BR-112019011445-B1 - Nucleic acid molecule, vector, construct, bacterial host cell and methods for transformation, selection, production and protection of herbicide-tolerant transgenic cells and plants.

Abstract

The present invention relates to compositions and methods for improving transformation frequency. The compositions, synthetic selection marker genes, are used in transformation methods and result in higher transformation frequency.

Inventors

• Sivamani Elumalai
• Qiudeng Que
• Michael Schweiner

Assignees

• SYNGENTA CROP PROTECTION AG

Dates

Publication Date

20260317

Application Date

20171128

Priority Date

20161208

Claims (7)

1. 1. Nucleic acid molecule, characterized by the fact that it is SEQ ID NO 13.
2. 2. Vector or construct, characterized in that it comprises the nucleic acid molecule, as defined in claim 1.
3. 3. Transgenic host cell, characterized in that it contains the nucleic acid molecule as defined in claim 1, wherein the cell is a bacterial cell.
4. 4. Improved method for plant transformation, characterized in that it comprises the steps of: (a) providing the nucleic acid molecule according to claim 1; (b) introducing into a plant, tissue culture or plant cell the nucleic acid molecule of step (a) to obtain a transformed plant, transformed tissue culture or transformed cell expressing PAT of the nucleic acid molecule of step (a); and (c) selecting transformants using a herbicide concentration that allows the growth of cells expressing PAT of the nucleic acid molecule of step (a), while simultaneously killing or inhibiting the growth of cells that do not comprise said PAT gene, wherein said herbicide comprises phosphinothricin or glufosinate, wherein more transformants are recovered compared to a method that does not use the nucleic acid molecule, as defined in claim 1.
5. 5. An improved method for selecting a transgenic plant cell, characterized in that said method comprises supplying the nucleic acid molecule, as defined in claim 1, to a plurality of plant cells, and growing said plurality of cells in a concentration of a herbicide that allows the growth of cells expressing the PAT gene of said vector, while simultaneously killing or inhibiting the growth of cells that do not comprise said PAT gene, wherein said herbicide comprises phosphinothricin or glufosinate, and wherein more transgenic plant cells are recovered compared to a method that does not utilize the nucleic acid molecule, as defined in claim 1.
6. 6. Improved method for producing a transgenic plant that is tolerant to the herbicidal activity of a glutamine synthetase inhibitor, including phosphinothricin or a compound with a phosphinothricin moiety, characterized in that it comprises the steps of: (a) producing a transgenic plant cell comprising the nucleic acid molecule as defined in claim 1; and (b) regenerating a transgenic plant from said cell, wherein more transgenic plant cells comprising a PAT gene are recovered compared to a method that does not use the nucleic acid molecule as defined in claim 1.
7. 7. Method for protecting a group of herbicide-tolerant transgenic plants grown in a field by destroying weeds, characterized in that said plants comprise the nucleic acid molecule as defined in claim 1, and said weeds are destroyed by applying a herbicide comprising a glutamine synthetase inhibitor as an active ingredient.

Description

RELATED ORDERS [0001] This application claims the benefit of U.S. Provisional Application No. 62/431,664, filed December 8, 2016, and incorporated by reference in its entirety herein. SEQUENCE LISTING [0002] A Sequence Listing in ASCII text format, submitted under 37 C.F.R. § 1.821, entitled “81157_ST25.txt”, 139 kilobytes in size, generated on October 25, 2017 and deposited via EFS-Web is provided instead of a paper copy. This Sequence Listing is thus incorporated by reference in the descriptive report for your disclosures. FIELD OF THE INVENTION [0003] The present invention relates generally to the field of plant biotechnology. More specifically, the present invention relates to compositions and methods for improving transformation frequency. Specifically, the invention includes compositions and methods for using enhanced selection markers to improve transformation frequency. BACKGROUND OF THE INVENTION [0004] Cultivated crops such as maize, soybeans, and cotton have substantial commercial value worldwide. The development of useful scientific methods for improving the quantity and quality of important crops is therefore of significant commercial interest. Significant effort has been expended to improve the quality of cultivated crop species through conventional plant breeding. Conventional methods for crop and horticultural breeding utilize artificial selection techniques to identify plants with desirable characteristics. However, such artificial selection techniques have several disadvantages, namely the fact that these techniques are often very labor-intensive and result in plants that often contain heterogeneous genetic components that may not always result in the desired characteristic being transmitted from the parent plants. Advances in molecular biology have allowed humans to modify the germplasm of animals and plants. Plant genetic engineering involves the isolation and manipulation of genetic material (typically in the form of DNA) and the subsequent introduction of this genetic material into the genome of a plant. This technology has the ability to impart improved economic, agronomic, or horticultural characteristics to crops or plants. [0005] The introduction of foreign genetic material into a plant's genome is typically accomplished by one of two methods, although other methods are known to experts in the art. The first is biolistic particle bombardment, whereby a metal particle is coated with the foreign DNA, or “transgene,” which is then released into plant tissue. Some of this foreign genetic material is taken up by plant cells, which are thus “transformed.” The second method is by Agrobacterium-mediated transformation, which involves exposing plant cells and tissues to a suspension of Agrobacterium cells containing specific DNA plasmids. In both methods, the foreign DNA typically encodes a selection marker that allows plant cells to grow in the presence of a selection agent, for example, an antibiotic or herbicide. These cells can be further manipulated to regenerate, forming complete fertile transgenic plants. [0006] Glutamine synthetase (GS) is one of the essential enzymes for the development and life of most plant cells. GS is known to convert glutamate to glutamine. GS is involved in an efficient pathway in most plants for the detoxification of ammonia released by nitrate reduction, amino acid degradation, or photorespiration. Therefore, potent GS inhibitors are highly toxic to plant cells and can be used as broad-spectrum herbicides. One class of herbicides, which includes phosphinothricin (PPT) and glufosinate, consists of GS inhibitors. Transgenic plants have been made tolerant to this class of herbicides through the introduction of a gene encoding a phosphinothricin acetyltransferase (PAT). Such plants are said to be herbicide-tolerant. In these transgenic plants, PAT detoxifies PPT by acetylation of the free amino group of PPT. The PAT gene is derived from Streptomyces viridochromogenes and confers tolerance to GS inhibitors. (U.S. Patents Nos. 5,531,236, 5,646,024, 5,648,477 and 5,276,268). [0007] In addition to the PAT gene functioning as a herbicide tolerance trait gene for GS-inhibiting herbicides, it can also be used as a selection marker in the transformation of monocotyledonous and dicotyledonous plant species. In cereal transformation, its use is more widespread than other selection markers. However, despite the PAT gene having been successfully used as a selection marker, the transformation frequency is relatively low, so its use as a selection marker requires many resources. An enhanced PAT, which can confer an increase in the transformation frequency, is needed to improve the usefulness of PAT as a selection marker. SUMMARY OF THE INVENTION [0008] The present invention provides an optionally isolated nucleic acid molecule that is at least 90% identical to any of SEQ ID NO: 1 to 20. The present invention also provides a nucleic acid molecule, a chimeric nucleic acid mole