Compositions and methods for silencing gene expression

• US 10,240,161 B2
• Filed: 05/23/2013
• Issued: 03/26/2019
• Est. Priority Date: 05/24/2012
• Status: Active Grant

• Current Assignee: Monsanto Technology LLC (Bayer AG)
• Original Assignee: A.B. Seed Ltd.
• Inventors: Avniel, Amir, Lidor-Nili, Efrat, Maor, Rudy, Meir, Ofir, Noivirt-Brik, Orly, Yanai-Azulay, Osnat
• Primary Examiner(s): Rosen, Jason Deveau
• Application Number:
  US13/901,326
• Publication Number:
  US 20130318657A1
• Time in Patent Office: 2,133 Days
• Field of Search: None
• CPC Class Codes: C12N 15/8201 Methods for introducing genetic material into plant cells, e.g. DNA, RNA, stable or transient incorporation, tissue culture methods adapted for transformation
• CPC Class Codes: C12N 15/8206 by physical or chemical, i.e. non-biological, means, e.g. electroporation, PEG mediated
• CPC Class Codes: C12N 15/8216 Methods for controlling, regulating or enhancing expression of transgenes in plant cells
• CPC Class Codes: C12N 15/8218 Antisense, co-suppression, viral induced gene silencing [VIGS], post-transcriptional induced gene silencing [PTGS]
• CPC Class Codes: C12N 15/825 involving pigment biosynthesis
• CPC Class Codes: C12N 15/8261 with agronomic (input) traits, e.g. crop yield
• CPC Class Codes: C12N 15/8266 Abscission; Dehiscence; Senescence
• CPC Class Codes: C12N 15/8267 Seed dormancy, germination or sprouting
• CPC Class Codes: C12N 15/8271 for stress resistance, e.g. heavy metal resistance
• CPC Class Codes: C12N 15/8279 for biotic stress resistance, pathogen resistance, disease resistance
• CPC Class Codes: C12N 15/8282 for fungal resistance
• CPC Class Codes: C12N 15/8283 for virus resistance
• CPC Class Codes: Y02A 40/146 Genetically Modified [GMO] plants, e.g. transgenic plants

Abstract

A method of introducing naked dsRNA into a seed is provided. The method comprising contacting the seed with the naked dsRNA under conditions which allow penetration of the dsRNA into the seed, thereby introducing the dsRNA into the seed.

Claims

1. A method of introducing into a seed naked dsRNA that is capable of down-regulating a target gene, the method comprising shaking the seed in a solution comprising a final concentration of 0.013-100 μ

g/μ

L of the naked dsRNA from 1 hour up to 60 hours, thereby introducing the dsRNA into the seed, wherein the naked dsRNA is not associated with a cationic oligopeptide.

7. The method of claim 1, wherein said introducing comprises penetration to an endosperm, and alternatively or additionally, an embryo of said seed.

9. The method of claim 1, wherein said introducing results in the presence of said dsRNA in the plant for at least 10 days following germination.

11. The method of claim 1, further comprising priming the seed prior to said shaking.

12. The method of claim 11, wherein said priming is effected by:

(i) washing the seed prior to said shaking; and

(ii) drying the seed following step (i).

13. The method of claim 12, wherein said washing is effected for 2-6 hours.

14. The method of claim 12, wherein said drying is effected at 25-30°

C. for 10-16 hours.

15. The method of claim 1, wherein said final concentration of the naked dsRNA is 0.013-0.5 μ

g/μ

l.

16. The method of claim 1, wherein said shaking is effected prior to breaking of seed dormancy and embryo emergence.

17. The method of claim 1, further comprising coating the seed with an agent selected from the group consisting of a pesticide, a fungicide, an insecticide, a fertilizer, a coating agent, and a coloring agent.

19. The method of claim 1, wherein said final concentration of the naked dsRNA is 0.013-3 μ

g/μ

l.

21. The method of claim 1, wherein said shaking is from 1 to 24 hours.

2. A method of down-regulating expression of a target gene, the method comprising:

(a) shaking a seed in a solution comprising a final concentration of 0.013-100 μ

g/μ

L of a naked dsRNA from 1 hour up to 60 hours, wherein the naked dsRNA down-regulates expression of the target gene, and wherein the naked dsRNA is not associated with a cationic oligopeptide; and

optionally

(b) generating a plant of the seed.

3. The method of claim 2, wherein said down-regulating expression of the target gene improves tolerance of the plant to abiotic or biotic stress.

4. The method of claim 2, further comprising drying said seed following said shaking.

5. The method of claim 2, wherein said target gene is an endogenous plant gene.

6. The method of claim 2, wherein said target gene is a gene of a viral pathogen.

8. The method of claim 2, wherein said naked dsRNA does not integrate into the genome of said seed.

10. The method of claim 6 further comprising observing reduced infectability or replicability of said viral pathogen.

18. The method of claim 2, further comprising coating the seed with an agent selected from the group consisting of a pesticide, a fungicide, an insecticide, a fertilizer, a coating agent, and a coloring agent.

20. The method of claim 2, wherein said final concentration of the naked dsRNA is 0.013-3 μ

g/μ

l.

22. The method of claim 2, wherein said shaking is from 1 to 24 hours.