Methods of introducing dsRNA to plant seeds for modulating gene expression

• US 10,683,505 B2
• Filed: 12/30/2013
• Issued: 06/16/2020
• Est. Priority Date: 01/01/2013
• Status: Expired due to Fees

• Current Assignee: Monsanto Technology LLC (Bayer AG)
• Original Assignee: Monsanto Technology LLC (Bayer AG)
• Inventors: Avniel, Amir, Lidor-Nili, Efrat, Maor, Rudy, Meir, Ofir, Noivirt-Brik, Orly
• Primary Examiner(s): Fan, Weihua
• Application Number:
  US14/143,836
• Publication Number:
  US 20140230090A1
• Time in Patent Office: 2,360 Days
• 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 15/1131 against viruses
• CPC Class Codes: C12N 15/1137 against enzymes viral enzymes C12N15/1131; receptors C12N15/1138
• 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
• CPC Class Codes: C12N 15/8283 for virus resistance
• CPC Class Codes: C12N 15/8286 for insect resistance
• CPC Class Codes: C12N 2310/14 interfering N.A.
• CPC Class Codes: C12N 2320/32 Special delivery means, e.g. tissue-specific
• CPC Class Codes: Y02A 40/146 Genetically Modified [GMO] plants, e.g. transgenic plants

Abstract

A method of introducing an exogenous non-transcribable polynucleotide trigger, for example dsRNA, molecule into a seed is provided. The method comprises contacting the seed with the exogenous non-transcribable polynucleotide trigger, for example dsRNA, molecule under conditions which allow penetration of the exogenous non-transcribable polynucleotide trigger, for example dsRNA, molecule into the seed, thereby introducing the exogenous non-transcribable polynucleotide trigger, for example dsRNA, molecule into the seed.

Claims

1. A method of providing a maize, soybean, rice, wheat, tomato, or cucumber plant having improved resistance to an insect pest selected from the group consisting of a Spodoptera spp., Diabrotica virgifera virgifera, and Leptinotarsa decemlineata, comprising:

growing said plant from a seed,

wherein said seed has been soaked in a solution comprising a concentration of 0.005-1.5 μ

g/μ

L exogenous non-transcribable double-stranded RNA (dsRNA) molecule comprising a nucleic acid sequence that is identical or complementary to at least 21 contiguous nucleotides of an essential gene of said insect pest or to the sequence of an RNA transcribed from said essential gene, wherein said seed is shaken in said solution for up to 24 hours,

wherein said plant exhibits improved resistance to said insect pest relative to a control plant, and

wherein said control plant is grown from a seed not contacted with said exogenous dsRNA molecule.

2. The method of claim 1, wherein said insect pest is Spodoptera littoralis.

3. The method of claim 1, wherein said insect pest gene is selected from the group consisting of V-ATPase, NADPH Cytochrome P450 Oxidoreductase, Inhibitor of Apoptosis (IAP), Chitin Synthase, EF1α

, and β

-actin.

4. The method of claim 1, wherein said exogenous non-transcribable dsRNA molecule further comprises a nucleic acid sequence that is at least 80% identical to at least 25 consecutive bases of a nucleic acid sequence of an endogenous maize, soybean, rice, wheat, tomato, or cucumber gene.

5. The method of claim 1, wherein said seed is further treated with an agent selected from the group consisting of a pesticide, a fungicide, an insecticide, a fertilizer, a coating agent, and a coloring agent.

6. A method for generating a maize, soybean, rice, wheat, tomato, or cucumber plant having insect resistance-comprising:

a) soaking an ungerminated seed in a solution comprising a non-transcribable double-stranded RNA (dsRNA) molecule at a concentration of 0.005-1.5 μ

g/μ

L,

b) shaking said ungerminated seed in said solution for up to 24 hours,

wherein said non-transcribable dsRNA molecule is introduced into said ungerminated seed,

wherein said non-transcribable dsRNA molecule comprises at least one polynucleotide strand comprising at least one segment comprising a first nucleic acid sequence identical or complementary to at least 21 contiguous nucleotides of an essential insect pest gene in either anti-sense or sense orientation,

wherein said insect pest is selected from the group consisting of a Spodoptera spp., Diabrotica virgifera virgifera, and Leptinotarsa decemlineata; and

c) germinating said ungerminated seed to generate said maize, soybean, rice, wheat, tomato, or cucumber plant exhibiting insect resistance after emerging from the seed.

7. The method of claim 6, wherein said plant does not comprise detectable levels of DNA having a nucleic acid sequence of said the nucleic acid molecule after emerging from the seed.

8. The method of claim 6, wherein said insect pest gene is selected from the group consisting of V-ATPase, NADPH Cytochrome P450 Oxidoreductase, Inhibitor of Apoptosis (IAP), Chitin Synthase, EF1α

, and β

-actin.

9. The method of claim 6, wherein said insect resistance is resistance to Spodoptera littoralis infestation.

10. The method of claim 6, wherein said non-transcribable nucleic acid molecule further comprises a second segment having a second nucleic acid sequence that is at least 80% identical to at least 25 consecutive bases to an endogenous maize, soybean, rice, wheat, tomato, or cucumber gene.

11. The method of claim 6, further comprising priming said seed prior to introducing said non-transcribable nucleic acid molecule.

12. The method of claim 11, wherein said priming comprises:

(i) washing said seed prior to introducing said nucleic acid molecule; and

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