Polynucleotide molecules for gene regulation in plants

• US 9,988,634 B2
• Filed: 08/30/2013
• Issued: 06/05/2018
• Est. Priority Date: 03/08/2010
• Status: Active Grant

• Current Assignee: Monsanto Technology LLC (Bayer AG)
• Original Assignee: Monsanto Technology LLC (Bayer AG)
• Inventors: Sammons, Robert D, Ivashuta, Sergey, Liu, Hong, Wang, Dafu, Feng, Paul C. C., Kouranov, Andrei Y, Andersen, Scott E
• Primary Examiner(s): UYENO, STEPHEN G
• Application Number:
  US14/015,785
• Publication Number:
  US 20140057789A1
• Time in Patent Office: 1,740 Days
• Field of Search: None
• CPC Class Codes: A01N 2300/00 Combinations or mixtures of active ingredients covered by classes A01N27/00 - A01N65/48 with other active or formulation relevant ingredients, e.g. specific carrier materials or surfactants, covered by classes A01N25/00 - A01N65/48
• CPC Class Codes: A01N 33/12 Quaternary ammonium compounds
• CPC Class Codes: A01N 57/16 containing heterocyclic radicals
• CPC Class Codes: A01N 57/20 containing acyclic or cycloaliphatic radicals
• CPC Class Codes: A01N 57/34 having phosphorus-to-halogen bonds; Phosphonium salts
• CPC Class Codes: A01N 63/60 Isolated nucleic acids
• CPC Class Codes: C12N 15/113 Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing when used in plants C12N15/8218
• CPC Class Codes: C12N 15/1137 against enzymes viral enzymes C12N15/1131; receptors C12N15/1138
• CPC Class Codes: C12N 15/8206 by physical or chemical, i.e. non-biological, means, e.g. electroporation, PEG mediated
• CPC Class Codes: C12N 15/8207 by mechanical means, e.g. microinjection, particle bombardment, silicon whiskers
• CPC Class Codes: C12N 15/8218 Antisense, co-suppression, viral induced gene silencing [VIGS], post-transcriptional induced gene silencing [PTGS]

Abstract

This invention provides polynucleotide molecules and methods for regulating genes in plants, e. g., by providing RNA for systemic regulation of genes. Various aspects of the invention provide polynucleotide molecules and methods for regulating endogenous genes and transgenes in a plant cell and polynucleotide molecules.

Claims

1. A method of delivering a double-stranded RNA (dsRNA) polynucleotide to the interior of a cell of a plant comprising:

applying the dsRNA polynucleotide and a transferring agent selected from an organosilicone surfactant and a cationic lipid to an exterior surface of the plant,

wherein the transferring agent facilitates permeation of the dsRNA polynucleotide from the exterior surface of the plant to the interior of the plant cell without the aid of a physical abrasive, and

wherein the dsRNA polynucleotide is 21 to 700 base pairs in length and comprises a nucleotide sequence that is identical or complementary to at least 21 contiguous nucleotides of an endogenous gene or a transcribed RNA of the plant.

2. The method of claim 1, wherein the plant is growing in a field.

3. The method of claim 1, wherein the plant is a pigweed, velvetleaf, waterhemp, prickly lettuce, dandelion, alfalfa, corn, soybean, canola, cotton, sugar beet, sugarcane, wheat, or rice plant.

4. The method of claim 1, wherein the plant is a weed or volunteer plant.

5. The method of claim 1, wherein the plant is resistant to one or more of glyphosate, dicamba and sulfonylurea.

6. The method of claim 1, wherein the cell is a leaf cell.

7. The method of claim 1, wherein the dsRNA polynucleotide and the transferring agent are applied separately.

8. The method of claim 1, wherein the dsRNA polynucleotide and the transferring agent are applied concurrently.

9. The method of claim 1, wherein the organosilicone surfactant is a silicone polyether copolymer.

10. The method of claim 9, wherein the silicone polyether copolymer is a copolymer of polyalkylene oxide modified heptamethyl trisiloxane and allyloxypolypropylene glycol methylether.

11. The method of claim 1, further comprising applying an organic or an inorganic salt.

12. The method of claim 11, wherein the salt is an ammonium salt.

13. The method of claim 12, wherein the ammonium salt is ammonium sulfate.

14. The method of claim 1, wherein the dsRNA polynucleotide and the transferring agent are applied onto the exterior surface of the plant by a spray apparatus.

15. The method of claim 1, wherein the endogenous gene:

(i) is an essential gene, (ii) encodes a protein that provides herbicide resistance, or (iii) transcribes to an RNA regulatory agent.

16. The method of claim 1, wherein the endogenous gene encodes a protein selected from the group consisting of a 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS), an acetohydroxyacid synthase, an acetolactate synthase (ALS), an acetyl-coenzyme A carboxylase (ACCase), a dihydropteroate synthase, a phytoene desaturase (PDS), a protoporphyrin IX oxygenase (PPO), a hydroxyphenylpyruvate dioxygenase (HPPD), a para-aminobenzoate synthase, a glutamine synthase (GS), a glufosinate-tolerant glutamine synthase, a 1-deoxy-D-xylulose 5-phosphate (DOXP) synthase, a dihydropteroate (DHP) synthase, a phenylalanine ammonia lyase (PAL), a glutathione S-transferase (GST), a D1 protein of photosystem II, a mono-oxygenase, a cytochrome P450, a cellulose synthase, a beta-tubulin, and a serine hydroxymethyltransferase.

17. The method of claim 1, wherein the endogenous gene is a native gene or a recombinant transgene.

18. The method of claim 1, further comprising applying a non-polynucleotide herbicide.

19. The method of claim 18, wherein the non-polynucleotide herbicide is selected from the group consisting of glyphosate, dicamba, phosphinothricin, bromoxynil, ioxynil and chlorsulfuron.