# US 9,433,217 B2 · Methods and compositions for introduction of exogenous dsRNA into plant cells

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- Number: US 9,433,217 B2
- Document ID: US9433217B2
- Status: Granted
- Kind: B2
- Application number: 13/585,947
- Filed: 2012-08-15
- Published: 2013-02-21
- Granted: 2016-09-06
- Earliest claimed: 2011-08-16
- Assignees: Syngenta Participations AG
- Applicants: Guo-Qing Tang
- Inventors: Guo-Qing Tang
- Web page: https://patentgrep.com/patent/US9433217B2

## CPC

- A01N 57/16: containing heterocyclic radicals
- C12N 15/8206: {by physical or chemical, i.e. non-biological, means, e.g. electroporation, PEG mediated}
- C12N 15/8218: {Antisense, co-suppression, viral induced gene silencing [VIGS], post-transcriptional induced gene silencing [PTGS]}
- A01N: PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF (preservation of food or foodstuff A23); BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES (preparations for medical, dental or toiletry purposes which kill or prevent the growth or proliferation of unwanted organisms A61K); PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)

## Abstract

This invention provides a method to silence an endogenous target gene expression in plants by applying a specific dsRNA onto the exterior surface of a plant. Application, such as by spraying or brushing a plant with dsRNA is done without wounding the plant tissue and cells such as by mechanical-type wounding, particle bombardment or mechanical infection with viral vectors. The present invention enables the regulation of gene expression in plants. In some embodiments of the invention, the dsRNA is directed to an essential gene of a plant pathogen or pest, whereby the pathogen and/or pest damage is controlled, resulting in desired agronomic performance.

## Claims

### Claim 1

A method of integrating dsRNA into a plant cell to silence an endogenous target gene of a plant comprising:
a) providing a formulation comprising a gene-specific dsRNA, water, and a plant hormone Brassinosteroid, wherein the Brassinosteroid is at 1.0 micromolar, and wherein the gene-specific dsRNA is derived from the endogenous target gene encoding a glutamine synthetase;
b) applying the formulation to the leaf surface of a live plant, wherein the dsRNA is assimilated from the external leaf surface into cells of the plant leaf, wherein the silencing efficiency of the endogenous target gene is increased five to eight fold compared to a formulation containing the dsRNA without the plant hormone Brassinosteroid, and wherein the plant is a maize plant.

### Claim 2 (depends on 1)

The method of claim 1 wherein the step of applying the formulation is to a live plant about 12 days from germination.

### Claim 3 (depends on 1)

The method of claim 1 wherein the gene-specific dsRNA is derived from the glutamine synthetase cDNA sequence depicted in SEQ ID NO: 2.

### Claim 4

A method of integrating dsRNA into a plant cell to silence an endogenous target gene of a plant comprising:
a) providing a formulation comprising a gene-specific dsRNA, water, and a plant hormone Brassinosteroid, wherein the dsRNA is at about 250 nanograms per microliter, wherein the Brassinosteroid is at 1.0 micromolar, and wherein the gene specific dsRNA is derived from the endogenous target gene encoding a glutamine synthetase; and
b) applying the formulation to the leaf surface of a live plant, wherein the dsRNA is assimilated from the external leaf surface into cells of the plant leaf, wherein the silencing efficiency of the endogenous target gene is increased five to eight fold compared to a formulation containing the dsRNA without the plant hormone Brassinosteroid, and wherein the plant is a maize plant.

### Claim 5 (depends on 4)

The method of claim 4 wherein the step of applying the formulation is to a live plant about 12 days from germination.

### Claim 6 (depends on 4)

The method of claim 4 wherein the gene-specific dsRNA is derived from the glutamine synthetase cDNA sequence depicted in SEQ ID NO: 2.

### Claim 7

A method of integrating dsRNA into a plant cell to silence an endogenous target gene of a plant comprising:
a) providing a formulation comprising a gene-specific dsRNA, water, and a plant hormone Brassinosteroid, wherein the Brassinosteroid is at 1.0 micromolar, and wherein the gene-specific dsRNA is derived from the glutamine synthetase cDNA sequence depicted in SEQ ID NO: 2; and
b) applying the formulation to the leaf surface of a live plant about 12 days from germination, wherein the dsRNA is assimilated from the external leaf surface into cells of the plant leaf, wherein the silencing efficiency of the endogenous target gene is increased five to eight fold compared to a formulation containing the dsRNA without the plant hormone Brassinosteroid, and wherein the plant is a maize plant.