Plant transformation without selection

Patent metadata

Field	Value
Publication number	US-10233455-B2
Application number	US-201715464287-A
Country	US
Kind code	B2
Filing date	Mar 20, 2017
Priority date	Aug 31, 2006
Publication date	Mar 19, 2019
Grant date	Mar 19, 2019

Abstract

The invention provides methods for identifying regenerated transformed plants and differentiated transformed plant parts, obtained without subjecting plant cells to selective conditions prior to regenerating the cells to obtain differentiated tissues. In particular embodiments, the plant cells are corn plant cells. Methods for growing and handling plants, including identifying plants that demonstrate specific traits of interest are also provided.

First claim

What is claimed is:

1. A method for identifying transgenic monocot plants, comprising:
(a) obtaining monocot plant cells transformed with a DNA segment comprising a nucleic acid sequence of interest;
(b) regenerating a plurality of monocot plants or differentiated monocot plant parts from the cells without first selecting for the presence of said DNA segment;
(c) identifying at least a first transgenic monocot plant or transgenic differentiated plant part from the plurality of monocot plants or differentiated monocot plant parts;
wherein the plants are regenerated by growth solely on liquid media prior to identifying the transgenic monocot plant or transgenic differentiated plant part.

2. The method of claim 1 , wherein the DNA segment does not comprise a selectable marker or visual marker gene.

3. The method of claim 1 , wherein the transformation frequency of cells grown solely in liquid media subsequent to contacting the cells with a gene of interest (GOI) and prior to identification of transgenic plants or transgenic plant parts is enhanced relative to the transformation frequency observed when cells are grown in solid media, semi-solid media, soil, or a combination of solid media, semi-solid media, liquid media, and/or soil, subsequent to contacting the cells with a GOI and prior to identification of transgenic plants or transgenic plant parts.

4. The method of claim 1 , wherein the plant cells are immature corn embryo cells.

5. The method of claim 4 , wherein the immature corn embryos are from about 1.5 mm to about 3.5 mm in length.

6. The method of claim 5 , wherein the immature corn embryos are from about 1.9 mm to about 2.3 mm in length.

7. The method of claim 1 , further comprising, between steps (b) and (c):
(1) placing the plurality of monocot plants or differentiated plant parts in culture tubes or growth plugs comprising a growth medium or water while maintaining the individual identity of the monocot plants; and
(2) subjecting the plants or plant parts to at least a first assay for the presence of the DNA segment to identify one or more plant or plant part as transgenic based on results from the assay.

8. The method of claim 7 , wherein the assay is selected from the group consisting of Southern hybridization, PCR, DNA sequencing, northern blotting, western blotting, an immunoassay, and an assay for the enzymatic activity encoded by the DNA segment.

9. The method of claim 7 , wherein the assay is performed prior to placing the regenerated plants into soil.

10. The method of claim 8 , wherein putatively transformed monocot plants or differentiated plant parts lacking the nucleic acid sequence of interest are identified, wherein the assay is performed on plant tissue comprising pooled subsets of nucleic acids from said plurality of monocot plants or differentiated plant parts.

11. The method of claim 1 , wherein the monocot plants or monocot plant parts are regenerated not later than 6 weeks after the DNA segment is transformed into the monocot plant cells.

12. The method of claim 1 , wherein the monocot plants or monocot plant parts are regenerated not later than 4 weeks after the DNA segment is transformed into the monocot plant cells.

13. The method of claim 1 , wherein the monocot plants or monocot plant parts are regenerated not later than 3 weeks after the DNA segment is transformed into the monocot plant cells.

14. The method of claim 1 , wherein the monocot plants or monocot plant parts are regenerated not later than 2 weeks after the DNA segment is transformed into the monocot plant cells.

15. The method of claim 1 , wherein the monocot plants or monocot plant parts are regenerated not later than 1 week after the DNA segment is transformed into the monocot plant cells.

16. The method of claim 1 , wherein the DNA segment is introduced into the monocot plant cell by bacterially-mediated transformation, electroporation, PEG-mediated transformation, or particle bombardment.

17. The method of claim 11 , wherein the bacterially-mediated transformation is mediated by a bacterial cell selected from the group consisting of an Agrobacterium cell, a Rhizobium cell, a Sinorhizobium cell, and a Mesorhizobium cell.

18. The method of claim 1 , further comprising the step of subjecting a monocot plant or plant part derived from the first monocot plant cell to culture conditions that select for, or allow screening for, the presence or absence of the nucleic acid sequence of interest after regeneration of a plant or plant part.

19. The method of claim 7 , wherein the growth medium is a solid medium.

20. The method of claim 19 , wherein the growth medium is soil.

21. The method of claim 1 , wherein the regenerated plant or differentiated plant part is uniform with respect to the presence of the DNA segment.

22. The method of claim 1 , wherein the monocot plant is a corn plant and wherein the DNA segment does not comprise a selectable marker or visual marker gene.

23. The method of claim 1 , wherein the monocot plant is a corn plant and wherein the regenerated plant or differentiated plant part is uniform with respect to the presence of the DNA segment.

24. The method of claim 1 , wherein the monocot plant is a corn plant and wherein regeneration occurs solely on liquid media prior to identifying the transgenic corn plant or transgenic differentiated corn plant part.

25. A method for identifying transgenic corn plants, comprising:
(a) obtaining corn plant cells transformed with a DNA segment comprising a nucleic acid sequence of interest;
(b) regenerating a plurality of corn plants or differentiated corn plant parts from the cells without first selecting for the presence of said DNA segment;
(c) identifying at least a first transgenic corn plant or transgenic differentiated plant part from the plurality of corn plants or differentiated corn plant parts;
further wherein the method comprises, between steps (b) and (c):
(1) placing the plurality of corn plants or differentiated plant parts in culture tubes or growth plugs comprising a growth medium or water while maintaining the individual identity of the corn plants; and
(2) subjecting the plants or plant parts to at least a first assay for the presence of the DNA segment to identify one or more plant or plant part as transgenic based on results from the assay;
wherein the plants are regenerated by growth solely on liquid media prior to identifying the transgenic corn plant or transgenic differentiated corn plant part, and wherein the assay is performed prior to placing the regenerated plants into soil.

Assignees

• Monsanto Technology Llc

Inventors

• Rout Jyoti R

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

• C12N15/8209

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

• C12N15/8205 Primary

  Agrobacterium mediated transformation · CPC title

• C12N15/8265

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

• C12N15/8212

  Colour markers, e.g. beta-glucoronidase [GUS], green fluorescent protein [GFP], carotenoid · CPC title