Surface application of polynucleotide molecules for improving the yield traits of plants

Abstract

A composition comprising: (i) a dsRNA molecule of at least 18 consecutive nucleotides that is substantially identical or substantially complementary to a plant gene or a transcript of said plant gene; and (ii) a transfer agent that modulates the plant surface to enable the penetration of the dsRNA molecule into plant cells; wherein the penetration of the dsRNA molecule into plant cells causes a transient decrease in gene expression, and wherein the transient decrease in gene expression causes a change in a plant yield-related trait.

Description

BACKGROUND OF THE INVENTION

[0002] According to data from the United Nations Food and Agricultural Organization (UN FAO), by 2050, the world's population will exceed 9.6 billion, which will require significant improvements in agriculture to meet the growing food demand. At the same time, resource conservation, reducing the use of fertilizers, pesticides, and herbicides, and environmental sustainability are increasingly important factors in how food is grown. There is a need to improve agricultural plants and farming practices so that increased plant yields can be achieved using fewer resources and more environmentally sustainable inputs.

[0003] Yield is affected by various factors, such as the number and size of plant organs, plant architecture (e.g., the number of branches), seed filling, seed number, drought tolerance, shattering, the number of flowers and tillers, etc.

[0004] Currently, crop performance is mainly optimized through techniques targeting the interaction between crop genotypes (e.g., plant breeding, genetically modified (GM) crops) and their surrounding environment (e.g., fertilizers, synthetic herbicides, pesticides). While these approaches have helped double global food production in the past 50 years, the yield growth rate of many major crops has stalled, and there is an urgent need for new solutions to increase crop yields. In addition to their long development and regulatory timelines, public concerns about GM crops and synthetic chemicals have challenged their use in many major crops and countries, leading to a lack of acceptance of many transgenic traits and the exclusion of GM crops and many synthetic chemicals from some global markets. Therefore, there is a significant need for innovative, effective, environmentally sustainable, and publicly acceptable methods to increase yields. SUMMARY OF THE INVENTION

[0006] The present invention provides compositions and methods for providing increased yield in plants by inhibiting the expression of yield-related genes in plants, by providing to the plant surface a composition comprising a polynucleotide molecule capable of hybridizing to a yield-related gene or gene transcript and a transfer agent that conditions the plant surface to permit penetration of the polynucleotide molecule into plant cells, thereby improving yield-related traits in plants. Non-limiting examples of yield-related traits in plants include: increased branching in plants, grain size, increased number of panicles, increased number of tillers, increased seeds, increased size of plant siliques, increased seed filling, increased number of seeds, increased heading, improved drought tolerance, reduced breakage, reduced abscission tissue formation, reduced petals in plants, late / early flowering, shortened / extended flowering period, delayed senescence, increased oil content, improved oil composition, starch content, starch composition, carbohydrate content, carbohydrate composition, increased protein content, improved protein composition, and any combination thereof. Each possibility is a separate embodiment.

[0007] According to some embodiments, penetration of the polynucleotide molecule can cause a transient reduction in gene expression, resulting in non-permanent spatial and temporal effects on the plant and not leading to or requiring integration of exogenous polynucleotides into the plant's chromosomes. This method has several advantages. First, it circumvents the need for GMO legislation. Additionally, it is a more refined and efficient method because it is dynamic and allows the user to determine the application based on real-time needs, the timing of trait improvement, and / or the environmental conditions to be addressed. As non-limiting examples, during a drought, a farmer can decide to transiently inhibit the expression of a gene / transcript to improve resilience to water stress and stop the inhibition once the weather changes. As another non-limiting example, a farmer can decide to transiently inhibit a gene / transcript associated with early flowering in plants in the event of unexpected rainfall, temperature changes, etc.

[0008] Advantageously, the technology is applicable for use in a variety of crops, including but not limited to maize, rice, soybean, cotton, canola, oilseed rape, tomato, potato, etc., and is particularly applicable to crops with complex genomes, such as wheat, strawberry, or fruit trees.

[0009] According to some embodiments, the polynucleotide molecule is provided in a composition that is permeable or absorbable into living plant tissue to initiate systemic gene silencing or regulation. In certain embodiments of the present invention, the polynucleotide molecule ultimately provides to the plant an RNA (e.g., dsRNA) or RNA-like molecule that is capable of hybridizing to RNA transcribed from an endogenous target gene in the plant cell under physiological conditions in the plant cell, thereby affecting (silencing or inhibiting) the expression of the target gene.

[0010] According to some embodiments, silencing / inhibition of a target gene can directly improve yield-related traits of a plant. Optionally, silencing / inhibition of the target gene can indirectly improve yield-related traits of a plant. For example, silencing or inhibiting the target gene can alter (increase or decrease) the expression of another gene that improves yield-related traits of the plant.

[0011] As another important practical advantage, surface application of a composition comprising an exogenous polynucleotide and a transfer agent does not require the exogenous polynucleotide to be physically bound to particles, such as in the biolistic-mediated introduction of polynucleotides associated with gold or tungsten particles into the interior portions of plants, plant parts, or plant cells.

[0012] According to some embodiments, the polynucleotide molecule targets the mRNA of a plant gene. According to some embodiments, the polynucleotide molecule targets the translation region of the mRNA. According to some embodiments, the polynucleotide molecule targets the untranslated region of the mRNA.

[0013] According to some embodiments, there is provided a composition comprising: (i) a polynucleotide molecule comprising at least 18 consecutive nucleotides that are substantially identical or substantially complementary to a plant gene or a transcript of a plant gene; and (ii) a transfer agent that conditions the plant surface to enable the polynucleotide molecule to penetrate into plant cells; wherein penetration of the polynucleotide molecule into plant cells causes a transient decrease in gene expression, and wherein the transient decrease in gene expression causes a change in plant yield-related traits.

[0014] According to some embodiments, the yield-related traits of a plant are selected from the group consisting of: increased grain / seed size, increased number of grains, increased number of spikes / siliques, increased number of tillers, increased branching, increased seed size, increased seed filling, increased number of seeds, increased heading, improved drought tolerance, reduced shattering, reduced abscission tissue formation, late flowering, early flowering, increased shattering, increased abscission tissue formation, reduced petals in the plant, increased plant protein content, increased plant carbohydrate content, increased plant oil content, improved plant oil composition, starch content, starch composition, carbohydrate content, carbohydrate composition, and any combination thereof. Each possibility is a separate embodiment.

[0015] According to some embodiments, there is provided a composition suitable for surface application to a plant, the composition comprising a dsRNA molecule comprising at least 18 consecutive nucleotides that are substantially identical or substantially complementary to a portion of a plant gene or a transcript of a plant gene; and a transfer agent configured to facilitate penetration of the dsRNA molecule into the cells of the plant, wherein penetration of the dsRNA molecule into plant cells causes a transient decrease in gene expression.

[0016] Transient reduction of gene expression causes changes in plant traits.

[0017] The plant traits are selected from the group consisting of: increased branching, increased grain filling, increased trehalose-6-phosphate (T6P) level, increased panicle number, increased seed filling, increased seed number, increased seed size, reduced shattering, reduced abscission tissue formation, increased tiller number, increased heading in plants, reduced petals, increased silique size, late flowering or early flowering, delayed senescence, and any combination thereof; or selected from the group consisting of: increased branching, increased grain filling, increased panicle number, increased seed filling, increased seed number, increased seed size, reduced shattering, reduced abscission tissue formation, increased tiller number, increased heading in plants, reduced petals, increased silique size, and any combination thereof; or selected from the group consisting of: increased branching, increased grain filling, increased panicle number, increased seed filling, increased seed number, reduced shattering, reduced abscission tissue formation, increased tiller number, reduced petals, increased silique size, and any combination thereof. Each possibility is a separate embodiment.

[0018] According to some embodiments, the plant gene is selected from ADPG1, PTL, CKX2, BRC1, KIN1, SKIN1, PIN5b, JAG1, BS1, PLDα1, and / or any homolog or combination thereof. Each possibility is a separate embodiment. According to some embodiments, the plant gene is selected from ADPG1, PTL, CKX2, BRC1, and / or any homolog or combination thereof. Each possibility is a separate embodiment.

[0019] According to some embodiments, the plant is a Brassica napus plant, and the dsRNA molecule comprises at least 18 consecutive nucleotides that are substantially identical or substantially complementary to a part of the sequence encoding any of the amino acid sequences listed in SEQ ID NO:599, SEQ ID NO:650, SEQ ID NO:522, and SEQ ID NO:365. Each possibility is a separate embodiment. According to some embodiments, the dsRNA molecule has at least 80% homology with any of the sequences listed in SEQ ID NO:729, SEQ ID NO:733, SEQ ID NO:731, and SEQ ID NO:730. Each possibility is a separate embodiment. According to some embodiments, the dsRNA molecule has at least 90% homology with any of the sequences listed in SEQ ID NO:729, SEQ ID NO:733, SEQ ID NO:731, and SEQ ID NO:730. Each possibility is a separate embodiment.

[0020] According to some embodiments, the plant is a soybean plant and the dsRNA molecule comprises at least 18 consecutive nucleotides that are substantially identical or substantially complementary to a portion of a sequence encoding any one of the amino acid sequences listed in SEQ ID NO: 379, SEQ ID NO: 603, SEQ ID NO: 655, SEQ ID NO: 564, SEQ ID NO: 517, SEQ ID NO: 480, and SEQ ID NO: 488. Each possibility is a separate embodiment.

[0021] According to some embodiments, the plant is a soybean plant and the dsRNA molecule comprises at least 18 consecutive nucleotides that are substantially identical or substantially complementary to a portion of a sequence encoding any one of the amino acid sequences listed in SEQ ID NO: 379, SEQ ID NO: 517, SEQ ID NO: 480, and SEQ ID NO: 488. Each possibility is a separate embodiment. According to some embodiments, the dsRNA molecule has at least 80% homology to any of the sequences listed in SEQ ID NO: 734 - 741. Each possibility is a separate embodiment. According to some embodiments, the dsRNA molecule has at least 90% homology to any of the sequences listed in SEQ ID NO: 734 - 741. Each possibility is a separate embodiment.

[0022] According to some embodiments, the plant is a rice plant and the dsRNA molecule comprises at least 18 consecutive nucleotides that are substantially identical or substantially complementary to a portion of a sequence encoding any one of the amino acid sequences listed in SEQ ID NO: 407, SEQ ID NO: 610, SEQ ID NO: 659, SEQ ID NO: 589, SEQ ID NO: 416, and SEQ ID NO: 450. Each possibility is a separate embodiment.

[0023] According to some embodiments, the plant is a rice plant and the dsRNA molecule comprises at least 18 consecutive nucleotides that are substantially identical or substantially complementary to a portion of a sequence encoding any one of the amino acid sequences listed in SEQ ID NO: 407, SEQ ID NO: 416, and SEQ ID NO: 450. Each possibility is a separate embodiment. According to some embodiments, the dsRNA molecule has at least 80% homology to any of the sequences listed in SEQ ID NO: 742 - 747. Each possibility is a separate embodiment. According to some embodiments, the dsRNA molecule has at least 90% homology to any of the sequences listed in SEQ ID NO: 742 - 747. Each possibility is a separate embodiment.

[0024] According to some embodiments, the dsRNA molecule has a length of at least about 50 bases. According to some embodiments, the dsRNA molecule has a length of at least about 200 bases.

[0025] According to some embodiments, the transfer agent includes N,N-dimethyldecanamide, cocamidopropyl dimethylamine, silicone polyalkylene oxide copolymer, EM-30, dimethylamide of C8 / C10 fatty acids, esterification copolymer of glycerol, trisiloxane ethoxylate, or any combination thereof. Each possibility is a separate embodiment.

[0026] According to some embodiments, the transfer agent can be any transfer agent listed in Table 1.

[0027] According to some embodiments, a method is provided for surface applying the compositions substantially disclosed herein to the surface of a plant.

[0028] According to some embodiments, the application includes spraying the composition onto the surface of the plant. According to some embodiments, the composition is sprayed onto the surface of the plant using a boom extending over the crop, a boomless sprayer, an agricultural sprayer, a crop dusting aircraft, a pressurized backpack sprayer, a track sprayer, or a laboratory sprayer / submersion device. Each possibility is a separate embodiment.

[0029] According to some embodiments, the application includes providing the composition through an irrigation system.

[0030] According to some embodiments, the plant surface is the surface of one or more plant parts selected from the group consisting of hypocotyl, cotyledon, leaf, flower, stem, tassel, meristem, pollen, ovule, and fruit. Each possibility is a separate embodiment.

[0031] According to some embodiments, the method further includes scheduling the application of the composition at a desired developmental stage of the plant, as substantially explained herein, for example, in Table 2.

[0032] Certain embodiments of the present disclosure may include some of the above advantages, all of the above advantages, or may not include the above advantages. One or more technical advantages may be apparent to those skilled in the art from the accompanying drawings, the specification, and the claims included herein. In addition, while specific advantages have been listed above, various embodiments may include all of the listed advantages, some of the listed advantages, or may not include the listed advantages.

[0033] In addition to the exemplary aspects and embodiments described above, additional aspects and embodiments will become apparent by reference to the drawings and by studying the following detailed description. Brief Description of the Drawings

[0035] The present invention will now be described with reference to the accompanying illustrative drawings, in conjunction with certain examples and embodiments, so that the present invention can be more fully understood.

[0036] Figure 1 Shows the change in contact angle (indicating penetration) over time after application of a mixture of targeting agents on the leaves of Brassica napus plants.

[0037] Figure 2 Shows exemplary photographs of the flower morphology of Brassica napus plants (here Brassica napus plants) ectopically sprayed with 10 μg / ml of dsRNA listed in SEQ ID NO: 733 targeting the sequence listed in SEQ ID NO: 286. The plants in the control treatment have normal petal morphology (left figure). The plants treated with BnPTL dsRNA result in flowers with altered petal morphology (right figure).

[0038] Figure 3 Shows exemplary photographs of Brassica napus plants (here Brassica napus plants) ectopically sprayed with 10 μg / ml of dsRNA listed in SEQ ID NO: 730 targeting the BnBRC1 sequence listed in SEQ ID NO: 1 compared to control plants (Ctrl). The total number of branches per component is depicted.

[0039] Figure 4 Shows the average number of branches per Brassica napus plant (here Brassica napus plants) ectopically sprayed with 1 μg / ml or 10 μg / ml of dsRNA listed in SEQ ID NO: 730 targeting the BnBRC1 sequence listed in SEQ ID NO: 1 or plants sprayed with only surfactant solution. * Indicates a significant change (P < 0.1) of the treated plants compared to the control plants (Ctrl).

[0040] Figure 5A Shows the average seed weight / 0.8 m of Brassica napus plants (here Brassica napus plants) sprayed with dsRNA listed in SEQ ID NO: 733 targeting the sequence listed in SEQ ID NO: 286 obtained from the first field 2 . * Indicates a significant change (P < 0.1) of the treated plants compared to the control plants (Ctrl).

[0041] Figure 5B Shows the average seed weight / 0.8 m of Brassica napus plants (here Brassica napus plants) sprayed with dsRNA listed in SEQ ID NO: 733 targeting the sequence listed in SEQ ID NO: 286 obtained from the second field2 。*Indicates a significant change (P<0.1) in the treated plants compared to the control plants (Ctrl).

[0042] Figure 6A Shows the percentage of average oil content of Brassica napus plants (here Brassica napus plants) sprayed with dsRNA listed in SEQ ID NO:733 targeting the sequence listed in SEQ ID NO:286 obtained from the first field. *Indicates a significant change (P<0.1) in the treated plants compared to the control plants (Ctrl).

[0043] Figure 6B Shows the percentage of average oil content of Brassica napus plants (here Brassica napus plants) sprayed with dsRNA listed in SEQ ID NO:733 targeting the sequence listed in SEQ ID NO:286 obtained from the second field. *Indicates a significant change (P<0.1) in the treated plants compared to the control plants (Ctrl).

[0044] Figure 7 Shows the average number of branches of Brassica napus plants (Brassica napus plants) ectopically sprayed with 1 μg / ml or 10 μg / ml of dsRNA listed in SEQ ID NO:730 targeting the sequence listed in SEQ ID NO:1 or plants sprayed with only surfactant solution. *Indicates a significant change (P<0.1) in the treated plants compared to the control plants (Ctrl).

[0045] Figure 8A Shows the average seed weight / 0.8m of Brassica napus plants (Brassica napus plants) sprayed with dsRNA listed in SEQ ID NO:731 targeting the sequence listed in SEQ ID NO:158 obtained from the first field 2 。*Indicates a significant change (P<0.1) in the treated plants compared to the control plants (Ctrl).

[0046] Figure 8B Shows the average seed weight / 0.8m of Brassica napus plants (Brassica napus plants) sprayed with dsRNA listed in SEQ ID NO:731 targeting the sequence listed in SEQ ID NO:158 obtained from the second field 2 。*Indicates a significant change (P<0.1) in the treated plants compared to the control plants (Ctrl).

[0047] Figure 9AShows the average seed weight / 0.8m of Brassica napus plants (oilseed rape plants) sprayed with the dsRNA listed in SEQ ID NO:729 targeting the sequence listed in SEQ ID NO:235 obtained from the first field 2 . *Indicates a significant change (P<0.1) of the treated plants compared to the control plants (Ctrl).

[0048] Figure 9B Shows the average seed weight / 0.8m of Brassica napus plants (oilseed rape plants) sprayed with the dsRNA listed in SEQ ID NO:729 targeting the sequence listed in SEQ ID NO:235 obtained from the second field 2 . *Indicates a significant change (P<0.1) of the treated plants compared to the control plants (Ctrl).

[0049] Figure 10A Shows the percentage of average oil content of Brassica napus plants (oilseed rape plants) sprayed with the dsRNA listed in SEQ ID NO:729 targeting the sequence listed in SEQ ID NO:235 obtained from the first field. *Indicates a significant change (P<0.1) of the treated plants compared to the control plants (Ctrl).

[0050] Figure 10B Shows the percentage of average oil content of Brassica napus plants (oilseed rape plants) sprayed with the dsRNA listed in SEQ ID NO:729 targeting the sequence listed in SEQ ID NO:235 obtained from the second field. *Indicates a significant change (P<0.1) of the treated plants compared to the control plants (Ctrl).

[0051] Figure 11 Shows the average number of tillers per rice plant (Oryza sativa) treated with 1 μg / ml or 10 μg / ml of the dsRNA listed in SEQ ID NO:742 targeting the sequence listed in SEQ ID NO:43 or plants sprayed with only the surfactant solution. *Indicates a significant change (P<0.1) of the treated plants compared to the control plants (Ctrl).

[0052] Figure 12 Shows the average number of branches per soybean plant (Glycine max) treated with 1 μg / ml or 10 μg / ml of the dsRNA listed in SEQ ID NO:734 targeting the sequence listed in SEQ ID NO:15 or plants sprayed with only the surfactant solution. *Indicates a significant change (P<0.1) of the treated plants compared to the control plants (Ctrl).

[0053] Detailed Description

[0054] In the following description, various aspects of the present disclosure will be described. For purposes of illustration, specific configurations and details are set forth in order to provide a thorough understanding of the different aspects of the present disclosure. However, it will also be apparent to those skilled in the art that the present disclosure may be practiced without the specific details presented herein. Additionally, well-known features may be omitted or simplified so as not to obscure the present disclosure.

[0055] The following definitions and methods are provided to better define the present invention and to guide one of ordinary skill in the art in practicing the present invention. Unless otherwise noted, terms shall be understood according to the ordinary usage of one of ordinary skill in the relevant art.

[0056] In cases where a term is provided in the singular, the inventors also contemplate aspects of the present invention described in the plural form of that term.

[0057] As used herein, the terms "polynucleotide molecule" and "polynucleotide" may be used interchangeably and refer to any polynucleotide of 18 or more nucleotides that are covalently joined in a chain and capable of hybridizing to DNA and RNA molecules under physiological conditions. According to some embodiments, the polynucleotide may be a synthetic and / or artificial polynucleotide molecule. According to some embodiments, the polynucleotide molecule is a biopolymer. According to some embodiments, the biopolymer is a DNA (deoxyribonucleic acid) or RNA (ribonucleic acid) molecule.

[0058] According to some embodiments, the polynucleotide molecule targets the mRNA of a plant gene. According to some embodiments, the polynucleotide molecule targets the translation region of the mRNA. According to some embodiments, the polynucleotide molecule targets the untranslated region (UTR) of the mRNA.

[0059] As used herein, the terms "DNA", "DNA molecule", and "DNA polynucleotide molecule" refer to single-stranded or double-stranded DNA molecules of genomic or synthetic origin, such as polymers of deoxyribonucleotide bases or DNA polynucleotide molecules.

[0060] As used herein, the terms "DNA sequence", "DNA nucleotide sequence", and "DNA polynucleotide sequence" refer to the nucleotide sequence of a DNA molecule.

[0061] As used herein, the term "gene" refers to any portion of a nucleic acid that provides for the expression of a transcript or encodes a transcript. Thus, "gene" includes, but is not limited to, a promoter region, a 5' untranslated region, a transcript coding region that may include intron regions, and a 3' untranslated region.

[0062] As used herein, the terms "RNA", "RNA molecule", and "RNA polynucleotide molecule" refer to single-stranded or double-stranded RNA molecules of genomic or synthetic origin, such as polymers of ribonucleic acid bases that contain single-stranded or double-stranded regions or any other structural elements.

[0063] Unless otherwise indicated, nucleotide sequences in the text of this specification are given in the 5' to 3' direction when read from left to right. The nomenclature used herein is the nomenclature required by 37 CFR § 1.822 of the United States Code of Federal Regulations and is set forth in the tables in WIPO Standard ST.25 (1998), Appendix 2, Tables 1 and 3.

[0064] As used herein, "plant surface" refers to any external part of a plant. Thus, the plant surface includes, but is not limited to, the surfaces of flowers, stems, tubers, fruits, anthers, pollen, leaves, roots, or seeds. The plant surface can be on a part of the plant that is attached to other parts of the plant or on a part of the plant that is separated from the plant.

[0065] As used herein, the phrase "a polynucleotide is not operably linked to a promoter" means a polynucleotide that is not covalently linked to a polynucleotide promoter sequence that is specifically recognized by a DNA-dependent RNA polymerase II protein or a viral RNA-dependent RNA polymerase in such a way that the polynucleotide will be transcribed by the DNA-dependent RNA polymerase protein or the viral RNA-dependent RNA polymerase. A polynucleotide that is not operably linked to a promoter can be transcribed by a plant RNA-dependent RNA polymerase.

[0066] As used herein, SEQ ID NO: 1-364 and SEQ ID NO: 729-747, although presented in the sequence listing in the form of ssDNA, include dsDNA equivalents, dsRNA equivalents, ssRNA equivalents, ssRNA complementary sequences, the ssDNA shown, and ssDNA complementary sequences.

[0067] As used herein, the term "transfer agent" can refer to any agent that renders a plant receptive to a polynucleotide when the agent is applied to the plant surface. According to some embodiments, the transfer agent is an agent that modulates the surface of plant tissue (e.g., seeds, leaves, stems, roots, flowers, or fruits) to allow penetration of the polynucleotide molecule into plant cells. Chemical agents for modulation or transfer include (a) wetting agents, (b) surfactants, (c) organic solvents or aqueous solutions or aqueous mixtures of organic solvents, (d) oxidizing agents, (e) acids, (f) bases, (g) oils, (h) enzymes, or combinations thereof.

[0068] According to some embodiments, the transfer agent can be selected from N,N-dimethyldecanamide, coconut amide propyl dimethylamine, silicone polyalkylene oxide copolymer, EM-30, dimethylamides of C8 / C10 fatty acids, esterification copolymers of glycerol, trisiloxane ethoxylates, or any combination thereof.

[0069] Non-limiting examples of suitable transfer agents include silicone compounds.

[0070] As used herein, the term "organosilicone preparation" refers to a liquid comprising one or more silicone compounds, wherein the liquid or components contained therein, when combined with a polynucleotide in a composition applied to the surface of a target plant, better enable the polynucleotide to enter plant cells. Exemplary organosiloxane preparations include, but are not limited to, preparations sold under the trade name or In certain embodiments, the organosilicone preparation can better enable the polynucleotide to enter plant cells in a manner that allows polynucleotide-mediated inhibition of target gene expression in plant cells.

[0071] Non-limiting examples of specific suitable transfer agents include L-77, which is a modified trisiloxane that combines a very low molecular weight trisiloxane with a polyether group. It is characterized by significant interfacial activity, which can result in a significantly reduced water surface tension, excellent spreading or leveling, and stable foaming. All of these can be achieved using fractions of typical concentration levels of organic or fluorocarbon surfactants.

[0072] Another non-limiting example of a specific suitable transfer agent includes GENAGEN TM 4166 (Clariant, material number: 10783626892). GENAGEN TM 4166 is a dimethylamide based on fatty acids of natural origin.

[0073] Another non-limiting example of a specific suitable transfer agent includes GL 5: (Clariant, material number: 20072326894). GL5 is an adjuvant based on polyglycerol esters, which is a TAE-free surfactant derived from renewable resources. Another non-limiting example of a specific suitable transfer agent includes GENAGEN TM 4296 (Clariant, material number: 10783926892). GENAGEN TM 4296 is a dimethylamide based on fatty acids of natural origin.

[0074] Another non-limiting example of a specific suitable transfer agent includes GA: (Clariant, material number: 27251626894). GA is a bio - enhancer of salts of novel agrochemicals based on alkyl glucamides. It is a glycosyl surfactant with a renewable carbon index (RCI) above 95% and thus has an excellent ecological profile.

[0075] Another non - limiting example of a suitable transfer agent includes GENAGEN TM SC 35 (Clariant, material number: 25923226892). GENAGEN TM SC 35 is a basic surfactant mixture of sodium alkyl diglycol ether sulfate and coconut fatty acid monoethanolamide.

[0076] Another non - limiting example of a suitable transfer agent includes 1306 (Clariant, material number: 13326826900). 1306 is an anionic emulsifier used in the emulsion polymerization of monomers such as pure acrylates, styrene - acrylates, and vinyl acetate.

[0077] Another non - limiting example of a suitable transfer agent includes SURFECO PLUS TM (Latro). SURFECO PLUS TM is a silicone - based adjuvant used to modify the physical properties of agrochemicals and enhance their biological activity.

[0078] Additional suitable transfer agents and their chemical properties are summarized in Table 1 below.

[0079] Table 1 - Transfer Agents

[0080]

[0081] As used herein, the phrase "increased yield" refers to any measurable increase in yield. In certain embodiments, the increase in yield of a plant or plant part can be determined by comparison with a control plant or plant part that has not been treated with a composition comprising a polynucleotide. When used herein, a control plant is a plant that has not been treated with a polynucleotide and a transfer agent. Such control plants will include, but are not limited to, untreated plants or mock - treated plants.

[0082] Non - limiting examples of traits affected by the compositions disclosed herein include: increased branching, increased seed filling, increased seed number, improved drought tolerance, reduced shattering, reduced abscission tissue formation, late / early flowering, and any combination thereof. Each possibility is a separate embodiment.

[0083] Non-limiting examples of rice plant traits affected by the compositions disclosed herein include: increased grain size, increased number of panicles, increased number of tillers, increased heading, and any combination thereof. Each possibility is a separate embodiment.

[0084] Non-limiting examples of Brassica napus traits affected by the compositions disclosed herein include: increased grain size, increased number of panicles, increased number of tillers, increased branching, increased seed filling, increased number of seeds, increased heading, improved drought tolerance, reduced shattering, reduced abscission tissue formation, late / early flowering, shortened / extended flowering period, and any combination thereof. Each possibility is a separate embodiment.

[0085] According to some embodiments, the plant can be any cultivated plant, such as but not limited to Brassica napus, rapeseed plant, rice, wheat, barley, soybean, peanut, cotton, corn, sorghum, sugarcane, beet, legumes, sunflower, potato, sweet potato, alfalfa, banana, apricot, grape, apple, peach, plum, citrus, date palm, palm oil plant, pepper, tomato, broccoli, onion, melon, watermelon, yam, cassava. Each possibility is a separate embodiment.

[0086] According to some embodiments, the plant can be a soybean plant, a rice plant, or a Brassica napus plant. Each possibility is a separate embodiment. According to some embodiments, the soybean plant can be a plant of the species Glycine max. According to some embodiments, the rice plant can be a plant of the species Oryza sativa. According to some embodiments, the Brassica napus plant can be Brassica napus. Each possibility is a separate embodiment.

[0087] According to some embodiments, the gene targeted by the polynucleotide molecule can be referred to by the scientific name used in one species (e.g., in Arabidopsis thaliana). However, those of ordinary skill in the art understand that aliases and homologs of another species referred to by another name (alias / homolog) are included within the scientific name. As a non-limiting example, when the gene targeted by the polynucleotide molecule is called BRC1, it includes the alias / homolog TB1 / FC1.

[0088] According to some embodiments, the target gene can have a nucleotide sequence selected from any of the nucleotide sequences listed in SEQ ID NOs: 1-364. Each possibility is a separate embodiment. According to some embodiments, the target gene can encode an amino acid sequence selected from any of the amino acid sequences listed in SEQ ID NOs: 365-728. Each possibility is a separate embodiment. According to some embodiments, the polynucleotide (i.e., dsRNA) can have a nucleotide sequence listed in SEQ ID NOs: 729-747. Each possibility is a separate embodiment.

[0089] According to some embodiments, the polynucleotide molecule is a dsRNA having a polynucleotide sequence that is substantially the same as the sequence listed in any one of SEQ ID NOs: 729-747 or a major portion thereof. As used herein, the term "major portion thereof" in reference to a dsRNA means that the dsRNA is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, or at least 98% identical to at least 18-20 consecutive base pairs of the sequence listed in any one of SEQ ID NOs: 729-747. Each possibility is a separate embodiment.

[0090] As used herein, the term "substantially the same as" in reference to a dsRNA means a dsRNA sequence that has at least 80%, at least 90%, at least 95% or at least 98% homology to a portion of the nucleotide sequence listed in SEQ ID NOs: 1-364. As used herein, the term "portion of the nucleotide sequence listed" means a portion of the nucleotide sequence targeted by the dsRNA that has a length substantially the same as the dsRNA. As a non-limiting example, if the dsRNA has a length of 18 bp, the portion of the nucleotide sequence targeted by the dsRNA has a length of about 18 bp. As another non-limiting example, if the dsRNA has a length of 200 bp, the portion of the nucleotide sequence targeted by the dsRNA has a length of about 200 bp.

[0091] As used herein, the terms "about" and "approximately" mean + / - 10%, or + / - 5% or + / - 2% relative to the range to which they refer. Each possibility is a separate embodiment.

[0092] According to some embodiments, the dsRNA targets BnADPG1 (SEQ ID NO: 235) of Brassica napus plants and has the polynucleotide sequence set forth in SEQ ID NO: 729.

[0093] According to some embodiments, the dsRNA targets BnBRC1 (SEQ ID NO: 1) of Brassica napus plants and has the polynucleotide sequence set forth in SEQ ID NO: 730.

[0094] According to some embodiments, the dsRNA targets BnCKX2 (SEQ ID NO: 158) of Brassica napus plants and has the polynucleotide sequence set forth in SEQ ID NO: 731.

[0095] According to some embodiments, the dsRNA targets BnKIN10 (SEQ ID NO: 62) of Brassica napus plants and has the polynucleotide sequence set forth in SEQ ID NO: 732.

[0096] According to some embodiments, the dsRNA targets BnPTL (SEQ ID NO: 286) of Brassica napus plants and has the polynucleotide sequence set forth in SEQ ID NO: 733.

[0097] According to some embodiments, the dsRNA targets GmBRC1 (SEQ ID NO: 15) of soybean plants and has the polynucleotide sequence set forth in SEQ ID NO: 734 or 735.

[0098] According to some embodiments, the dsRNA targets GmBS1 (SEQ ID NO: 116) of soybean plants and has the polynucleotide sequence set forth in SEQ ID NO: 736 or 737.

[0099] According to some embodiments, the dsRNA targets GmJAG1 (SEQ ID NO: 153) of soybean plants and has the polynucleotide sequence set forth in SEQ ID NO: 738 or 739.

[0100] According to some embodiments, the dsRNA targets GmPLDα1 (SEQ ID NO: 124) of soybean plants and has the polynucleotide sequence set forth in SEQ ID NO: 740 or 741.

[0101] According to some embodiments, the dsRNA targets OsBRC1 (SEQ ID NO: 43) of rice plants and has the polynucleotide sequence set forth in SEQ ID NO: 742 or 743.

[0102] According to some embodiments, the dsRNA targets OsPIN5b (SEQ ID NO:86) of a rice plant and has a polynucleotide sequence listed in SEQ ID NO:744 or 745.

[0103] According to some embodiments, the dsRNA targets OsSKIN1 (SEQ ID NO:52) of a rice plant and has a polynucleotide sequence listed in SEQ ID NO:746 or 747.

[0104] According to some embodiments, the composition and the method for applying the composition may include scheduling the application of the composition to a time of a desired developmental trait of the plant. As a non-limiting example, the application of a composition comprising a polynucleotide configured to target gene BS1 (or other genes involved in regulation of seed filling) may be scheduled when the plant is in the seed filling stage (R3-R5 development (dev.) stage). As another non-limiting example, the application of a composition comprising a polynucleotide configured to target gene BRC1 (or other genes whose reduced expression causes an increase in the number of branches or tillers) may be scheduled to the bolting stage (R1-R2 development stage) of a soybean plant or the vegetative stage of axillary bud development of a rice plant. As another non-limiting example, the application of a composition comprising a polynucleotide configured to target gene JAG1 (or other genes that increase the number of seeds) may be scheduled when the plant is in the flowering stage (R1-R2 development stage). As another non-limiting example, the application of a composition comprising a polynucleotide configured to target gene SGR1 (or other target genes whose reduction increases drought resistance) may be scheduled to a period of unexpected drought. As another non-limiting example, the application of a composition comprising a polynucleotide configured to target gene AGL1 (or other target genes whose reduction causes reduced shattering) may be scheduled when the siliques of the plant are mature. As another non-limiting example, the application of a composition comprising a polynucleotide configured to target gene FT5a (or other target genes whose reduction is involved in flowering regulation) may be scheduled when the plant is in the flowering stage. As another non-limiting example, the application of a composition comprising a polynucleotide configured to target gene GNI1 (or other genes that regulate grain size) may be scheduled when the plant is in the seed filling stage.

[0105] According to some embodiments, the composition may comprise more than one polynucleotide sequence (different sequences), such as 2, 3, 4, 5 or more polynucleotide sequences. Each possibility is a separate embodiment.

[0106] According to some embodiments, two or more polynucleotide sequences can target the same target gene, i.e., they can be directed to different portions of the same target gene sequence.

[0107] According to some embodiments, two or more polynucleotide sequences can target different target genes.

[0108] According to some embodiments, different target genes can be involved in the same yield-related trait (e.g., reduced breakage). As a non-limiting example, two or more polynucleotide sequences can target AGL1 and PDH1. As another non-limiting example, two or more polynucleotide sequences can target two or more of JAG1, JAG2, CKX1, OTU1 (all affecting seed number).

[0109] According to some embodiments, different target genes can be involved in different yield-related traits (e.g., increased drought tolerance and seed filling). As a non-limiting example, the first of two or more polynucleotide sequences can target ERA1, SGR1, SGR2, ACO2, CER9, or CytG, while the second of two or more polynucleotide sequences can target BS1, PLD, ACO3, or PDHK.

[0110] As used herein, the phrase "reduced expression", when used in the context of a transcript or protein in a plant or plant part, refers to any measurable decrease in the level of the transcript or protein in the plant or plant part. In certain embodiments, the decrease in the level of the transcript or protein in the plant or plant part can be determined relative to a control plant or plant part not treated with the composition comprising the polynucleotide and the delivery agent.

[0111] As used herein, the phrase "wherein the plant does not contain a transgene" refers to a plant that lacks a DNA molecule comprising a promoter operably linked to a polynucleotide or lacks a recombinant viral vector.

[0112] As used herein, the term "transgene" describes a DNA segment that contains a gene sequence isolated from the DNA of one organism and introduced into a different organism.

[0113] As used herein, the phrase "inhibit expression" or "reduce expression", when used in the context of a gene, refers to any measurable decrease in the amount and / or activity of the product encoded by the gene. Thus, gene expression can be inhibited when the level of the transcript from the gene is decreased, the level of the protein encoded by the gene is decreased, the activity of the transcript from the gene is decreased, the activity of the protein encoded by the gene is decreased, any one of the foregoing conditions, or any combination of the foregoing conditions. In this document, the activity of a transcript includes, but is not limited to, the ability of the transcript to be translated into a protein and / or to exert any RNA-mediated biological or biochemical effect. In this document, the activity of a protein includes, but is not limited to, the ability of the protein to exert any protein-mediated biological or biochemical effect. When used herein, a control plant or plant part is a plant or plant part that has not been treated with a polynucleotide and a delivery agent.

[0114] As used herein, the term "transient", when used in the context of a decrease / inhibition of gene expression, refers to a time-limited decrease in gene expression that persists only as long as the polynucleotide that has been introduced into the cell has not been degraded, as opposed to long-term expression, which is commonly referred to as "stable expression".

[0115] As used herein, the term "transcript" corresponds to any RNA produced from a gene by the process of transcription. Thus, the transcript of a gene can comprise a primary transcript that may contain introns, or can comprise a mature RNA that lacks introns.

[0116] As used herein, the term "homolog" with respect to a polynucleotide molecule refers to the degree of sequence identity or similarity (homology) between nucleotide sequences that indicates a common ancestor. Two DNA segments can have a common ancestor due to a speciation event (inter-species homologs) or a duplication event (intra-species homologs). According to some embodiments, a homolog can refer to a polynucleotide having a sequence identity of essentially from about 70% to about 99%, or more preferably from about 80% to about 99%, or most preferably from about 90% to about 99%, or from about 95% to about 99% with a reference nucleotide sequence of a reference polynucleotide molecule. Each possibility is a separate embodiment.

[0117] As used herein, the terms "sequence identity", "sequence similarity", or "homology" are used to describe the sequence relationship between two or more nucleotide sequences. The percentage of "sequence identity" between two sequences is determined by comparing the two best-aligned sequences. A sequence that is identical at each position when compared to a reference sequence is said to be identical to the reference sequence, and vice versa. When a first nucleotide sequence as viewed in the 5' to 3' direction exhibits complete complementarity with a second or reference sequence as viewed in the 3' to 5' direction, the first nucleotide sequence is said to be the "complementary sequence" of or complementary to the second or reference nucleotide sequence. As used herein, a nucleic acid sequence molecule is said to exhibit "complete complementarity" when each nucleotide of one sequence as read from 5' to 3' is complementary to each nucleotide of another sequence as read from 3' to 5'. A nucleotide sequence that is complementary to a reference nucleotide sequence will exhibit the same sequence as the reverse complement of the reference nucleotide sequence. These terms and descriptions are well-defined in the art and readily understood by one of ordinary skill in the art.

[0118] According to some embodiments, the composition may further comprise a carrier. According to some embodiments, the carrier may be a liquid.

[0119] As used herein, the term "liquid" refers to homogeneous mixtures (such as solutions) and heterogeneous mixtures (such as suspensions, colloids, micelles, and emulsions). Each possibility is a separate embodiment.

[0120] According to some embodiments, the liquid may be an aqueous solution. According to some embodiments, the liquid may be an oil or an oil mixture.

[0121] According to some embodiments, the polynucleotide may be naked. As used herein, the term "naked" means that the polynucleotide is not encapsulated. However, a naked polynucleotide may be modified and / or conjugated.

[0122] According to other embodiments, the polynucleotide may be encapsulated.

[0123] According to some embodiments, the polynucleotide may be delivered by and / or encapsulated in a vehicle such as, but not limited to, nanoparticles, liposomes, micelles, etc.

[0124] The present disclosure provides certain methods and polynucleotide compositions that can be applied to living plant cells / tissues to inhibit the expression of target genes and provide this benefit to plants in need of increased yield. Also provided are plants and plant parts that exhibit increased yield and processed products of such plants or plant parts. The compositions can be applied topically to the surface of the plant, such as to the surface of the leaves. The compositions can be applied to a variety of plants, including but not limited to plants of the Brassicaceae, Fabaceae, or Poaceae families, such as but not limited to soybean plants, rice plants, Brassica napus plants, and / or rapeseed plants. Each possibility is a separate embodiment.

[0125] As used herein, "polynucleotide" refers to a DNA or RNA molecule containing multiple nucleotides, and generally refers to both "oligonucleotides" (polynucleotide molecules 18 - 25 nucleotides in length) and longer polynucleotides of 26 or more nucleotides. Embodiments of the invention include polynucleotides having a length of 18 - 25 nucleotides (18 - mers, 19 - mers, 20 - mers, 21 - mers, 22 - mers, 23 - mers, 24 - mers, or 25 - mers), or medium - length polynucleotides having a length of 26 or more nucleotides (26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, about 65, about 70, about 75, about 80, about 85, about 90, about 95, about 100, about 110, about 120, about 130, about 140, about 150, about 160, about 170, about 180, about 190, about 200, about 210, about 220, about 230, about 240, about 250, about 260, about 270, about 280, about 290, or about 300 nucleotides), or long polynucleotides having a length greater than about 300 nucleotides (e.g., between about 300 and about 400 nucleotides, between about 400 and about 500 nucleotides, between about 500 and about 600 nucleotides, between about 600 and about 700 nucleotides, between about 700 and about 800 nucleotides, between about 800 and about 900 nucleotides, between about 900 and about 1000 nucleotides, between about 300 and about 500 nucleotides, between about 300 and about 600 nucleotides, between about 300 and about 700 nucleotides, between about 300 and about 800 nucleotides, between about 300 and about 900 nucleotides, or about 1000 nucleotides in length, or even greater than about 1000 nucleotides in length, such as up to the entire length of a target gene, including the coding or non - coding portion of the target gene, or both the coding and non - coding portions). In the case where the polynucleotide is double - stranded, its length can be similarly described in base pairs.

[0126] The polynucleotide compositions used in various embodiments of the present invention include compositions comprising polynucleotides, said polynucleotides comprising: RNA or DNA or RNA / DNA hybrids or chemically modified polynucleotides or artificial polynucleotides or mixtures thereof. In certain embodiments, the polynucleotide can be a combination of ribonucleotides and deoxyribonucleotides, e.g., a synthetic polynucleotide consisting primarily of ribonucleotides but having one or more terminal deoxyribonucleotides, or a synthetic polynucleotide consisting primarily of deoxyribonucleotides but having one or more terminal dideoxyribonucleotides. In certain embodiments, the polynucleotide includes non-classical nucleotides such as inosine, thiouridine or pseudouridine. In certain embodiments, the polynucleotide includes chemically modified nucleotides. Examples of chemically modified oligonucleotides or polynucleotides are well known in the art. Illustrative examples include, but are not limited to, the phosphodiester backbone of naturally occurring polynucleotides, which can be modified in part or in whole with phosphorothioate, dithiophosphate or methylphosphonate internucleotide linkages, modified nucleobases or modified sugars can be used in polynucleotide synthesis, and the polynucleotide can be labeled with a fluorescent moiety (e.g., fluorescein or rhodamine) or other label (e.g., biotin).

[0127] According to some embodiments, dsRNA can be chemically modified on one or both strands to improve stability, extend the half-life of dsRNA in vivo, increase the biodistribution and pharmacokinetic properties of dsRNA, target dsRNA to specific cells, increase target binding affinity and / or improve drug delivery. As a non-limiting example, dsRNA can be modified to contain a methyl group at the 2'-position of the ribose ring of the second base of the dsRNA. As another non-limiting example, dsRNA can be modified to contain a 3'-overhang.

[0128] According to some embodiments, the modification can be included in the dsRNA. According to some embodiments, the modification does not prevent the dsRNA composition from being used as a substrate for Dicer. In one embodiment, one or more modifications are made to enhance the processing of the dsRNA by Dicer. In a second embodiment, one or more modifications are made to result in more efficient RNAi generation. In a third embodiment, one or more modifications are made to support a greater RNAi effect. In a fourth embodiment, one or more modifications are made to result in greater potency for each dsRNA molecule to be delivered to the cell. Modifications can be incorporated in the 3'-terminal region, the 5'-terminal region, both the 3'-terminal and 5'-terminal regions, or in some cases, at different positions within the sequence. Any number and combination of modifications can be incorporated into the dsRNA, subject to the above limitations. Where there is more than one modification, they can be the same or different. Modifications to the base, sugar moiety, phosphate backbone, and combinations thereof are contemplated. Any 5'-end can be phosphorylated.

[0129] Examples of modifications contemplated for the phosphate backbone include phosphonates, including methylphosphonate, phosphorothioate, and phosphotriester modifications such as alkyl phosphotriesters, etc. Examples of modifications contemplated for the sugar moiety include 2'-alkyl pyrimidines, such as 2'-O-methyl, 2'-fluoro, amino, and deoxy modifications, etc. (see, e.g., Amarzguioui et al., 2003). Examples of modifications contemplated for the base group include abasic sugars, 2-O-alkyl modified pyrimidines, 4-thiouracil, 5-bromouracil, 5-iodouracil, and 5-(3-aminoallyl)-uracil, etc. Locked nucleic acid (or LNA) can also be incorporated. Many other modifications are known and can be used as long as the above criteria are met.

[0130] The polynucleotide can be single-stranded or double-stranded RNA, single-stranded or double-stranded RNA with structural features, single-stranded or double-stranded DNA, double-stranded DNA / RNA hybrids, and their modified analogs. In certain embodiments of the present invention, the polynucleotide that provides single-stranded RNA in a plant cell can be: (a) a single-stranded RNA molecule (ssRNA), (b) a single-stranded RNA molecule that self-hybridizes to form a double-stranded RNA molecule, (c) a double-stranded RNA molecule (dsRNA), (d) a single-stranded DNA molecule (ssDNA), (e) a single-stranded DNA molecule that self-hybridizes to form a double-stranded DNA molecule, (f) a single-stranded DNA molecule containing a modified Pol III gene that transcribes into an RNA molecule, (g) a double-stranded DNA molecule (dsDNA), (h) a double-stranded DNA molecule containing a modified Pol III gene that transcribes into an RNA molecule, (i) a double-stranded hybrid RNA / DNA molecule, and (j) a single-stranded RNA molecule (ssRNA) that self-hybridizes to form a structural motif such as a stem-loop, or a combination thereof. In certain embodiments, these polynucleotides can contain both ribonucleic acid residues and deoxyribonucleic acid residues. In certain embodiments, these polynucleotides contain chemically modified nucleotides or non-classical nucleotides. In certain embodiments of the method, the polynucleotide includes double-stranded DNA formed by intramolecular hybridization, double-stranded DNA formed by intermolecular hybridization, double-stranded RNA formed by intramolecular hybridization, or double-stranded RNA formed by intermolecular hybridization. In certain embodiments where the polynucleotide is dsRNA, the antisense strand will contain at least 18 nucleotides that are substantially complementary to the target gene. In certain embodiments, the polynucleotide includes a single-stranded DNA or single-stranded RNA that self-hybridizes to form a hairpin structure having at least a partially double-stranded structure, and the single-stranded DNA or single-stranded RNA contains at least one segment that will hybridize to the RNA transcribed from the gene to be targeted for inhibition. Without being bound by any mechanism, it is believed that such polynucleotides are or will produce a single-stranded RNA having at least one segment that will hybridize to the RNA transcribed from the gene to be targeted for inhibition.

[0131] The polynucleotide molecules of the present invention are designed to regulate expression by inducing the regulation or inhibition of endogenous target genes in plants, and are designed to have a nucleotide sequence that is substantially the same as or substantially complementary to the nucleotide sequence of the endogenous target gene in the plant or the RNA sequence transcribed from the endogenous target gene in the plant, which can be a coding sequence or a non-coding sequence.

[0132] "Substantially the same" or "substantially complementary" means that the polynucleotide (or at least one strand of the double-stranded polynucleotide) has sufficient identity or complementarity with the endogenous gene or the RNA transcribed from the endogenous target gene (e.g., transcript) to inhibit the expression of the endogenous target gene (e.g., to achieve a reduction in the level or activity of the gene transcript and / or the encoded protein).

[0133] The polynucleotides of the methods and compositions provided herein need not have 100% identity or complementarity with an endogenous target gene or RNA transcribed from an endogenous target gene (i.e., a transcript) to inhibit the expression of the endogenous target gene (i.e., to effect a decrease in the level or activity of the gene transcript or encoded protein). Thus, in certain embodiments, the polynucleotide or a portion thereof is designed to be substantially identical or substantially complementary to a sequence of at least 18 or 19 contiguous nucleotides in the target gene or messenger RNA transcribed from the target gene (e.g., a transcript). In certain embodiments, a "substantially identical" polynucleotide has 100% sequence identity or at least about 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity when compared to a sequence of 18 or more contiguous nucleotides in an endogenous target gene or RNA transcribed from the target gene. In certain embodiments, a "substantially complementary" polynucleotide has 100% sequence complementarity or at least about 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence complementarity when compared to a sequence of 18 or more contiguous nucleotides in the target gene or RNA transcribed from the target gene.

[0134] In certain embodiments, the polynucleotides used in the methods and compositions provided herein can be substantially identical or substantially complementary to any of the following: i) a conserved region of a target gene of both monocotyledonous and dicotyledonous plants; ii) a conserved region of a target gene of monocotyledonous plants; or iii) a conserved region of a target gene of dicotyledonous plants. Such polynucleotides that are substantially identical or substantially complementary to such conserved regions can be used to improve delayed senescence and / or increase yield by inhibiting the expression of target genes in a variety of dicotyledonous plants.

[0135] Thus, polynucleotides that are mismatched to a target gene or transcript can be used in certain embodiments of the compositions and methods provided herein. In certain embodiments, a polynucleotide of 19 consecutive nucleotides that is substantially identical or substantially complementary to an endogenous target gene or RNA transcribed from a target gene (e.g., a transcript) can have 1 or 2 mismatches with the target gene or transcript. In certain embodiments, a polynucleotide of 20 or more nucleotides that has identity or complementarity to an endogenous target gene or RNA transcribed from a target gene over a continuous 19-nucleotide span can have 1 or 2 mismatches with the target gene or transcript. In certain embodiments, a polynucleotide of 21 consecutive nucleotides that is substantially identical or substantially complementary to an endogenous target gene or RNA transcribed from a target gene (e.g., a transcript) can have 1, 2, or 3 mismatches with the target gene or transcript. In certain embodiments, a polynucleotide of 22 or more nucleotides that has identity or complementarity to an endogenous target gene or RNA transcribed from a target gene over a continuous 21-nucleotide span can have 1, 2, or 3 mismatches with the target gene or transcript. When designing polynucleotides that have mismatches with an endogenous target gene or RNA transcribed from a target gene, certain types of mismatches and mismatches at certain positions can be used that are more likely to be tolerated.

[0136] According to some embodiments, the target gene can be any of the target genes listed in SEQ ID NO: 1-364 and interspecies homologous target genes obtainable from other crops.

[0137] According to some embodiments, the plant can be a Brassica napus plant, and the polynucleotide can reduce the level of a target gene having any of the nucleotide sequences listed in SEQ ID NO: 1-14, 49-51, 62-66, 79-84, 104-115, 149-151, 158-199, 235-238, 252-258, 275-279, 286-290, 299-303, 311-318, 332-341, and 360-361 or a major portion thereof. Each possibility is a separate embodiment.

[0138] According to some embodiments, the plant can be a Brassica napus plant, and the polynucleotide can reduce the level of a protein having any of the amino acid sequences listed in SEQ ID NO: 365-378, 413-415, 426-430, 443-448, 468-479, 513-515, 522-563, 599-602, 616-622, 639-643, 650-654, 663-667, 675-682, 696-705, and 724-725 or a major portion thereof. Each possibility is a separate embodiment.

[0139] According to some embodiments, the plant can be a Brassica napus plant, and the polynucleotide can reduce the level of a protein having any of the amino acid sequences listed in SEQ ID NO: 1, 2, 9, 49, 62, 79, 80, 104, 149, 150, 158, 235, 252, 275, 276, 286, 299, 311, 332, 333, and 360 or a major portion thereof. Each possibility is a separate embodiment.

[0140] According to some embodiments, the plant can be a Brassica napus plant, and the polynucleotide can reduce the level of a protein having any of the amino acid sequences listed in SEQ ID NO: 1, 2, 9, 49, 62, 79, 80, 104, 149, 150, 158, 235, 252 or a major portion thereof. Each possibility is a separate embodiment.

[0141] According to some embodiments, the plant can be a Brassica napus plant, and the polynucleotide can have a nucleotide sequence listed in SEQ ID NO: 729 - 733.

[0142] According to some embodiments, the plant can be a soybean plant, and the polynucleotide can reduce the level of a target gene having any of the nucleotide sequences listed in SEQ ID NO: 15 - 42, 67 - 72, 116 - 148, 152 - 157, 200 - 224, 239 - 245, 291 - 294, 304 - 310, 319 - 325, 342 - 351, and 362 or any major portion thereof. Each possibility is a separate embodiment.

[0143] According to some embodiments, the plant can be a soybean plant, and the polynucleotide can reduce the level of a target gene having any of the nucleotide sequences listed in SEQ ID NO: 15, 16, 22 - 31, 67 - 69, 116 - 124, 152 - 155, 200 - 204, 239, 240, 291 - 293, 304, 305, 319, 320, 342, 343, 345, and 362 or any major portion thereof. Each possibility is a separate embodiment.

[0144] According to some embodiments, the plant can be a soybean plant, and the polynucleotide can reduce the level of a target gene having any of the nucleotide sequences listed in SEQ ID NO: 15, 16, 67 - 69, 116 - 124, 152 - 155, 200 - 204, 239, 240 or any major portion thereof. Each possibility is a separate embodiment.

[0145] According to some embodiments, the plant can be a soybean plant, and the polynucleotide can reduce the level of a protein having any of the amino acid sequences listed in SEQ ID NOs: 379 - 406, 431 - 436, 480 - 512, 516 - 521, 564 - 588, 603 - 609, 655 - 658, 668 - 674, 683 - 689, 706 - 715, and 726 or any major portion thereof. Each possibility is a separate embodiment.

[0146] According to some embodiments, the plant can be a soybean plant, and the polynucleotide can have the nucleotide sequence listed in SEQ ID NOs: 734 - 741.

[0147] According to some embodiments, the plant can be a rice plant, and the polynucleotide can reduce the level of a target gene having any of the nucleotide sequences listed in SEQ ID NOs: 43 - 48, 52 - 61, 73 - 78, 85 - 103, 225 - 234, 246 - 251, 259 - 274, 280 - 285, 295 - 298, 326 - 331, 352 - 359, 363 - 364 or any major portion thereof. Each possibility is a separate embodiment.

[0148] According to some embodiments, the plant can be a rice plant, and the polynucleotide can reduce the level of a target gene having any of the nucleotide sequences listed in SEQ ID NOs: 43, 44, 52 - 57, 73 - 75, 85, 86, 225 - 227, 246 - 248, 259 - 264, 280, 281, 295 - 297, 326, 352, 353, and 363 or any major portion thereof. Each possibility is a separate embodiment.

[0149] According to some embodiments, the plant can be a rice plant, and the polynucleotide can reduce the level of a target gene having any of the nucleotide sequences listed in 43, 44, 52 - 57, 73 - 75, 85, 86, 225 - 227, 246 - 248, 259 - 264 or any major portion thereof. Each possibility is a separate embodiment.

[0150] According to some embodiments, the plant can be a rice plant, and the polynucleotide can reduce the level of a protein having any of the amino acid sequences listed in SEQ ID NOs: 407 - 412, 416 - 425, 437 - 442, 449 - 467, 589 - 598, 610 - 615, 623 - 638, 644 - 649, 659 - 662, 690 - 695, 716 - 723, 727, and 728 or any major portion thereof. Each possibility is a separate embodiment.

[0151] According to some embodiments, the plant can be a rice plant, and the polynucleotide (i.e., dsRNA) can have the nucleotide sequences listed in SEQ ID NO: 742 - 747.

[0152] In certain embodiments, the polynucleotide compositions and methods provided herein generally achieve regulation or modulation (e.g., inhibition) of gene expression during a period of days to weeks or more of the life of the treated plant and generally in a systemic manner. For example, within days of treating a plant leaf with the polynucleotide composition of the invention, primary siRNAs and transitive siRNAs can be detected in other leaves on the sides and above the treated leaf and in apical tissues. In certain embodiments, a method for systemically inhibiting the expression of a gene in a plant is provided, the method comprising treating the plant with a composition comprising at least one polynucleotide and a delivery agent, whereby the expression of the gene in the plant or its progeny is systemically inhibited as compared to a control plant not treated with the composition, wherein the polynucleotide comprises at least 18 or at least 19 consecutive nucleotides that are substantially identical or substantially complementary to a gene or transcript encoding the target gene of the plant.

[0153] A composition for inhibiting a target gene can comprise one or more polynucleotides that are substantially identical or substantially complementary to more than one gene or more than one fragment of one or more genes. In certain embodiments, a composition for inhibiting a target gene can comprise one or more polynucleotides that are substantially identical or substantially complementary to more than one contiguous segment of the target gene, more than one non - contiguous segment of the target gene, more than one allele of the target gene, or more than one target gene from one or more species.

[0154] In certain embodiments, the polynucleotide comprises two or more copies of a nucleotide sequence (a nucleotide sequence of 18 or more nucleotides), wherein the copies are arranged in tandem. In another embodiment, the polynucleotide comprises two or more copies of a nucleotide sequence (a nucleotide sequence of 18 or more nucleotides), wherein the copies are arranged in an inverted repeat manner (forming at least partially self - complementary strands). The polynucleotide can comprise both tandem copies and inverted repeat copies. Whether arranged in tandem or in inverted repeat, each copy can be directly adjacent to the next copy, or the copy pairs can be separated by a spacer region of one or more optionally nucleotides. The optional spacer region can be an unrelated sequence.

[0155] Although there is no upper limit on the concentration and dosage of polynucleotide molecules that can be used in the methods and compositions provided herein, lower effective concentrations and dosages will generally be sought for efficiency. The concentration can be adjusted according to the volume of spraying or treatment applied to the surface of plant leaves or other plant parts such as petals, stems, tubers, fruits, anthers, pollen, leaves, roots or seeds.

[0156] Embodiments of agents or treatments for conditioning plants to be permeable to polynucleotides include emulsions, inverse emulsions, liposomes, and other micelle-like compositions. Embodiments of agents or treatments for conditioning plants to be permeable to polynucleotides include counterions or other molecules known to associate with nucleic acid molecules, e.g., inorganic ammonium ions, alkylammonium ions, lithium ions, polyamines such as spermine, spermidine, or putrescine, and other cations. Organic solvents for conditioning plants to be permeable to polynucleotides include DMSO, DMF, pyridine, N-pyrrolidine, hexamethylphosphoramide, acetonitrile, dioxane, polypropylene glycol, and other solvents miscible with water or that will dissolve phosphonucleotides in non-aqueous systems such as for synthetic reactions. Oils of natural origin or synthetic oils with or without surfactants or emulsifiers can be used, e.g., oils of plant origin, crop oils.

[0157] In certain embodiments, as the silicone formulation commercially available as L-77 surfactant (having CAS number 27306-78-1 and EPA number: CAL.REG.NO.5905-50073-AA and currently available from Momentive Performance Materials, Albany, N.Y.) can be used to prepare polynucleotide compositions. In which the use of In certain embodiments of the pre - spraying treatment of L - 77 silicone formulations on plant leaves or other plant surfaces, freshly prepared concentrations in the range of about 0.015% to about 2% (wt%) (e.g., about 0.01 wt%, 0.015 wt%, 0.02 wt%, 0.025 wt%, 0.03 wt%, 0.035 wt%, 0.04 wt%, 0.045 wt%, 0.05 wt%, 0.055 wt%, 0.06 wt%, 0.065 wt%, 0.07 wt%, 0.075 wt%, 0.08 wt%, 0.085 wt%, 0.09 wt%, 0.095 wt%, 0.1 wt%, 0.2 wt%, 0.3 wt%, 0.4 wt%, 0.5 wt%, 0.6 wt%, 0.7 wt%, 0.8 wt%, 0.9 wt%, 1.0 wt%, 1.1 wt%, 1.2 wt%, 1.3 wt%, 1.4 wt%, 1.5 wt%, 1.6 wt%, 1.7 wt%, 1.8 wt%, 1.9 wt%, 2.0 wt%, 2.1 wt%, 2.2 wt%, 2.3 wt%, 2.5 wt%) are effective in preparing the leaf or other plant surface to transfer the polynucleotide molecule from the surface application on the surface into plant cells. In certain embodiments of the methods and compositions provided herein, a composition is used or provided that comprises a polynucleotide molecule and a silicone formulation of L - 77.

[0158] According to some embodiments, the polynucleotide composition comprising the silicone formulation may comprise salts such as ammonium chloride, tetrabutylphosphonium bromide, and / or ammonium sulfate. Ammonium chloride, tetrabutylphosphonium bromide, and / or ammonium sulfate may be provided in the polynucleotide composition at a concentration of about 0.01% to about 5% (w / v).

[0159] According to some embodiments, other useful transfer agents or adjuvants for transfer agents that can be used in the polynucleotide compositions provided herein include surfactants and / or active molecules contained therein. Surfactants and / or active molecules contained therein include, but are not limited to, sodium or lithium salts of fatty acids (such as tallow or tallow amine or phospholipids) and silicone surfactants. In certain embodiments, the polynucleotide compositions containing the transfer agent are formulated with counterions or other molecules known to associate with nucleic acid molecules. Illustrative examples include tetraalkylammonium ions, trialkylammonium ions, sulfonium ions, lithium ions, and polyamines such as spermine, spermidine, or putrescine.

[0160] In certain embodiments, the polynucleotide composition further comprises glycerol. Glycerol can be provided in the composition at a concentration of about 0.1% to about 1% (w / v or v / v). A glycerol concentration of about 0.4% to 0.6% or about 0.5% (w / v or v / v) can also be used in the polynucleotide composition containing the transfer agent.

[0161] In certain embodiments, the polynucleotide composition further comprises an organic solvent. Non-limiting examples of suitable organic solvents include, but are not limited to, DMSO, DMF, pyridine, N-pyrrolidine, hexamethylphosphoramide, acetonitrile, dioxane, polypropylene glycol, and other solvents miscible with water or that will dissolve phosphonucleotides in non-aqueous systems (such as for synthetic reactions).

[0162] In certain embodiments, the polynucleotide composition further comprises an oil of natural origin or a synthetic oil with or without a surfactant and / or an emulsifier. Non-limiting examples of suitable oils include, but are not limited to, oils of plant origin, crop oils, paraffin oil, polyol fatty acid esters, or oils with short-chain molecules modified with amides or polyamines (such as polyethyleneimine or N-pyrrolidine).

[0163] The compositions and methods of the present invention can be used to modulate or inhibit the expression of endogenous target genes or transgenic target genes in plant cells or plants. In certain embodiments of the methods and compositions provided herein, the expression of the genes targeted by the polynucleotides disclosed herein can be completely, partially, and / or transiently inhibited to result in increased yields.

[0164] Target genes and plants containing these target genes can be obtained from: i) field crop plants; ii) vegetable plants; iii) culinary plants; iv) fruit plants; v) trees grown for ornamental or commercial purposes; or vi) trees in natural forests, or vii) ornamental plants. The methods and compositions provided herein can also be applied to plants produced by cutting, cloning, or grafting methods.

[0165] The compositions provided herein that contain polynucleotides and transfer agents can be surface-applied to plants or plant parts by any convenient method, such as in powder form, or sprayed or coated with a liquid composition including any of an emulsion, suspension, or solution. Such surface application by spraying or coating can be to the entire surface or any part of the surface of the plant or plant part. Similarly, in certain embodiments, compositions containing transfer agents or other pre-treatments can be applied to plants or plant parts by any convenient method (e.g., spraying or wiping a solution, emulsion, or suspension). The compositions provided herein that contain polynucleotides and transfer agents can be surface-applied to plant parts, including but not limited to roots, flowers, stems, tubers, meristems, ovules, fruits, anthers, pollen, leaves, or seeds.

[0166] According to some embodiments, the compositions can be provided by irrigation, such as using an existing or designated irrigation system.

[0167] The application of the compositions provided herein that contain polynucleotides and transfer agents to seeds is particularly provided. Seeds can be contacted with such compositions by spraying, atomizing, soaking, etc. According to some embodiments, the progeny plants and plant parts derived from the treated seeds will exhibit increased yields due to the inhibition of the expression of target genes.

[0168] Various methods of spraying the compositions on plants or plant parts can be used to surface-apply the compositions containing polynucleotides with transfer agents to the plant surface. In the field, the compositions can be applied using a boom that extends over the crop and delivers the composition to the plant surface or a boomless sprayer that distributes the composition over a wide area. In certain embodiments, agricultural sprayers suitable for directed, broadcast, or band spraying can also be used. Sprayers suitable for spraying specific parts of the plant (including but not limited to leaves, the undersurface of leaves, flowers, stems, male reproductive organs such as tassels, meristems, pollen, ovules, etc.) can also be used. The compositions can also be delivered aerially, such as by a crop-dusting airplane. In certain embodiments, the spray can be delivered using a pressurized backpack sprayer calibrated to deliver the composition at an appropriate rate.

[0169] In certain embodiments, plant parts can be sprayed before or after harvest to increase the yield of the plant parts. As described previously, the composition can be surface-applied to the plant parts attached to the plant by spraying. The composition can be surface-applied to the plant parts separated from the plant by spraying as described previously or by an alternative method. Alternative methods for applying the composition to the separated parts include but are not limited to passing the plant parts through a spray via a conveyor belt or trough, or immersing the plant parts in the composition.

[0170] Compositions comprising polynucleotides and transfer agents can be applied to plants or plant parts at one or more developmental stages as desired and / or as needed. In certain embodiments, application of the composition to pre-germination seeds and / or post-germination seedlings is provided. Seeds can be treated with the polynucleotide compositions provided herein by methods including, but not limited to, spraying, soaking, or any method providing coating of the polynucleotide composition on the seeds, imbibition, and / or uptake of the polynucleotide composition by the seeds. Seeds can be treated with the polynucleotide composition using a batch seed treatment system or a continuous flow treatment system. Seed treatment can also be applied in a laboratory or commercial scale treatment device such as a tumbler, mixer, or pan granulator. The polynucleotide composition for treating seeds can comprise one or more other desired components including, but not limited to, a liquid diluent, an adhesive serving as a polynucleotide matrix, a filler for protecting the seeds under stress conditions, and a plasticizer for improving the flexibility, adhesiveness, and / or spreadability of the coating. In addition, for oily polynucleotide compositions containing little or no filler, desiccants such as calcium carbonate, kaolin, or bentonite, perlite, diatomaceous earth, or any other adsorbent material can be added.

[0171] In certain embodiments, application of the composition at the early, middle, and late vegetative stages of plant development is provided. In certain embodiments, application of the composition at the early, middle, and late reproductive stages is also provided. Application of the composition to plant parts at different maturity stages is also provided.

[0172] The following examples are included to demonstrate examples of certain preferred embodiments of the invention. Those skilled in the art should understand that the techniques disclosed in the following examples represent methods that the inventors have found function well in the practice of the invention and thus can be considered examples of preferred modes for its practice. However, those skilled in the art should understand that, in light of the present disclosure, many changes can be made in the specific embodiments disclosed and still obtain similar or like results without departing from the spirit and scope of the invention. Examples

[0173] Example 1 - Verification of improved plant yield traits.

[0174] The timing of application of the composition (e.g., by irrigation or spraying) is arranged according to the transcription of the target gene and the desired trait it affects. Examples of appropriate timing are outlined in Table 2, which shows selected examples based on the targeted gene (here Brassica napus) and the appropriate timing for indicating the application of the composition. The dsRNA mixture is applied according to the timing of specific gene expression, i.e., one week before the expected expression peak, at the expected expression peak, and one week after its expression peak. After treatment, the plant phenotypes related to the targeted yield traits are examined, as outlined in Table 2 for example, which shows selected examples of traits and their related genes.

[0175] Example 2 - Testing the permeability of the penetrant

[0176] To test the permeability of the penetrant, a 0.01% to 1% or 0.1 - 10 mg / ml penetrant solution was sprayed on the leaves, and the contact angle was evaluated using standard methods.

[0177] The efficiency of the tested penetrants is shown in Figure 1 which shows the change in contact angle (indicating penetration) over time after application on the leaves of the plant. As observed, all the tested penetrants significantly reduced the contact angle compared to when no penetrant was applied.

[0178] Example 3 - Increased branching in Brassica napus by targeting BRC1

[0179] The dsRNA molecule (SEQ ID NO: 730) targeting BnBRC1 (SEQ ID NO: 1) of Brassica napus (Brassica napus L.) was applied at a dsRNA concentration of 1 ng / ml to 1 mg / ml diluted in 0.01% to 1% or 0.1 - 10 mg / ml surfactant using a nebulizer, where the surfactant is a silicone polyalkylene oxide copolymer ( L - 77AG), however, other surfactants such as those listed in Table 1 can also be used. The BnBRC1 (SEQ ID NO: 730) dsRNA was applied from the start of bolting until the emergence of the second inflorescence.

[0180] After treatment, the branching of the plants was evaluated by visual inspection. As Figure 3 shown in, which shows illustrative images of Brassica napus plants after surface application of dsRNA, the branching of the plants increased significantly (5 - 6 branches per plant in the control plants treated with mock treatment (left figure), while 9 - 10 branches per plant in the plants treated with BnBRC1 dsRNA (right figure), where the dsRNA contains the sequence listed in SEQ ID NO: 730).

[0181] Example 4 - Ectopic application of dsRNA affects petal architecture in greenhouse and field Brassica napus.

[0182] The dsRNA molecule (SEQ ID NO: 733) targeting the Brassica napus gene BnPTL (SEQ ID NO: 286) was applied using a hand - held sprayer at a dsRNA concentration of 1 ng / ml to 1 mg / ml diluted in 0.01% to 1% or 0.1 - 10 mg / ml surfactant, where the surfactant is a siloxane polyalkylene oxide copolymer ( L - 77AG). However, other surfactants such as those listed in Table 1 can also be used. The dsRNA was applied when the plants reached 70% to full stem length.

[0183] After treatment, the number of petals was evaluated by visual inspection. Figure 2 Preliminary illustrative images of Brassica napus plants (here Brassica napus) after topical application of BnPTL dsRNA are shown. In mock - treated plants (left panel), normal petal architecture was observed, while BnPTL dsRNA - treated plants (right panel) showed abnormal petal architecture (three petals per flower or asymmetric flowers), indicating the ability of topically applied dsRNA to affect petal architecture in Brassica napus plants.

[0184] Example 5 - Ectopic application of dsRNA for yield increase - Brassica napus

[0185] Seeds of the Brassica napus cultivar Belinda (Brassica napus) were sown in 3 - liter pots in a controlled greenhouse, one plant per pot, and watered and fertilized once a day. Plants were treated with a dsRNA molecule (SEQ ID NO: 733) targeting the BnPTL gene (SEQ ID NO: 286) (hereinafter referred to as treatment 'A' in all other descriptions) and a dsRNA molecule (SEQ ID NO: 730) targeting the BnBRC1 gene (SEQ ID NO: 1) (hereinafter referred to as treatment 'B'). According to the appropriate developmental stages of the plants listed in Table 2 below, the treatments were applied at a concentration of 1 μg / ml and 10 μg / ml of dsRNA diluted in surfactant ( L - 77AG), 10 ml per plant.

[0186] For the flower morphology of 'A' (BnPTL dsRNA) and the number of branches of 'B' (BnBRC1 dsRNA), phenotypic evaluations were carried out one month after application of the treatments, and the results were compared with Ctrl (surfactant only).

[0187] Method

[0188] Brassica napus seeds were sown in a 2x1,000 m field in the Sharon region of Israel (32°10′1.55″N 34°52′33.96″E), divided into 12 rows, each row being 52 m x 0.8 m, with a clear lane of 40 cm between each row. 2 The seeds were sown using a push seeder at a distance of approximately 15 cm between each seed and a depth of 2 cm.

[0189] Each row was divided into blocks of 0.8 m x 2 m, separated by 1 m of untreated plants as an interval between adjacent different treatments, with approximately 70 plants per plot.

[0190] Five dsRNAs were tested, namely:

[0191] 1) A dsRNA molecule (SEQ ID NO: 733) targeting the BnPTL gene (SEQ ID NO: 286) of Brassica napus - hereinafter referred to as treatment 'A',

[0192] 2) A dsRNA molecule (SEQ ID NO: 730) targeting the BnBRC1 gene (SEQ ID NO: 1) of Brassica napus - hereinafter referred to as treatment 'B',

[0193] 3) A dsRNA molecule (SEQ ID NO: 731) targeting the BnCKX2 gene (SEQ ID NO: 158) of Brassica napus, hereinafter referred to as treatment 'C';

[0194] 4) A dsRNA molecule (SEQ ID NO: 732) targeting the BnKIN10 gene (SEQ ID NO: 62) of Brassica napus - hereinafter referred to as treatment 'D';

[0195] 5) A dsRNA molecule (SEQ ID NO: 729) targeting the BnADPG gene (SEQ ID NO: 235) of Brassica napus - hereinafter referred to as treatment 'E'.

[0196] For each plot, the dsRNA was sprayed with 200 ml of water supplemented with dsRNA and surfactant. The dsRNA treatment was applied during the plant growth stage according to the expected peak expression time of each selected gene (as listed in Table 2).

[0197] Table 2: Developmental stages of Brassica napus plants for dsRNA application for each gene and its phenotypic evaluation.

[0198]

[0199]

[0200] ​

[0201] For each treatment, two dsRNA doses (1 μg / ml or 10 μg / ml) and two spraying regimens (1 or 5 times in field plot 1, and 1 or 3 times in field plot 2) were conducted, as listed in Table 3. The interval between subsequent treatments was one week.

[0202] Table 3 - Field trial parameters.

[0203]

[0204] Surfactant only (without dsRNA) at the same time point was used as a control (ctrl).

[0205] Each treatment was replicated 10 times on different plots of land and sprayed using a "Solo" 2-liter manual sprayer with the smallest droplet size.

[0206] At the end of the growing season, the plots of land were harvested manually, dried for one week, processed through a thresher (Classic ST, Wintersteiger, Germany), and the net seed weight for each plot / each treatment was weighed.

[0207] All field data were statistically analyzed to compare each dsRNA treatment with its associated Ctrl (P < 0.1).

[0208] The weight of 1000 seeds was measured using a specified seed counter (Contador, Pfeuffer, Germany), with 5 replicates per plot of land, combined with the total weight of a fixed volume. The oil content was measured using hexane as a solvent according to the "Soxhlet" extraction method at the Biotechnology Engineering Faculty, Ben-Gurion University, Be’er Sheva, Israel.

[0209] Result

[0210] Treatment A

[0211] Flower morphology

[0212] For treatment 'A', flower morphology was evaluated three weeks after the application of dsRNA.

[0213] As from Figure 2As observed, alterations in flower morphology were only observed in plants treated with dsRNA. Compared to the control, at least one flower in each inflorescence lacked a petal. Additionally, compared to the control, inflorescence development and flowering were delayed by approximately 2 weeks in dsRNA-treated plants.

[0214] Light penetration percentage :

[0215] Three weeks after spraying with dsRNA (SEQ ID NO:733), the percentage of light penetration at the base of the inflorescence was measured. The measurement was conducted after the peak of flowering. For all treatments applied, compared to the control, dsRNA treatment caused a significant increase in light penetration, namely: 1-1 (1-1 = 1 μg / ml, 1 treatment), 10-1 (10-1 = 10 μg / ml, 1 treatment), 1-5 (1-5 = 1 μg / ml, 5 treatments), and 10-5 (10-5 = 10 μg / ml, 5 treatments) caused increases in light penetration of 42.3%, 45.1%, 47.6%, and 50%, respectively.

[0216] These results indicate that reducing BnPTL expression (SEQ ID NO:286) by ectopic application of dsRNA molecules targeting PTL reduced the number of petals, which in turn led to increased light penetration to the lower part of the plant and increased total photosynthesis efficiency and yield.

[0217] Seed weight:

[0218] In Field 1, treatments 1-5 and 10-5 increased seed weight by 4.9% and 9.4% respectively ( Figure 5A ). In Field 2, treatment 10-1 increased seed weight by 17.5% ( Figure 5B ).

[0219] These results indicate that reducing BnPTL expression by ectopic application of dsRNA targeting BnPTL can increase seed weight.

[0220] Oil content:

[0221] In Field 1, a consistent increase in oil content was observed in dsRNA-treated plants, 1.4%, 1.9%, and 1.1% (for treatments 1-1, 1-5, and 10-1 respectively —— Figure 6A ).

[0222] In Field 2, larger increases were observed, 6%, 2.4%, 5.9%, and 2.3% (for treatments 1-1, 1-3, 10-1, and 10-3 respectively —— Figure 6B ).

[0223] These results indicate that reducing BnPTL expression by ectopic application of dsRNA targeting BnPTL can increase the oil content of Brassica napus plants.

[0224] Treatment B

[0225] Number of branches:

[0226] For treatment 'B', the number of branches was evaluated three weeks after the application of dsRNA.

[0227] As observed from Figure 3 As a result of the dsRNA treatment (10 μg / ml), the number of branches increased, and as further observed from Figure 4 the increase in the number of branches was dose-dependent (P < 0.1).

[0228] As observed from Figure 7 field results showed that compared with the control, the expression of BnBRC1 (SEQ ID NO: 1) targeted by ectopic application of dsRNA (SEQ ID NO: 730 targeting BnBRC1) led to an increase in the number of branches in treatments 1-1, 1-5, and 10-5 by 8.2%, 5.9%, and 16.4%, respectively.

[0229] Treatment C

[0230] Seed weight:

[0231] As observed from Figure 8A and Figure 8B the expression of BnCKX2 (SEQ ID NO: 158) targeted by ectopic application of dsRNA (SEQ ID NO: 731) targeting CKX2 led to an increase in seed weight in both of the two tested fields. In field 1, for treatments 1-1, 1-5, 10-1, and 10-5, the increases were 2.1%, 1.3%, 1.3%, and 4% ( Figure 8A ). In field 2, for treatments 1-1, 10-1, and 10-3, the seed weight increased by 1.2%, 15.6%, and 9% respectively ( Figure 8B ).

[0232] 1000-seed weight and seed size:

[0233] In addition, compared with Ctrl, the expression of BnCKX2 targeted by ectopic application of dsRNA targeting BnCKX2 led to an increase in the 1000-seed weight in treatments 1-1, 1-5, and 10-1 in field 1 by 3.9%, 3.5%, and 1.6%. The seed size in treatments 1-1, 1-5, and 10-5 also increased by 1.1%, 5.1%, and 1.2%, respectively.

[0234] In the second field, targeting BnCKX2 by ectopic application of dsRNA targeting BnCKX2 increased the 1000-seed weight of treatments 1-3, 10-1, and 10-3 by 5.8%, 1.5%, and 1.1%, respectively. The seed size in treatments 1-3, 10-1, and 10-3 also increased by 4.1%, 0.7%, and 3.8%, respectively.

[0235] These results clearly show that targeting the expression of BnCKX2 by ectopic application of dsRNA targeting BnCKX2 increased the weight and size of Brassica napus seeds.

[0236] Treatment E

[0237] Seed weight:

[0238] As observed in Figure 9A and Figure 9B targeting the expression of BnADPG1 (SEQ ID NO: 235) by ectopic application of dsRNA targeting BnADPG1 (SEQ ID NO: 729) led to an increase in seed weight in both of the tested fields. In the first field, for treatments 1-1, 1-5, and 10-1, the increases were 9.5%, 9.4%, and 12%, respectively. In the second field, treatments 1-3, 10-1, and 10-3 increased the seed weight by 11.4%, 14.2%, and 10.1%, respectively.

[0239] 1000-seed weight and seed size:

[0240] Targeting ADPG1 by ectopic application of dsRNA targeting BnADPG1 led to a 4.5%, 7.7%, 6%, and 7% increase in the 1000-seed weight of treatments 1-1, 1-5, 10-1, and 10-5 in the first field relative to Ctrl. The seed size in treatments 1-1, 1-5, 10-1, and 10-5 also increased by 6.8%, 8.2%, 1.1%, and 8.1%, respectively.

[0241] In the second field, targeting BnADPG1 caused a 14.7%, 3%, and 13.3% increase in the 1000-seed weight of treatments 1-3, 10-1, and 10-3, respectively. The seed size in treatments 1-1, 1-3, 10-1, and 10-3 also increased by 2%, 13.3%, 2.2%, and 9.6%, respectively.

[0242] These results clearly show that targeting the expression of BnADPG1 by ectopic application of dsRNA targeting BnADPG can increase the weight and size of Brassica napus seeds.

[0243] Oil content:

[0244] In the first plot, an increase in oil content was observed in plants treated with BnADPG1-dsRNA (1.5%, 2.2% and 1.5% for treatments 1-1, 10-1 and 10-5 respectively - Figure 10A ). Similarly, in the second plot, increases in oil content of 2.1%, 1.5% and 4.1% were observed in treatments 1-3, 10-1 and 10-3 respectively compared to the control ( Figure 10B ).

[0245] Example 6 - Ectopic Application of dsRNA for Yield Increase - Rice (Oryza sativa)

[0246] Materials and Methods:

[0247] Rice (Oryza sativa spp.) seeds were sown in 3-litre pots in a greenhouse, one seed per pot, and watered and fertilized once a day.

[0248] The plants were treated with the dsRNAs listed below at 10 μg / ml (in water and surfactant):

[0249] 1) A dsRNA molecule (SEQ ID NO: 742) against OsBRC1 (SEQ ID NO: 43) of rice,

[0250] 2) A dsRNA molecule (SEQ ID NO: 744) against OsPIN5b (SEQ ID NO: 86) of rice,

[0251] 3) A dsRNA molecule (SEQ ID NO: 746) against OsSKIN1 (SEQ ID NO: 52) of rice,

[0252] The dsRNAs were applied according to the plant developmental stage, as listed in Table 4 below.

[0253] Table 4: Developmental stages of rice (O. sativa) plants for specific dsRNA treatments.

[0254] dsRNA Developmental stage of treatment SEQ ID NO:742 Axillary bud emergence SEQ ID NO:744 Plant at maximum tiller number SEQ ID NO:746 Plant at seed filling stage

[0255] The dsRNAs were applied as a single spray application at a concentration of 1 μg / ml or 10 μg / ml diluted in surfactant ( L-77AG), 10 ml per plant, 6 pots / 6 plants per treatment.

[0256] The treatments were evaluated 1 month after application of dsRNA and compared to the Ctrl. For OsBRC1, the number of axillary tillers was evaluated, for OsPIN5b, the number of panicles was evaluated, and for OsSKIN1, the seed size was evaluated. The total seed weight of all treatments was measured.

[0257] Result

[0258] At 1 month after treatment, the total number of tillers of plants treated with dsRNA (SEQ ID NO: 742) targeting OsBRC1 of rice plants was counted and compared to the control. Interestingly, treating plants with a low concentration of OsBRC1-dsRNA caused a slight increase in the number of tillers, while a high concentration caused a decrease in the number of tillers ( Figure 11 ). This indicates that dsRNA targeting OsBRC1 can be used to control the number of tillers in rice.

[0259] At 3 months after treatment, the number of panicles of rice plants treated with dsRNA (SEQ ID NO: 744 and SEQ ID NO: 745) targeting OsPIN5b was evaluated compared to the control.

[0260] At 3 months after treatment, the total seed weight of all treatments was measured.

[0261] Example 7 - Ectopic Application of dsRNA for Yield Increase - Soybean (Glycine max)

[0262] Materials and Methods:

[0263] Soybean (Glycine max, cultivar Williams82) seeds were sown in 3-liter pots in a greenhouse, one seed per pot, and watered and fertilized once a day or as needed.

[0264] Several examples of dsRNA applications aimed at causing phenotypic changes and increasing yield were tested (as specified below):

[0265] 1) SEQ ID NO: 734 targeting GmBRC1 (SEQ ID NO: 15) of soybean.

[0266] 2) SEQ ID NO: 738 targeting GmJAG1 (SEQ ID NO: 153) of soybean.

[0267] 3) SEQ ID NO: 736 targeting GmBS1 (SEQ ID NO: 116) of soybean.

[0268] 4) SEQ ID NO: 740 for GmPLDα1 (SEQ ID NO: 124) in soybean.

[0269] Apply dsRNA according to the plant development stage, as listed in Table 5 below.

[0270] Table 5: Plant development stages of soybean (G. max) for specific dsRNA treatments.

[0271] dsRNA Developmental stage of treatment SEQ ID NO:734 Bolting stage SEQ ID NO:738 Flowering stage SEQ ID NO:736 Seed filling stage SEQ ID NO:740 Seed filling stage

[0272] dsRNA was applied in two sprays. The first spray was at the assumed peak of gene expression time, and the second spray was two weeks later. dsRNA was applied at a concentration of 1 μg / ml or 10 μg / ml diluted in a surfactant ( L-77AG), 10 ml per portion, covering the entire plant surface, six pots / six plants per treatment. Each dsRNA experiment was conducted with six replicates.

[0273] The treatments were evaluated 1 month after the application of dsRNA and compared with the Ctrl. For GmBRC1 targeting, the number of axillary branches was evaluated; for GmJAG1 targeting, the number of seeds per pod was evaluated; for GmBS1 targeting, the seed size was evaluated; and for GmPLDα1, the seed weight / grouting was evaluated. In addition, the total seed weight of all treatments was measured.

[0274] Result

[0275] One month after treatment, the total number of axillary branches of plants treated with dsRNA targeting GmBRC1 was evaluated. As observed in Figure 12 , plants treated with dsRNA had a higher number of branches compared to Ctrl plants. Interestingly, the largest increase (54.1%) was observed for the lower dsRNA concentration.

[0276] One month after treatment with dsRNA targeting GmJAG1, the total number of seeds per pod of the plants was evaluated.

[0277] After the pods of the plants were completely dry, the seed size of plants treated with dsRNA targeting GmBS1 was evaluated, and the total seed weight of plants treated with dsRNA targeting GmPLDα1 was evaluated.

[0278] Although certain embodiments of the present invention have been illustrated and described, it should be clear that the present invention is not limited to the embodiments described herein. Many modifications, changes, variations, substitutions, and equivalents that do not depart from the spirit and scope of the present invention as described in the appended claims will be apparent to those skilled in the art.

Claims

1. A composition comprising: a dsRNA molecule comprising at least 18 consecutive nucleotides that are substantially identical to a portion of the sequence encoding the amino acid sequence set forth in SEQ ID NO: 407 of a rice plant; and a delivery agent configured to facilitate the penetration of the dsRNA molecule into the cells of the rice plant, wherein: the penetration of the dsRNA molecule into the cells of the rice plant causes a transient reduction in the gene expression; and wherein the transient reduction affects a trait of the rice plant, resulting in an increase in the yield of the plant, the trait of the plant being selected from the group consisting of increased branching, increased grain filling, increased number of panicles, increased number of seeds, increased number of tillers, increased heading of the plant, and any combination thereof; and wherein the dsRNA molecule consists of the nucleotide sequence set forth in SEQ ID NO:

742.

2. The composition according to claim 1, wherein the dsRNA molecule comprises at least 18 consecutive nucleotides that are substantially identical to a portion of the sequence set forth in SEQ ID NO:

43.

3. The composition according to claim 1, wherein the dsRNA molecule has a length of at least about 50 base pairs.

4. The composition according to claim 3, wherein the dsRNA molecule has a length of at least about 200 base pairs.

5. The composition according to claim 1, wherein the transfer agent comprises N,N-dimethyldecanamide, coconut amide propyl dimethylamine, silicone polyalkylene oxide copolymer, EM-30, dimethylamide of C8 / C10 fatty acid, esterification copolymer of glycerol, trisiloxane ethoxylate or any combination thereof.

6. A method of transiently affecting a trait of a rice plant, the method comprising topically applying the composition according to any one of claims 1-5 to the surface of the rice plant.

7. The method according to claim 6, wherein the application comprises spraying the composition onto the surface of the rice plant.

8. The method according to claim 7, wherein the composition is sprayed onto the surface of the rice plant using a boom that extends over the crop, a boomless sprayer, an agricultural sprayer, a crop dusting aircraft, a pressurized backpack sprayer, a track sprayer, or a laboratory sprayer / submersion device.

9. The method according to claim 6, wherein the application comprises providing the composition through an irrigation system.

10. The method according to claim 6, wherein the surface is one or more parts of the rice plant selected from the group consisting of hypocotyl, cotyledon, leaf, flower, stem, spikelet, meristem, pollen, ovule, and fruit.

11. The method according to claim 6, further comprising scheduling the application of the composition at a desired developmental stage of the rice plant.

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