Assignee: Monsanto Technology LLC (Bayer AG)
• Original Assignee: Monsanto Technology LLC (Bayer AG)
• Inventors: Somers, David, Petersen, Michael, Williams, Edward, Ye, Xudong, Martinell, Brian, Wan, Yuechun
• Primary Examiner(s): Fox; David T
• Application Number:
  US12/045,562
• Publication Number:
  US 20090138985A1
• Time in Patent Office: 1,303 Days
• Field of Search: None
• US Class Current: 800/294
• 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 method for producing a transgenic plant containing at least two heterologous nucleic acid sequences, comprising:

(a) providing an explant comprising a first heterologous nucleic acid sequence that confers resistance to an herbicide, an aminoglycoside, or hygromycin;

(b) inoculating the explant with Agrobacterium comprising a vector which comprises a second heterologous nucleic acid sequence comprising a selectable marker gene conferring spectinomycin resistance;

(c) co-cultivating the explant with the Agrobacterium to obtain an explant that exhibits spectinomycin resistance; and

(d) regenerating the explant that exhibits spectinomycin resistance into a transgenic plant containing at least the two heterologous nucleic acid sequences;

wherein the explant is treated with a cytokinin at least during steps (b) and (c).

2. The method of claim 1, wherein the explant comprises an embryonic meristem.

3. The method of claim 1, wherein the first heterologous nucleic acid sequence confers resistance to glyphosate, bialaphos, phosphinothricin, Basta, glufosinate, 2,4-D, kanamycin, an acetyl-coA carboxylase inhibitor, an oxygen radical generator, or dicamba.

4. The method of claim 1, wherein the explant is a soybean, corn, cotton, or canola explant.

5. The method of claim 1, wherein the explant is a soybean or cotton explant.

6. The method of claim 1, wherein the transgenic plant arises from transformation of a meristem that results in transformation of germline tissue.

7. The method of claim 1, wherein the resulting plant is non-chimeric.

8. The method of claim 1, wherein the resulting plant is chimeric.

9. The method of claim 1, 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).

10. The method of claim 1, further comprising treatment of the explant with a cytokinin prior to step (b).