Methods and compositions for introducing nucleic acids into plants

• US 10,655,136 B2
• Filed: 06/02/2016
• Issued: 05/19/2020
• Est. Priority Date: 06/03/2015
• Status: Active Grant

• Current Assignee: Monsanto Technology LLC (Bayer AG)
• Original Assignee: Monsanto Technology LLC (Bayer AG)
• Inventors: Huang, Shihshieh, Iandolino, Alberto B., Peel, Gregory J.
• Primary Examiner(s): Zheng, Li
• Application Number:
  US15/579,120
• Publication Number:
  US 20180163219A1
• Time in Patent Office: 1,447 Days
• Field of Search: 800279
• CPC Class Codes: A01H 1/06 Processes for producing mutations, e.g. treatment with chemicals or with radiation specific mutations prepared by genetic engineering on plant cell or plant tissues C12N15/00 ; process for producing transgenic plants C12N15/82
• CPC Class Codes: C12N 15/8207 by mechanical means, e.g. microinjection, particle bombardment, silicon whiskers
• CPC Class Codes: C12N 15/8213 Targeted insertion of genes into the plant genome by homologous recombination
• CPC Class Codes: C12N 15/8218 Antisense, co-suppression, viral induced gene silencing [VIGS], post-transcriptional induced gene silencing [PTGS]
• CPC Class Codes: C12N 15/8279 for biotic stress resistance, pathogen resistance, disease resistance

Abstract

The present disclosure provides compositions and methods for introducing nucleic acids into plants. More specifically, disclosed herein are methods and compositions for introducing a nucleic acid, such as a double-stranded RNA, for silencing a target gene in a plant. Also disclosed herein are plants exhibiting modification of a target gene, obtained through the use of such methods and compositions.

Claims

1. A method for silencing a target gene in a plant, comprising, in any order, the steps of:

(a) abrading a surface of a plant with a particulate of a size greater than about 2.5 micrometers;

(b) applying an RNA to the surface of the plant, wherein the RNA comprises a sequence that is complementary or identical to 21 or more contiguous nucleotides in the sequence of a target gene in the plant;

wherein the particulate, the RNA, or both, are further applied with an osmolyte, and

whereby the target gene is silenced.

2. The method of claim 1, wherein (a) the target gene is an endogenous gene of the plant or a transgene expressed in the plant, (b) the target gene in the plant is a gene of a parasitic plant, fungus, or bacterium that is a pathogen or parasite of the plant, (c) the target gene is multiple target genes, or (d) the target gene is multiple target genes from more than one species.

3. The method of claim 1, wherein (a) the RNA is single-stranded RNA, (b) the RNA is double-stranded RNA, (c) the RNA is a miRNA precursor or a small RNA, (d) the RNA is non-transcribable, (e) the RNA is not self-replicating, (f) the RNA comprises 21-500 nucleotides or base pairs, or (g) the RNA is not contained in a viral vector nor encoded by a plasmid.

4. The method of claim 1, wherein (a) the particulate is selected from the group consisting of a mineral abrasive, a metal abrasive, a synthetic abrasive, and an organic abrasive, (b) the particulate is selected from the group consisting of aluminum oxide, silicon carbide, silicon dioxide, soda lime glass, diatomaceous silica (diatomaceous earth), flint, quartz, garnet, silicon dioxide, pumice, sand, feldspar, calcite, steel, tungsten, ceramic, boron carbide, tungsten carbide, (c) the particulate is of an average size range from about 2.5 micrometers to about 50 micrometers, or (d) the particulate is supported by, attached to, or embedded in a matrix.

5. The method of claim 1, wherein the particulate, the RNA, or both, are further applied with (a) at least one selected from the group consisting of water, a biocide, a chelator, a buffer, a nonionic surfactant, a zwitterionic surfactant, a defoamer, a rainfastness agent, and a photoprotectant, (b) at least one selected from the group consisting of a carrier agent, a surfactant, an organosilicone, a polynucleotide herbicidal molecule, a non-polynucleotide herbicidal molecule, a non-polynucleotide pesticide, a safener, an insect attractant, and an insect growth regulator, or (c) at least one pesticidal agent selected from the group consisting of a patatin, a plant lectin, a phytoecdysteroid, a Bacillus thuringiensis insecticidal protein, a Xenorhabdus insecticidal protein, a Photorhabdus insecticidal protein, a Bacillus laterosporous insecticidal protein, and a Bacillus sphaericus insecticidal protein.

6. A method for introducing a nucleic acid into a plant, comprising:

(a) in any order, the steps of (i) applying a composition comprising a nucleic acid to a surface of a plant, and (ii) contacting a matrix supporting an abrasive with the surface;

or,

(b) in any order, the steps of (i) abrading a surface of a plant with a particulate of a size greater than about 2.5 micrometers, and (ii) applying a composition comprising a nucleic acid to the surface of the plant, wherein the nucleic acid comprises a sequence that is complementary or identical to 21 or more contiguous nucleotides in the sequence of a target gene in the plant;

wherein the abrasive or the particulate, or the composition comprising a nucleic acid, or both, are further applied with an osmolyte, and

whereby the nucleic acid is introduced into the plant.

7. The method of claim 6, wherein (a) the nucleic acid is DNA, (b) the nucleic acid is DNA in the form of a plasmid or viral vector, (c) the nucleic acid is single-stranded RNA, (d) the nucleic acid is double -stranded RNA, (e) the nucleic acid is a combination of DNA and RNA, (f) the nucleic acid is a miRNA precursor or a small RNA, (g) the nucleic acid is a non-transcribable RNA, (h) the nucleic acid is a non-self-replicating DNA or RNA, (i) the nucleic acid is a DNA that comprises 21-500 nucleotides or base pairs, (j) the nucleic acid is an RNA that comprises 21-500 nucleotides or base pairs, (k) the nucleic acid is an RNA that is not contained in a viral vector nor encoded by a plasmid, (l) the nucleic acid is provided in a microbial cell or as a microbial fermentation product, or (m) the nucleic acid comprises at least 19 consecutive nucleotides having a sequence that is complementary or identical to the sequence of a target gene in the plant.

8. The method of claim 6, wherein (a) the target gene is an endogenous gene of the plant or a transgene expressed in the plant, (b) the target gene in the plant is a gene of a parasitic plant, fungus, or bacterium that is a pathogen or parasite of the plant, (c) the target gene is multiple target genes, or (d) the target gene is multiple target genes from more than one species.

9. The method of claim 6, wherein (a) the abrasive or the particulate is selected from the group consisting of a mineral abrasive, a metal abrasive, a synthetic abrasive, and an organic abrasive, (b) the abrasive or the particulate is selected from the group consisting of aluminum oxide, silicon carbide, silicon dioxide, soda lime glass, diatomaceous silica (diatomaceous earth), flint, quartz, garnet, silicon dioxide, pumice, sand, feldspar, calcite, steel, tungsten, ceramic, boron carbide, tungsten carbide, (c) the abrasive or the particulate is of an average size range from about 2.5 micrometers to about 50 micrometers, or (d) the abrasive or the particulate is supported by, attached to, or embedded in a matrix.

10. The method of claim 6, wherein the abrasive or the particulate, or the composition comprising a nucleic acid, or both, are further applied with (a) at least one selected from the group consisting of water, a biocide, a chelator, a buffer, a nonionic surfactant, a zwitterionic surfactant, a defoamer, a rainfastness agent, and a photoprotectant, (b) at least one selected from the group consisting of a carrier agent, a surfactant, an organosilicone, a polynucleotide herbicidal molecule, a non-polynucleotide herbicidal molecule, a non-polynucleotide pesticide, a safener, an insect attractant, and an insect growth regulator, or (c) at least one pesticidal agent selected from the group consisting of a patatin, a plant lectin, a phytoecdysteroid, a Bacillus thuringiensis insecticidal protein, a Xenorhabdus insecticidal protein, a Photorhabdus insecticidal protein, a Bacillus laterosporous insecticidal protein, and a Bacillus sphaericus insecticidal protein.