Compositions and methods for silencing gene expression

• US 10,240,162 B2
• Filed: 05/23/2013
• Issued: 03/26/2019
• Est. Priority Date: 05/24/2012
• Status: Expired due to Fees

• Current Assignee: Monsanto Technology LLC (Bayer AG)
• Original Assignee: AB Seeds Ltd (Bayer AG)
• Inventors: Avniel, Amir, Lidor-Nili, Efrat, Maor, Rudy, Meir, Ofir, Noivirt-Brik, Orly, Yanai-Azulay, Osnat
• Primary Examiner(s): Deveau Rosen, Jason
• Application Number:
  US14/403,491
• Publication Number:
  US 20150096079A1
• 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 naked dsRNA into a tomato seed, the method comprising shaking said tomato seed in a solution comprising a final concentration of 0.05-100 μ

g/μ

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

5. The method of claim 1, wherein said naked dsRNA is designed for down regulating expression of a gene of a viral pathogen.

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

7. The method of claim 1, wherein said naked dsRNA comprises siRNA.

8. The method of claim 1, further comprising priming said tomato seed prior to said shaking.

9. The method of claim 8, wherein said priming is effected by:

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

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

10. The method of claim 1, further comprising treating the tomato 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 following said contacting.

11. The method of claim 1, wherein said naked dsRNA is for downregulating expression of a coding gene.

12. The method of claim 1, wherein a concentration of the naked dsRNA is adjusted according to a parameter selected from the group consisting of, seed size, seed weight, seed volume, seed surface area, seed density and seed permeability.

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

15. 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 tomato seed a solution comprising a final concentration of 0.05-100 μ

g/μ

L of a naked dsRNA from 1 hour up to 60 hours, thereby introducing said naked dsRNA into said tomato seed, wherein said naked dsRNA down regulates expression of said target gene of said tomato seed, and wherein said naked dsRNA is not associated with a cationic oligopeptide; and

optionally

(b) generating a tomato plant from said tomato seed.

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

4. The method of claim 3, further comprising growing said tomato plant under abiotic or biotic stress following said generating.

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

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