Surface application of polynucleotide molecules for improving yield traits in plants

Abstract

The present application relates to the surface use of polynucleotide molecules for improving yield traits in plants. A composition comprising: (i) a dsRNA molecule of at least 18 contiguous nucleotides substantially identical or substantially complementary to a plant gene or a transcript of the plant gene; and (ii) a transfer agent that modulates the plant surface to permeate the dsRNA molecules into the plant cells; wherein permeation of the dsRNA molecule into the plant cell results in a transient reduction in gene expression, and wherein a transient reduction in gene expression results in a change in plant yield-related traits.

Description

[0001] This application is a divisional application of an application filed on March 15, 2021, with application number 202180021633.9, and invention name “Surface application of polynucleotide molecules for improving yield traits of plants”. Background of the Invention

[0002] According to 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 demand for food. At the same time, resource conservation, reduced use of fertilizers, pesticides and herbicides, and environmental sustainability are increasingly important factors in how food is grown. Improvements in agricultural plants and farming practices are needed to achieve increased plant yields using fewer resources and more environmentally sustainable inputs.

[0003] Yield is influenced by various factors such as the number and size of plant organs, plant architecture (eg number of branches), seed filling, seed number, drought resistance, shattering, flowering and number of tillers.

[0004] Currently, crop performance is primarily optimized through technologies that target 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 over the past 50 years, yield growth rates for many major crops have stagnated, and new solutions are urgently needed to increase crop yields. Public concerns about GM crops and synthetic chemicals, in addition to their lengthy development and regulatory timelines, have challenged their use in many major crops and countries, leading to a lack of acceptance of many genetically modified 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

[0005] Provided herein are compositions and methods for providing increased yield in plants by inhibiting the expression of yield-related genes in plants, by providing to the surface of the plant a composition comprising a polynucleotide molecule capable of hybridizing with a yield-related gene or gene transcript and a transfer agent that modulates the plant surface to allow the polynucleotide molecule to penetrate into plant cells, thereby improving the yield-related traits of the plant. Non-limiting examples of yield-related traits of plants include: increased branching, seed size, increased panicle number, increased tiller number, increased seeds, increased plant silique size, increased seed filling, increased seed number, increased heading, improved drought resistance, reduced shattering, reduced abscission tissue formation, reduced petals in the plant, 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.

[0006] According to some embodiments, the penetration of polynucleotide molecules can cause a transient reduction in gene expression, causing non-permanent spatial and temporal effects on plants, and does not result in nor require the integration of exogenous polynucleotides into the chromosomes of plants. This method has several advantages. First, it circumvents the need for GMO legislation. In addition, it is a more sophisticated and effective 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 environmental conditions that need to be addressed. As a non-limiting example, during droughts, farmers can decide to temporarily suppress the expression of genes / transcripts to improve the adaptability to water stress and stop suppressing once the weather changes. As another non-limiting example, farmers can decide to temporarily suppress genes / transcripts related to early flowering of plants under conditions such as unexpected rainfall, temperature changes, etc.

[0007] Advantageously, the technology is applicable for use in a variety of crops, including but not limited to corn, 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.

[0008] According to some embodiments, the polynucleotide molecules are provided in a composition that can penetrate or be absorbed into living plant tissues to initiate systemic gene suppression or regulation. In certain embodiments of the present invention, the polynucleotide molecules ultimately provide RNA (e.g., dsRNA) or RNA-like molecules to the plant that are capable of hybridizing with 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.

[0009] According to some embodiments, the silencing / suppression of the target gene can directly improve the yield-related traits of the plant. Alternatively, the silencing / suppression of the target gene can indirectly improve the yield-related traits of the plant. For example, silencing or suppressing the target gene can change (increase or decrease) the expression of another gene that improves the yield-related traits of the plant.

[0010] As another important practical advantage, topical application of a composition comprising an exogenous polynucleotide and a transfer agent does not require the exogenous polynucleotide to be physically bound to a particle, such as in biolistically mediated introduction of polynucleotides associated with gold or tungsten particles into an interior portion of a plant, plant part, or plant cell.

[0011] According to some embodiments, the polynucleotide molecule targets an mRNA of a plant gene. According to some embodiments, the polynucleotide molecule targets a translated region of an mRNA. According to some embodiments, the polynucleotide molecule targets an untranslated region of an mRNA.

[0012] According to some embodiments, a composition is provided comprising: (i) a polynucleotide molecule comprising at least 18 contiguous 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 modulates the surface of a plant to allow penetration of the polynucleotide molecule into plant cells; wherein penetration of the polynucleotide molecule into the plant cells causes a transient decrease in gene expression, and wherein the transient decrease in gene expression causes a change in a yield-related trait of the plant.

[0013] According to some embodiments, the yield-related traits of the plant are selected from the group consisting of: increased kernel / seed size, increased kernel number, increased ear / silique number, increased tiller number, increased branching, increased seed size, increased seed filling, increased seed number, increased heading, improved drought resistance, 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.

[0014] According to some embodiments, a composition suitable for topical application to a plant is provided, the composition comprising a dsRNA molecule comprising at least 18 contiguous nucleotides that are substantially identical or substantially complementary to a plant gene or a portion of a transcript of a plant gene; and a transfer agent configured to facilitate penetration of the dsRNA molecule into cells of the plant, wherein penetration of the dsRNA molecule into the plant cells causes a transient reduction in gene expression.

[0015] A transient reduction in gene expression results in changes in plant traits.

[0016] The trait of the plant is selected from the group consisting of increased branching, increased grain filling, increased trehalose-6-phosphate (T6P) levels, increased ear number, increased seed filling, increased seed number, increased seed size, reduced shattering, reduced abscission tissue formation, increased tiller number, increased heading in the plant, 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

[0014] The invention further provides a method for increasing the number of ears, increasing seed filling, increasing seed number, increasing seed size, decreasing shattering, decreasing abscission tissue formation, increasing tiller number, increasing heading in plants, reducing petals, increasing silique size, and any combination thereof; or is selected from the group consisting of: increasing branching, increasing kernel filling, increasing ear number, increasing seed filling, increasing seed number, decreasing shattering, decreasing abscission tissue formation, increasing tiller number, reducing petals, increasing silique size, and any combination thereof. Each possibility is a separate embodiment.

[0017] According to some embodiments, the plant gene is selected from ADPG1, PTL, CKX2, BRC1, KIN1, SKIN1, PIN5b, JAG1, BS1, PLDα1, and / or any homologs or combinations thereof. Each possibility is a separate embodiment. According to some embodiments, the plant gene is selected from ADPG1, PTL, CKX2, BRC1, and / or any homologs or combinations thereof. Each possibility is a separate embodiment.

[0018] According to some embodiments, the plant is a Brassica napus plant, and the dsRNA molecule is a sequence comprising 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 set forth 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 to any of the sequences set forth 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 to any of the sequences set forth in SEQ ID NO: 729, SEQ ID NO: 733, SEQ ID NO: 731, and SEQ ID NO: 730. Each possibility is a separate embodiment.

[0019] According to some embodiments, the plant is a soybean plant and the dsRNA molecule is a nucleotide sequence comprising at least 18 contiguous nucleotides that are substantially identical or substantially complementary to a portion of a sequence encoding any one of the amino acid sequences set forth 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.

[0020] According to some embodiments, the plant is a soybean plant, and the dsRNA molecule is a sequence comprising at least 18 consecutive nucleotides that are substantially identical or substantially complementary to a portion of a sequence encoding any 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.

[0021] According to some embodiments, the plant is a rice plant, and the dsRNA molecule is a nucleotide sequence comprising at least 18 contiguous nucleotides that are substantially identical or substantially complementary to a portion of a sequence encoding any one of the amino acid sequences set forth 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.

[0022] According to some embodiments, the plant is a rice plant, and the dsRNA molecule is a sequence comprising at least 18 consecutive nucleotides that are substantially identical or substantially complementary to a portion of a sequence encoding any 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.

[0023] According to some embodiments, the dsRNA molecule is at least about 50 bases in length. According to some embodiments, the dsRNA molecule is at least about 200 bases in length.

[0024] According to some embodiments, the transfer agent includes N,N-dimethyldecamide, cocamidopropyl dimethylamine, silicone polyalkylene oxide copolymer, EM-30, dimethylamide of C8 / C10 fatty acids, esterified copolymer of glycerol, trisiloxane ethoxylate, or any combination thereof. Each possibility is a separate embodiment.

[0025] According to some embodiments, the transfer agent can be any transfer agent listed in Table 1.

[0026] According to some embodiments, methods are provided for topically applying a composition substantially disclosed herein to a plant surface.

[0027] According to some embodiments, applying comprises 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 extended above the crop, a boomless sprayer, an agricultural sprayer, a crop spreading airplane, a pressurized knapsack sprayer, a track sprayer, or a laboratory sprayer / immersion device. Each possibility is a separate embodiment.

[0028] According to some embodiments, applying comprises providing the composition through an irrigation system.

[0029] 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.

[0030] According to some embodiments, the method further comprises timing the application of the composition to a desired developmental stage of the plant, as substantially explained herein, e.g., in Table 2.

[0031] Certain embodiments of the present disclosure may include some, all, or none of the advantages listed above. One or more of the technical advantages may be apparent to those skilled in the art based on the figures, description, and claims included herein. Furthermore, while specific advantages have been enumerated above, various embodiments may include all, some, or none of the enumerated advantages.

[0032] In addition to the exemplary aspects and embodiments described above, further aspects and embodiments will become apparent by reference to the drawings and by study of the following detailed description. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] The invention will now be described in conjunction with certain examples and embodiments with reference to the accompanying illustrative drawings so that it may be more fully understood.

[0034] Figure 1 shows the change in contact angle (indicative of penetration) over time after application of a targeting agent mixture on a Brassica napus plant leaf;

[0035] Figure 2 Exemplary photographs of flower morphology of Brassica napus plants (here, Brassica napus plants) sprayed ectopically with 10 μg / ml of the dsRNA set forth in SEQ ID NO: 733 targeting the sequence set forth in SEQ ID NO: 286. Control-treated plants have normal petal morphology (left panel). Plants treated with BnPTL dsRNA resulted in flowers with altered petal morphology (right panel).

[0036] Figure 3 Shown are exemplary photographs of Brassica napus plants (here Brassica napus plants) and control plants (Ctrl) sprayed ectopically with 10 μg / ml of the dsRNA set forth in SEQ ID NO: 730 targeting the BnBRC1 sequence set forth in SEQ ID NO: 1. The total number of branches per group is depicted.

[0037] Figure 4 Shown are the average number of branches per Brassica napus plant (here, Brassica napus plants) sprayed ectopically with 1 μg / ml or 10 μg / ml of the dsRNA set forth in SEQ ID NO: 730 targeting the BnBRC1 sequence set forth in SEQ ID NO: 1, or plants sprayed with only a surfactant solution. * indicates a significant change (P < 0.1) in treated plants compared to control plants (Ctrl).

[0038] Figure 5A The average seed weight / 0.8 m obtained for field 1 of the Brassica napus plants (here Brassica napus plants) sprayed with the dsRNA set forth in SEQ ID NO: 733 targeting the sequence set forth in SEQ ID NO: 286 is shown. 2 *Indicates significant changes (P<0.1) in treated plants compared to control plants (Ctrl).

[0039] Figure 5B The average seed weight / 0.8 m obtained for field 2 of Brassica napus plants (here Brassica napus plants) sprayed with the dsRNA set forth in SEQ ID NO: 733 targeting the sequence set forth in SEQ ID NO: 286 is shown.2 *Indicates significant changes (P<0.1) in treated plants compared to control plants (Ctrl).

[0040] Figure 6A Shown are the average percentage oil content of Brassica napus plants (here, Brassica napus plants) sprayed with the dsRNA set forth in SEQ ID NO: 733 targeting the sequence set forth in SEQ ID NO: 286, obtained for Field 1. *Indicates a significant change (P < 0.1) in treated plants compared to control plants (Ctrl).

[0041] Figure 6B Shown are the average percentage oil content of Brassica napus plants (here, Brassica napus plants) sprayed with the dsRNA set forth in SEQ ID NO: 733 targeting the sequence set forth in SEQ ID NO: 286, obtained for Field 2. *Indicates a significant change (P < 0.1) in treated plants compared to control plants (Ctrl).

[0042] Figure 7 Shown are the average number of branches per Brassica napus plant (Brassica napus plant) sprayed ectopically with 1 μg / ml or 10 μg / ml of the dsRNA set forth in SEQ ID NO: 730 targeting the sequence set forth in SEQ ID NO: 1, or sprayed with only a surfactant solution. *Indicates a significant change (P < 0.1) in treated plants compared to control plants (Ctrl).

[0043] Figure 8A Shown are the average seed weight / 0.8 m obtained for Field 1 of Brassica napus plants (Brassica napus plants) sprayed with the dsRNA set forth in SEQ ID NO: 731 targeting the sequence set forth in SEQ ID NO: 158. 2 *Indicates significant changes (P<0.1) in treated plants compared to control plants (Ctrl).

[0044] Figure 8B Shown are the average seed weight / 0.8 m obtained for Field 2 of Brassica napus plants (Brassica napus plants) sprayed with the dsRNA set forth in SEQ ID NO: 731 targeting the sequence set forth in SEQ ID NO: 158. 2 *Indicates significant changes (P<0.1) in treated plants compared to control plants (Ctrl).

[0045] Figure 9AShown are the average seed weight / 0.8 m obtained for Field 1 of Brassica napus plants (Brassica napus plants) sprayed with the dsRNA set forth in SEQ ID NO: 729 targeting the sequence set forth in SEQ ID NO: 235. 2 *Indicates significant changes (P<0.1) in treated plants compared to control plants (Ctrl).

[0046] Figure 9B Shown are the average seed weight / 0.8 m obtained for Field 2 of Brassica napus plants (Brassica napus plants) sprayed with the dsRNA set forth in SEQ ID NO: 729 targeting the sequence set forth in SEQ ID NO: 235. 2 *Indicates significant changes (P<0.1) in treated plants compared to control plants (Ctrl).

[0047] Figure 10A Shown are the average percentage oil content of Brassica napus plants (Brassica napus plants) sprayed with the dsRNA set forth in SEQ ID NO: 729 targeting the sequence set forth in SEQ ID NO: 235, obtained for Field 1. *Indicates a significant change (P < 0.1) in treated plants compared to control plants (Ctrl).

[0048] Figure 10B Shown are the average percentage oil content of Brassica napus plants (Brassica napus plants) sprayed with the dsRNA set forth in SEQ ID NO: 729 targeting the sequence set forth in SEQ ID NO: 235, obtained for Field 2. *Indicates a significant change (P < 0.1) in treated plants compared to control plants (Ctrl).

[0049] Figure 11 Shown are the average number of tillers per rice plant (Oryza sativa) treated with 1 μg / ml or 10 μg / ml of the dsRNA set forth in SEQ ID NO: 742 targeting the sequence set forth in SEQ ID NO: 43, or plants sprayed with only the surfactant solution. *Indicates a significant change (P < 0.1) in treated plants compared to control plants (Ctrl).

[0050] Figure 12 Shown are the average number of branches per soybean plant (Glycine max) treated with 1 μg / ml or 10 μg / ml of the dsRNA set forth in SEQ ID NO: 734 targeting the sequence set forth in SEQ ID NO: 15, or plants sprayed with only the surfactant solution. *Indicates a significant change (P < 0.1) in treated plants compared to control plants (Ctrl). Detailed description

[0051] In the following description, various aspects of the present disclosure will be described. For illustrative purposes, specific configurations and details are set forth 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 can be practiced without the specific details presented herein. In addition, well-known features may be omitted or simplified so as not to obscure the present disclosure.

[0052] The following definitions and methods are provided to better define the present invention and guide those skilled in the art to practice the present invention.Unless otherwise noted, terms should be understood according to conventional usage by those skilled in the relevant art.

[0053] Where a term is provided in the singular, the inventors also contemplate aspects of the invention described in the plural of that term.

[0054] As used herein, the terms "polynucleotide molecule" and "polynucleotide" are used interchangeably and refer to any polynucleotide comprising 18 or more nucleotides that are covalently bonded in a chain and capable of hybridizing with DNA and RNA molecules under physiological conditions. According to some embodiments, the polynucleotide can 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.

[0055] According to some embodiments, the polynucleotide molecule targets an mRNA of a plant gene. According to some embodiments, the polynucleotide molecule targets a translated region of an mRNA. According to some embodiments, the polynucleotide molecule targets an untranslated region (UTR) of an mRNA.

[0056] As used herein, the terms "DNA," "DNA molecule," and "DNA polynucleotide molecule" refer to single-stranded DNA or double-stranded DNA molecules of genomic or synthetic origin, such as polymers of deoxyribonucleotide bases or DNA polynucleotide molecules.

[0057] As used herein, the terms "DNA sequence," "DNA nucleotide sequence," and "DNA polynucleotide sequence" refer to the nucleotide sequence of a DNA molecule.

[0058] As used herein, the term "gene" refers to any portion of a nucleic acid that provides for transcript expression or encodes a transcript. Thus, a "gene" includes, but is not limited to, a promoter region, a 5' untranslated region, a transcript coding region that may include intronic regions, and a 3' untranslated region.

[0059] As used herein, the terms "RNA," "RNA molecule," and "RNA polynucleotide molecule" refer to single-stranded RNA or double-stranded RNA molecules of genomic or synthetic origin, such as polymers of ribonucleotide bases comprising single-stranded or double-stranded regions or any other structural elements.

[0060] Unless otherwise indicated, the nucleotide sequences in the text of this specification are given in the 5' to 3' direction when reading from left to right. The nomenclature used herein is the nomenclature required by 37 CFR §1.822 and is set forth in the tables of WIPO Standard ST.25 (1998), Appendix 2, Tables 1 and 3.

[0061] As used herein, "plant surface" refers to any external part of a plant. Thus, a plant surface includes, but is not limited to, the surface of a flower, stem, tuber, fruit, anther, pollen, leaf, root, or seed. A plant surface can be on a part of a plant that is connected to other parts of the plant or on a part of a plant that is separate from the plant.

[0062] As used herein, the phrase "a polynucleotide not operably linked to a promoter" refers to 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, such that the polynucleotide will be transcribed by the DNA-dependent RNA polymerase protein or the viral RNA-dependent RNA polymerase. A polynucleotide not operably linked to a promoter can be transcribed by a plant RNA-dependent RNA polymerase.

[0063] As used herein, SEQ ID NOs: 1-364 and SEQ ID NOs: 729-747, although presented in the sequence listing as ssDNA, include dsDNA equivalents, dsRNA equivalents, ssRNA equivalents, ssRNA complementary sequences, ssDNA as indicated, and ssDNA complementary sequences.

[0064] As used herein, the term "transfer agent" may refer to any agent that, when applied to a plant surface, renders the plant receptive to polynucleotides. 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 the polynucleotide molecules to penetrate into plant cells. Chemical agents used 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.

[0065] According to some embodiments, the transfer agent may be selected from N,N-dimethyldecylamide, cocamidopropyl dimethylamine, silicone polyalkylene oxide copolymer, EM-30, dimethylamide of C8 / C10 fatty acids, esterified copolymer of glycerol, trisiloxane ethoxylate, or any combination thereof.

[0066] Non-limiting examples of suitable transfer agents include organosilicon compounds.

[0067] As used herein, the term "organosilicone preparation" refers to a liquid comprising one or more organosilicon compounds, wherein the liquid or components contained therein, when combined with a polynucleotide in a composition that is topically applied to a target plant surface, enhance the entry of the polynucleotide into plant cells. Exemplary organosilicone preparations include, but are not limited to, those sold under the trade names or In certain embodiments, the organosilicon formulation can better enable polynucleotides to enter plant cells in a manner that allows for polynucleotide-mediated inhibition of target gene expression in the plant cells.

[0068] Non-limiting examples of specific suitable transfer agents include L-77 is a modified trisiloxane that combines an extremely low molecular weight trisiloxane with polyether groups. It is characterized by its significant interfacial activity, resulting in significantly reduced water surface tension, excellent spreading or leveling, and stable foam. All of this can be achieved using fractions of organic or fluorocarbon surfactants at typical concentration levels.

[0069] Another non-limiting example of a specific suitable transfer agent includes GENAGEN TM 4166 (Clariant, material number: 10783626892). GENAGEN TM 4166 is based on dimethylamides of naturally derived fatty acids.

[0070] Another non-limiting example of a specific suitable transfer agent includes GL 5: (Clariant, material number: 20072326894). GL5 is a polyglycerol ester based adjuvant that 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 based on dimethylamides of naturally derived fatty acids.

[0071] Another non-limiting example of a specific suitable transfer agent includes GA: (Clariant, material number: 27251626894). GA is a bioenhancer based on salts of a new agrochemical class of alkylglucamides. It is a sugar-based surfactant with a Renewable Carbon Index (RCI) above 95% and therefore has an excellent ecological profile.

[0072] Another non-limiting example of a specific suitable transfer agent includes GENAGEN TM SC 35 (Clariant, material number: 25923226892). GENAGEN TM SC 35 is an alkaline surfactant mixture of sodium alkyl diglycol ether sulfate and coconut fatty acid monoethanolamide.

[0073] Another non-limiting example of a specific 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.

[0074] Another non-limiting example of a specific suitable transfer agent includes SURFECO PLUS TM (Latro). SURFECO PLUS TM Silicone-based adjuvants used to modify the physical properties of agrochemicals and enhance their biological activity.

[0075] Additional suitable transfer agents and their chemical properties are summarized in Table 1 below.

[0076] Table 1 - Transfer Agents

[0077]

[0078]

[0079] 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 to a control plant or plant part that has not been treated with a composition comprising a polynucleotide. As used herein, a control plant is a plant that has not been treated with a polynucleotide and a transfer agent. Such control plants would include, but are not limited to, untreated plants or mock-treated plants.

[0080] Non-limiting examples of traits affected by the compositions disclosed herein include: increased branching, increased seed filling, increased seed number, improved drought resistance, reduced shattering, reduced abscission tissue formation, late / early flowering, and any combination thereof. Each possibility is a separate embodiment.

[0081] Non-limiting examples of rice plant traits affected by the compositions disclosed herein include: increased kernel size, increased panicle number, increased tiller number, increased heading, and any combination thereof. Each possibility is a separate embodiment.

[0082] Non-limiting examples of Brassica napus traits affected by the compositions disclosed herein include: increased kernel size, increased number of ears, increased number of tillers, increased branching, increased seed filling, increased seed number, increased heading, improved drought resistance, reduced shattering, reduced abscission tissue formation, late / early flowering, shortened / extended flowering period, and any combination thereof. Each possibility is a separate embodiment.

[0083] According to some embodiments, the plant can be any cultivated plant, such as, but not limited to, rapeseed, rice, wheat, barley, soybean, peanut, cotton, corn, sorghum, sugarcane, sugar beet, beans, sunflower, potato, sweet potato, alfalfa, banana, apricot, grape, apple, peach, prune, citrus, date palm, palm oil plant, pepper, tomato, broccoli, onion, melon, watermelon, yam, cassava. Each possibility is a separate embodiment.

[0084] 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.

[0085] According to some embodiments, a gene targeted by a polynucleotide molecule may be referred to by a scientific name used in one species (e.g., in Arabidopsis thaliana). However, one of ordinary skill in the art understands that aliases and homologs of another species referred to by another name (alias / homolog) are encompassed by the scientific name. As a non-limiting example, when a gene targeted by a polynucleotide molecule is referred to as BRC1, it includes the aliases / homologs TB1 / FC1.

[0086] According to some embodiments, the target gene can have a nucleotide sequence selected from any nucleotide sequence listed in SEQ ID NO: 1-364. Each possibility is a separate embodiment. According to some embodiments, the target gene can encode an amino acid sequence selected from any amino acid sequence listed in SEQ ID NO: 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 NO: 729-747. Each possibility is a separate embodiment.

[0087] According to some embodiments, the polynucleotide molecule is a dsRNA having a polynucleotide sequence substantially identical to the sequence set forth in any one of SEQ ID NOs: 729-747, or a substantial portion thereof. As used herein, the term "substantial portion thereof" in reference to a dsRNA means that the dsRNA is at least 80% identical to at least 18-20 consecutive base pairs of a sequence set forth in any one of SEQ ID NOs: 729-747, the dsRNA is at least 85% identical to at least 18-20 consecutive base pairs of a sequence set forth in any one of SEQ ID NOs: 729-747, the dsRNA is at least 90% identical to at least 18-20 consecutive base pairs of a sequence set forth in any one of SEQ ID NOs: 729-747, the dsRNA is at least 95% identical to at least 18-20 consecutive base pairs of a sequence set forth in any one of SEQ ID NOs: 729-747, or the dsRNA is at least 98% identical to at least 18-20 consecutive base pairs of a sequence set forth in any one of SEQ ID NOs: 729-747. Each possibility is a separate embodiment.

[0088] As used herein, the term "substantially identical to" with respect to a dsRNA refers to a dsRNA sequence that is at least 80%, at least 90%, at least 95%, or at least 98% homologous to a portion of a nucleotide sequence listed in SEQ ID NOs: 1-364. As used herein, the term "a portion of a listed nucleotide sequence" refers to a portion of a nucleotide sequence targeted by a dsRNA that is substantially the same length 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 approximately 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 approximately 200 bp.

[0089] 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.

[0090] According to some embodiments, the dsRNA targets BnADPG1 (SEQ ID NO:235) of the Brassica napus plant and has the polynucleotide sequence set forth in SEQ ID NO:729.

[0091] According to some embodiments, the dsRNA targets BnBRC1 (SEQ ID NO: 1) of the Brassica napus plant and has the polynucleotide sequence set forth in SEQ ID NO:730.

[0092] According to some embodiments, the dsRNA targets BnCKX2 (SEQ ID NO: 158) of the Brassica napus plant and has the polynucleotide sequence set forth in SEQ ID NO: 731.

[0093] According to some embodiments, the dsRNA targets BnKIN10 (SEQ ID NO:62) of the Brassica napus plant and has the polynucleotide sequence set forth in SEQ ID NO:732.

[0094] 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.

[0095] According to some embodiments, the dsRNA targets GmBRC1 (SEQ ID NO: 15) of the soybean plant and has the polynucleotide sequence set forth in SEQ ID NO: 734 or 735.

[0096] According to some embodiments, the dsRNA targets GmBS1 (SEQ ID NO: 116) of the soybean plant and has the polynucleotide sequence set forth in SEQ ID NO: 736 or 737.

[0097] According to some embodiments, the dsRNA targets GmJAG1 (SEQ ID NO: 153) of the soybean plant and has the polynucleotide sequence set forth in SEQ ID NO: 738 or 739.

[0098] According to some embodiments, the dsRNA targets GmPLDα1 (SEQ ID NO: 124) of the soybean plant and has the polynucleotide sequence set forth in SEQ ID NO: 740 or 741.

[0099] According to some embodiments, the dsRNA targets OsBRC1 (SEQ ID NO:43) of the rice plant and has the polynucleotide sequence set forth in SEQ ID NO:742 or 743.

[0100] According to some embodiments, the dsRNA targets OsPIN5b (SEQ ID NO: 86) of the rice plant and has the polynucleotide sequence set forth in SEQ ID NO: 744 or 745.

[0101] According to some embodiments, the dsRNA targets OsSKIN1 (SEQ ID NO: 52) of the rice plant and has the polynucleotide sequence set forth in SEQ ID NO: 746 or 747.

[0102] According to some embodiments, the compositions and methods for applying the compositions can include timing the application of the compositions to coincide with the desired developmental traits of the plants. As a non-limiting example, the application of a composition comprising a polynucleotide configured to target the gene BS1 (or other genes involved in regulating seed filling) can be timed to coincide with the plant's seed filling stage (R3-R5 developmental (dev.) stage). As another non-limiting example, the application of a composition comprising a polynucleotide configured to target the gene BRC1 (or other genes whose expression is reduced to increase the number of branches or tillers) can be timed to coincide with the bolting stage (R1-R2 developmental stage) of soybean plants or the vegetative stage of axillary bud development in rice plants. As another non-limiting example, the application of a composition comprising a polynucleotide configured to target the gene JAG1 (or other genes that increase seed quantity) can be timed to coincide with the plant's flowering stage (R1-R2 developmental stage). As another non-limiting example, the application of a composition comprising a polynucleotide configured to target the gene SGR1 (or other target genes whose reduction increases resistance to drought) can be timed to coincide with periods of unexpected drought. As another non-limiting example, application of a composition comprising a polynucleotide configured to target the gene AGL1 (or other target gene whose reduction results in reduced breakage) can be timed to when the plant's siliques are ripe. As another non-limiting example, application of a composition comprising a polynucleotide configured to target the gene FT5a (or other target gene whose reduction is involved in flowering regulation) can be timed to when the plant is in the flowering stage. As another non-limiting example, application of a composition comprising a polynucleotide configured to target the gene GNI1 (or other gene that regulates seed size) can be timed to when the plant is in the seed filling stage.

[0103] 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.

[0104] According to some embodiments, two or more polynucleotide sequences may target the same target gene, ie, they may be directed against different parts of the same target gene sequence.

[0105] According to some embodiments, two or more polynucleotide sequences may target different target genes.

[0106] According to some embodiments, different target genes may be involved in the same yield-related trait (e.g., reduced shattering). As a non-limiting example, two or more polynucleotide sequences may target AGL1 and PDH1. As another non-limiting example, two or more polynucleotide sequences may target two or more of JAG1, JAG2, CKX1, and OTU1 (all of which affect seed number).

[0107] According to some embodiments, different target genes can be involved in different yield-related traits (e.g., increased drought resistance and seed filling). As non-limiting examples, a first of the two or more polynucleotide sequences can target ERA1, SGR1, SGR2, ACO2, CER9 or CytG, while a second of the two or more polynucleotide sequences can target BS1, PLD, ACO3 or PDHK.

[0108] As used herein, the phrase "reduction in 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 a transcript or protein in a plant or plant part. In certain embodiments, the decrease in transcript or protein level in a plant or plant part can be determined as compared to a control plant or plant part that has not been treated with a composition comprising a polynucleotide and a transfer agent.

[0109] As used herein, the phrase "wherein the plant does not comprise 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.

[0110] As used herein, the term "transgene" describes a segment of DNA comprising a gene sequence isolated from one organism and introduced into the DNA of a different organism.

[0111] As used herein, the phrases "inhibit expression" or "reduce expression," when used in the context of a gene, refer to any measurable decrease in the amount and / or activity of a product encoded by a gene. Thus, expression of a gene can be inhibited when the level of a transcript from the gene is reduced, the level of a protein encoded by the gene is reduced, the activity of a transcript from the gene is reduced, the activity of a protein encoded by the gene is reduced, any one of the foregoing conditions, or any combination of the foregoing conditions occurs. As used herein, 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. As used herein, the activity of a protein includes, but is not limited to, the ability of a protein to exert any protein-mediated biological or biochemical effect. As used herein, a control plant or plant part is a plant or plant part that has not been treated with a polynucleotide and a transfer agent.

[0112] As used herein, the term "transient" when used in the context of reduction / inhibition of gene expression refers to a time-limited reduction in gene expression that lasts only as long as the polynucleotide permeated into the cell is not degraded, as opposed to long-term expression, which is often referred to as "stable expression."

[0113] As used herein, the term "transcript" corresponds to any RNA produced from a gene by the process of transcription. Thus, a transcript of a gene may comprise a primary transcription product that may contain introns, or may comprise a mature RNA that lacks introns.

[0114] As used herein, the term "homolog" relating to a polynucleotide molecule refers to the degree of sequence identity or similarity (homology) between nucleotide sequences indicating a common ancestor. Two DNA segments may have a common ancestor due to a speciation event (interspecific homologs) or a duplication event (intraspecific homologs). According to some embodiments, a homolog may refer to a polynucleotide having substantially from about 70% to about 99% sequence identity, or more preferably from about 80% to about 99% sequence identity, or most preferably from about 90% to about 99% sequence identity, or from about 95% to about 99% sequence identity to a reference nucleotide sequence of a reference polynucleotide molecule. Each possibility is a separate embodiment.

[0115] 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 "sequence identity" percentage between two sequences is determined by comparing the two best aligned sequences. Compared to a reference sequence, a sequence that is identical at every position is referred to as being identical to the reference sequence, and vice versa. When a first nucleotide sequence observed in a 5' to 3' direction shows complete complementarity with a second or reference sequence observed in a 3' to 5' direction, the first nucleotide sequence is referred to as being the "complement" of the second or reference nucleotide sequence or is complementary thereto. As used herein, a nucleic acid sequence molecule is referred to as showing "complete complementarity" when each nucleotide of a sequence read from 5' to 3' is complementary to each nucleotide of another sequence when read from 3' to 5'. A nucleotide sequence that is complementary to a reference nucleotide sequence will show a sequence identical to the reverse complementary sequence of the reference nucleotide sequence. These terms and descriptions are well defined in the art and are readily understood by those of ordinary skill in the art.

[0116] According to some embodiments, the composition may further comprise a carrier. According to some embodiments, the carrier may be a liquid.

[0117] As used herein, the term "liquid" refers to both homogeneous mixtures, such as solutions, and heterogeneous mixtures, such as suspensions, colloids, micelles, and emulsions. Each possibility is a separate embodiment.

[0118] 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.

[0119] According to some embodiments, the polynucleotide can be naked. As used herein, the term "naked" refers to a polynucleotide that is not encapsulated. However, naked polynucleotides can be modified and / or conjugated.

[0120] According to other embodiments, the polynucleotide may be encapsulated.

[0121] According to some embodiments, the polynucleotide may be delivered via and / or encapsulated in a vehicle such as, but not limited to, nanoparticles, liposomes, micelles, and the like.

[0122] Provided herein are some methods and polynucleotide compositions, which can be applied to living plant cells / tissues to suppress the expression of target genes, and provide this benefit for plants that need to increase yield. Also provided herein are plants and plant parts showing the yield increased, and processed products of such plants or plant parts. Compositions can be surface-applied to the surface of plants, such as being applied to the surface of leaves. Compositions can be applied to various plants, including but not limited to Cruciferae (Brassicaceae) plants, Leguminosae (Fabaceae) plants or Gramineae (Poaceae) plants, such as but not limited to soybean plants, rice plants, Brassica napus plants and / or rapeseed plants. Every possibility is a separate embodiment.

[0123] As used herein, "polynucleotide" refers to a DNA or RNA molecule comprising a plurality of nucleotides, and generally refers to both "oligonucleotides" (polynucleotide molecules of 18-25 nucleotides in length) and longer polynucleotides of 26 or more nucleotides. Embodiments of the present 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 intermediate-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, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109 , 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 nucleotides, between about 400 to about 500 nucleotides, between about 500 to about 600 nucleotides, between about 600 to about 700 nucleotides, between about 700 to about 800 nucleotides, between about 800 to about 900 nucleotides, between about 1000 to about 1100 nucleotides, or about 1200 to about 1300 nucleotides. In some embodiments, the polynucleotide may be 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, e.g., up to the entire length of the target gene, including the coding or non-coding portions, or both, of the target gene. Where the polynucleotide is double-stranded, its length can similarly be described in terms of base pairs.

[0124] The polynucleotide compositions used in various embodiments of the present invention include compositions comprising polynucleotides, and the polynucleotides include: RNA or DNA or RNA / DNA hybrids or chemically modified polynucleotides or artificial polynucleotides or their mixtures. In certain embodiments, polynucleotides can be a combination of ribonucleotides and deoxyribonucleotides, for example, mainly consisting of ribonucleotides but having a synthetic polynucleotide of one or more terminal deoxyribonucleotides, or mainly consisting of deoxyribonucleotides but having a synthetic polynucleotide of one or more terminal dideoxyribonucleotides. In certain embodiments, polynucleotides include non-classical nucleotides, such as inosine, thiouridine or pseudouridine. In certain embodiments, polynucleotides include chemically modified nucleotides. The example of chemically modified oligonucleotides or polynucleotides is well known in the art. Illustrative examples include, but are not limited to, the phosphodiester backbone of naturally occurring polynucleotides, which can be partially or fully modified with phosphorothioate, phosphorodithioate, or methylphosphonate internucleotide linkage modifications, modified nucleobases or modified sugars can be used in polynucleotide synthesis, and polynucleotides can be labeled with fluorescent moieties (e.g., fluorescein or rhodamine) or other labels (e.g., biotin).

[0125] According to some embodiments, dsRNA can be chemically modified on one or both strands to improve stability, extend the half-life of the dsRNA in vivo, increase the biodistribution and pharmacokinetic properties of the dsRNA, target the dsRNA to specific cells, increase target binding affinity and / or improve drug delivery. As a non-limiting example, the dsRNA can be modified to include a methyl group at the 2' position of the ribosyl ring of the second base of the dsRNA. As another non-limiting example, the dsRNA can be modified to include a 3' overhang.

[0126] According to some embodiments, modification can be included in dsRNA. According to some embodiments, modification will not prevent dsRNA composition from being used as a substrate for Dicer. In one embodiment, one or more modifications are made to enhance Dicer's processing of dsRNA. In a second embodiment, one or more modifications are made to cause more effective RNAi generation. In a third embodiment, one or more modifications are made to support a larger RNAi effect. In a fourth embodiment, one or more modifications are made to cause a larger effectiveness of each dsRNA molecule to be delivered to a cell. Modifications can be incorporated into the 3'-terminal region, the 5'-terminal region, the 3'-terminal region, and the 5'-terminal region, or in some cases, modifications can be incorporated into different positions within the sequence. Under the premise of considering the above-mentioned restrictions, any number and combination of modifications can be incorporated into the dsRNA. In the presence of more than one modification, they can be the same or different. Modifications to bases, sugar moieties, phosphate backbones, and combinations thereof are envisioned. Any 5'-end can be phosphorylated.

[0127] It is envisioned that the example of modification for the phosphate backbone includes phosphonate, including methylphosphonate, phosphorothioate and phosphotriester modification, such as alkylphosphotriester etc. It is envisioned that the example of modification for the sugar moiety includes 2'-alkyl pyrimidine, such as 2'-O-methyl, 2'-fluoro, amino and deoxy modification etc. (see, for example, Amarzguioui et al., 2003). It is envisioned that the example of modification for the base group includes pyrimidine, 4-thiouracil, 5-bromouracil, 5-iodouracil and 5-(3-aminoallyl)-uracil, etc., without basic sugar, 2-O-alkyl modification. Locked nucleic acid (or LNA) can also be incorporated. Many other modifications are known, and as long as the above-mentioned criteria are met, it is possible to use.

[0128] 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 modified analogs thereof. In certain embodiments of the present invention, the polynucleotide that provides single-stranded RNA in plant cells 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 comprising a modified Pol III gene that is transcribed into an RNA molecule, (g) a double-stranded DNA molecule (dsDNA), (h) a double-stranded DNA molecule comprising a modified Pol III gene that is transcribed into an RNA molecule, (i) a double-stranded hybridized 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 comprise both ribonucleic acid residues and deoxyribonucleic acid residues. In certain embodiments, these polynucleotides include chemically modified nucleotides or non-classical nucleotides. In certain embodiments of the method, the polynucleotides include 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 polynucleotides are dsRNAs, the antisense strand will include at least 18 nucleotides substantially complementary to the target gene. In certain embodiments, the polynucleotides include single-stranded DNA or single-stranded RNA that form a hairpin structure with at least part of the double-stranded structure through self-hybridization, and the single-stranded DNA or single-stranded RNA include at least one segment that will hybridize with the RNA transcribed from the gene that is suppressed by targeting. Without intending to be bound by any mechanism, it is believed that such polynucleotides are or will produce single-stranded RNAs that have at least one segment that will hybridize with the RNA transcribed from the gene that is suppressed by targeting.

[0129] The polynucleotide molecules of the present invention are designed to modulate expression by inducing regulation or repression of an endogenous target gene in a plant, and are designed to have a nucleotide sequence that is substantially identical or substantially complementary to the nucleotide sequence of an endogenous target gene of a plant or an RNA sequence transcribed from an endogenous target gene of a plant, which can be a coding sequence or a non-coding sequence.

[0130] "Substantially identical" or "substantially complementary" means that the polynucleotide (or at least one strand of a double-stranded polynucleotide) has sufficient identity or complementarity to an endogenous gene or RNA transcribed from an endogenous target gene (e.g., a transcript) to inhibit the expression of the endogenous target gene (e.g., to achieve a decrease in the level or activity of the gene transcript and / or encoded protein).

[0131] The polynucleotides of the methods and compositions provided herein do not need to have 100% identity or complementarity to suppress the expression of endogenous target genes (that is, to achieve the level of gene transcripts or encoded proteins or the reduction of activity). Therefore, in certain embodiments, polynucleotides or a portion thereof are designed to be substantially identical or substantially complementary to the sequence of at least 18 or 19 continuous nucleotides in the messenger RNA (for example, transcript) transcribed from the target gene or from the target gene. In certain embodiments, when compared with the sequence of 18 or more continuous nucleotides in the RNA (for example, transcript) transcribed from the target gene, the polynucleotides of "substantially identical" have 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. In certain embodiments, a polynucleotide that is "substantially complementary" 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.

[0132] In certain embodiments, the polynucleotides used in the methods and compositions provided herein can be substantially identical to or substantially complementary to any of the following: i) a conserved region of a target gene in both monocots and dicots; ii) a conserved region of a target gene in monocots; or iii) a conserved region of a target gene in dicots. Such polynucleotides that are substantially identical to 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 various dicots.

[0133] In some embodiments, the polynucleotides comprising a mispairing of a target gene or a transcript may be used in some embodiments of the compositions and methods provided herein. In certain embodiments, the polynucleotides comprising 19 consecutive nucleotides substantially identical to or essentially complementary to an endogenous target gene or an RNA transcribed from a target gene (e.g., transcript) may have 1 or 2 mispairings with a target gene or a transcript. In certain embodiments, the polynucleotides comprising 20 or more nucleotides of identity or complementarity for 19 consecutive nucleotide spans with an endogenous target gene or an RNA transcribed from a target gene may have 1 or 2 mispairings with a target gene or a transcript. In certain embodiments, the polynucleotides comprising 21 consecutive nucleotides substantially identical to or essentially complementary to an endogenous target gene or an RNA transcribed from a target gene may have 1, 2, or 3 mispairings with a target gene or a transcript. In certain embodiments, the polynucleotides comprising 22 or more nucleotides of identity or complementarity for 21 consecutive nucleotide spans with an endogenous target gene or an RNA transcribed from a target gene may have 1, 2, or 3 mispairings with a target gene or a transcript. When designing polynucleotides with mismatches to an endogenous target gene or RNA transcribed from a target gene, it may be advantageous to use certain types of mismatches and mismatches at certain positions that are more likely to be tolerated.

[0134] According to some embodiments, the target gene can be any target gene listed in SEQ ID NO: 1-364 as well as interspecific homologous target genes available from other crops.

[0135] 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 NOs: 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 substantial portion thereof. Each possibility is a separate embodiment.

[0136] 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 NOs: 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 substantial portion thereof. Each possibility is a separate embodiment.

[0137] 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 set forth in SEQ ID NOs: 1, 2, 9, 49, 62, 79, 80, 104, 149, 150, 158, 235, 252, 275, 276, 286, 299, 311, 332, 333, and 360, or a substantial 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 set forth in SEQ ID NOs: 1, 2, 9, 49, 62, 79, 80, 104, 149, 150, 158, 235, 252, or a substantial portion thereof. Each possibility is a separate embodiment.

[0139] According to some embodiments, the plant may be a Brassica napus plant, and the polynucleotide may have a nucleotide sequence set forth in SEQ ID NOs: 729-733.

[0140] 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 NOs: 15-42, 67-72, 116-148, 152-157, 200-224, 239-245, 291-294, 304-310, 319-325, 342-351 and 362, or any substantial portion thereof. Each possibility is a separate embodiment.

[0141] 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 NOs: 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 substantial portion thereof. Each possibility is a separate embodiment.

[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 set forth in SEQ ID NOs: 15, 16, 67-69, 116-124, 152-155, 200-204, 239, 240, or any substantial 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 protein having any of the amino acid sequences set forth 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 substantial portion thereof. Each possibility is a separate embodiment.

[0144] According to some embodiments, the plant can be a soybean plant, and the polynucleotide can have the nucleotide sequence set forth in SEQ ID NOs: 734-741.

[0145] 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.

[0146] 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 substantial portion thereof. Each possibility is a separate embodiment.

[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 43, 44, 52-57, 73-75, 85, 86, 225-227, 246-248, 259-264, or any substantial 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 protein having any of the amino acid sequences set forth 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 substantial portion thereof. Each possibility is a separate embodiment.

[0149] According to some embodiments, the plant can be a rice plant, and the polynucleotide (ie, dsRNA) can have the nucleotide sequence set forth in SEQ ID NOs: 742-747.

[0150] In certain embodiments, the polynucleotide compositions and methods provided herein typically achieve regulation or modulation (e.g., inhibition) of gene expression over a period of days to weeks or longer of the life of the treated plant and typically in a systemic manner. For example, within a few days of treating a plant leaf with a polynucleotide composition of the present invention, primary siRNAs and transitive siRNAs can be detected in other leaves on the sides and above the treated leaf, as well as in apical tissue. 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 transfer agent, whereby expression of the gene in the plant or its progeny is systemically inhibited 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 a target gene of the plant.

[0151] The composition for inhibiting a target gene may 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, the composition for inhibiting a target gene may comprise one or more polynucleotides that are substantially identical or substantially complementary to more than one continuous segment of a target gene, more than one non-contiguous segment of a target gene, more than one allele of a target gene, or more than one target gene from one or more species.

[0152] 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 series. 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 mode (forming a chain that is at least partially self-complementary). The polynucleotide can comprise both tandem copies and inverted repeat copies. No matter whether arranged in series or inverted repeat mode, each copy can be directly adjacent to the next copy, or the copy pair can be separated by an optional spacer of one or more nucleotides. The optional spacer can be an unrelated sequence.

[0153] Although there is no upper limit to the concentration and dosage of the polynucleotide molecules that can be used in the methods and compositions provided herein, for efficiency, lower effective concentrations and dosages will generally be sought. The concentration can be adjusted based on the volume of the spray or treatment applied to the plant leaves or other plant part surfaces (such as petals, stems, tubers, fruits, anthers, pollen, leaves, roots or seeds).

[0154] For regulating plant so that its agent that is penetrated by polynucleotide or the embodiment of processing comprise emulsion, reverse emulsion, liposome and other micelle-like compositions.For regulating plant so that its agent that is penetrated by polynucleotide or the embodiment of processing comprise known counter ion or other molecules associated with nucleic acid molecules, for example, inorganic ammonium ion, alkylammonium ion, lithium ion, polyamines (such as spermine, spermidine or putrescine) and other cations.For regulating plant so that its organic solvent that is penetrated by polynucleotide comprises DMSO, DMF, pyridine, N-pyrrolidine, hexamethylphosphoramide, acetonitrile, dioxane, polypropylene glycol, miscible with water or other solvents (such as for building-up reactions) that can dissolve nucleotide phosphate in non-aqueous system.Can use the oil or synthetic oil of the natural origin with or without surfactant or emulsifying agent, for example, oil, crop oil of plant origin.

[0155] In certain embodiments, as L-77 surfactant (having CAS No. 27306-78-1 and EPA No.: CAL.REG.NO. 5905-50073-AA and currently available from Momentive Performance Materials, Albany, NY) is a commercially available silicone formulation that can be used to prepare polynucleotide compositions. In certain embodiments of the L-77 silicone formulation as a pre-spray treatment for plant leaves or other plant surfaces, the freshly prepared L-77 silicone formulation is in the range of about 0.015% to about 2% by weight (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 %, 0.09 %, 5wt%, 0.1wt%, 0.2wt%, 0.3wt%, 0.4wt%, 0.5wt%, 0.6wt%, 0.7wt%, 0.8wt%, 0.9wt%, 1.0wt%, 1.1wt%, 1.2wt%, 1.3wt%, 1.4wt%, 1.5wt%, 1.6wt%, 1.7wt%, 1.8wt%, 1.9wt%, 2.0wt%, 2.1wt%, 2.2wt%, 2.3wt%, 2.5wt%) are effective in preparing leaves or other plant surfaces for transfer of polynucleotide molecules from surface application on the surface to plant cells. In certain embodiments of the methods and compositions provided herein, a composition comprising a polynucleotide molecule and comprising a polynucleotide molecule in the range of about 0.015% to about 2% by weight (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 %, 0.085 wt %, 0.090 wt %, 0.100 wt %, 0.115 wt %, 0.125 wt %, 0.135 wt %, 0.140 wt %, 0.150 wt %, 0.160 wt %, 0.175 wt %, 0.180 wt %, 0.190 wt %, 0.200 wt %, 0.210 wt %, 0.220 wt %, 0.230 wt %, 0.240 wt %, 0.250 wt %, 0.260 wt %, 0.270 wt %, 0.280 %, 8wt%, 0.085wt%, 0.09wt%, 0.095wt%, 0.1wt%, 0.2wt%, 0.3wt%, 0.4wt%, 0.5wt%, 0.6wt%, 0.7wt%, 0.8wt%, 0.9wt%, 1.0wt%, 1.1wt%, 1.2wt%, 1.3wt%, 1.4wt%, 1.5wt%, 1.6wt%, 1.7wt%, 1.8wt%, 1.9wt%, 2.0wt%, 2.1wt%, 2.2wt%, 2.3wt%, 2.5wt%) L-77 silicone formulation.

[0156] According to some embodiments, the polynucleotide composition comprising an organosilicon formulation may comprise a salt such as ammonium chloride, tetrabutylphosphonium bromide and / or ammonium sulfate. The 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).

[0157] According to some embodiments, other useful transfer agents or adjuvants of transfer agents that can be used in the polynucleotide compositions provided herein include surfactants and / or the effective molecules contained therein. Surfactants and / or the effective molecules contained therein include, but are not limited to, sodium salts or lithium salts of fatty acids (such as tallow or tallow amine or phospholipids) and organosilicon surfactants. In certain embodiments, the polynucleotide compositions comprising transfer agents are formulated together with known counterions or other molecules associated with nucleic acid molecules. Illustrative examples include tetraalkylammonium ions, trialkylammonium ions, sulfonium ions, lithium ions and polyamines such as spermine, spermidine or putrescine.

[0158] 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 polynucleotide compositions comprising a transfer agent.

[0159] In certain embodiments, the polynucleotide composition also 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, other solvents (such as for building-up reactions) that are miscible with water or can dissolve nucleotide phosphates in non-aqueous systems.

[0160] In certain embodiments, the polynucleotide composition also comprises oil or synthetic oil of natural origin with or without surfactant and / or emulsifying agent.The limiting examples of suitable oil include but not limited to oil, crop oil, paraffin oil, polyol fatty acid ester or the oil with the short-chain molecule of modification with amide or polyamine (such as polyethyleneimine or N-pyrrolidine) of plant origin.

[0161] 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 genes targeted by the polynucleotides disclosed herein can be completely, partially and / or transiently inhibited to result in increased yields.

[0162] The target genes and plants comprising these target genes can be obtained from: i) row 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 cuttings, cloning or grafting methods.

[0163] The compositions comprising polynucleotides and transfer agents provided herein can be applied to plants or plant parts by any convenient method surface, for example, with powder, or sprayed or coated with any liquid composition comprising an emulsion, a suspension or a solution. Such surface-applied spraying or coating can be all or any part of the surface of a plant or plant part. Similarly, in certain embodiments, the compositions comprising transfer agents or other pretreatments can be applied to plants or plant parts by any convenient method (for example, spraying or wiping a solution, an emulsion or a suspension). The compositions comprising polynucleotides and transfer agents provided herein can be applied to plant parts by surface, including but not limited to roots, flowers, stems, tubers, meristems, ovules, fruits, anthers, pollen, leaves or seeds.

[0164] According to some embodiments, the composition may be provided by irrigation, for example using an existing or designated irrigation system.

[0165] The present invention particularly provides the application of a composition comprising a polynucleotide and a transfer agent to seeds. The seeds can be contacted with such a composition by spraying, atomizing, dipping, etc. According to some embodiments, the offspring plants or plant parts derived from the treated seeds will exhibit increased yield due to the inhibition of expression of the target gene.

[0166] The various methods of spraying compositions on plants or plant parts can all be used for applying the composition surface comprising the polynucleotide containing transfer agent to the plant surface. In the field, the composition can be applied with a spray boom that extends above the crop and delivers the composition to the plant surface or with a boomless sprayer that distributes the composition over a wide area. In certain embodiments, agricultural sprayers suitable for directional, dispersing or band spraying can also be used. Sprayers suitable for spraying plant specific parts (including but not limited to leaves, the lower surface of leaves, flowers, stems, male reproductive organs such as spike-shaped male flowers, meristems, pollen, ovules, etc.) can also be used, and the composition can also be delivered aerially, such as by a crop broadcasting aircraft. In certain embodiments, the spray can be delivered using a pressurized backpack sprayer calibrated to deliver the composition at an appropriate rate.

[0167] In certain embodiments, can be before gathering in the crops or after gathering in the crops, spray plant part, to improve the output of plant part.As described above, said composition can be applied to the plant part that is connected to plant by spraying surface.Composition can be applied to the plant part that is separated from plant by spraying as described above or by alternative method surface.Alternative method that is used for composition being applied to the part that is separated includes but not limited to, by conveyer belt or groove, plant part is passed through spraying, or plant part is immersed in composition.

[0168] The composition comprising polynucleotide and transfer agent can be applied to plant or plant part at one or more developmental stages according to expectation and / or as needed.In certain embodiments, the application of composition to seed before germination and / or seedling after germination is provided.Can be processed seed with polynucleotide composition provided herein by the following method: include but not limited to spraying, dipping or providing polynucleotide composition to the coating of seed, seed to any method of imbibition and / or taking in polynucleotide composition.Can use seed batch processing system or continuous flow processing system to process seed with polynucleotide composition.Seed treatment can also be applied in laboratory or commercial scale processing equipment (such as drum (tumbler), mixer, or disc granulator).The polynucleotide composition for processing seed can comprise one or more other required components, include but not limited to liquid diluent, as the adhesive of polynucleotide matrix, for protecting the filler of seed under stress conditions and for improving the toughness, adhesion and / or spreadability plasticizer of coating. Additionally, for oily polynucleotide compositions containing little or no fillers, a desiccant such as calcium carbonate, kaolin or bentonite clay, perlite, diatomaceous earth, or any other adsorbent material may be added.

[0169] In certain embodiments, the use of the composition in the early, middle, and late vegetative stages of plant development is provided. In certain embodiments, the use of the composition in the early, middle, and late reproductive stages is also provided. The use of the composition to plant parts at different stages of maturity is also provided.

[0170] The following examples are included to illustrate examples of certain preferred embodiments of the present invention. It will be appreciated by those skilled in the art that the techniques disclosed in the following examples represent methods that the inventors have found to work well in the practice of the present invention, and therefore can be considered to constitute examples of preferred modes for its practice. However, it will be appreciated by those skilled in the art that, in light of this disclosure, many changes can be made in the specific embodiments disclosed without departing from the spirit and scope of the present invention, and still achieve similar or similar results. Example

[0171] Example 1 - Validation of improved plant yield traits.

[0172] The timing of applying the composition (e.g., by irrigation or spraying) is arranged according to the transcription of the target gene and the desired proterties that it affects. Examples of appropriate timing are summarized in Table 2, which shows selected examples of appropriate timings for applying the composition based on the gene (here, Brassica napus) targeted and its indication. The dsRNA mixture is applied one week before, during, and after the expected peak expression of the specific gene according to the timing of expression of the specific gene. After treatment, the plant phenotype relevant to the yield trait of the target is checked, as for example summarized in Table 2, which shows selected examples of proterties and their associated genes.

[0173] Example 2 - Testing Transfer Agent Permeability

[0174] To test the penetration of the transfer agent, 0.01% to 1% or 0.1-10 mg / ml transfer agent solutions were sprayed on leaves and the contact angles were evaluated using standard methods.

[0175] The efficiency of the tested transfer agents was Figure 1 , which shows the change in contact angle (indicative of penetration) over time after application on plant leaves. As observed, all tested transfer agents significantly reduced the contact angle compared to when no transfer agent was applied.

[0176] Example 3 - Increased branching by targeting BRC1 in Brassica napus.

[0177] A dsRNA molecule (SEQ ID NO: 730) directed against BnBRC1 (SEQ ID NO: 1) of Brassica napus (Brassica napus) was applied using a 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 may also be used, such as those listed in Table 1. BnBRC1 (SEQ ID NO: 730) dsRNA was applied from the start of bolting until the emergence of the second inflorescence.

[0178] After treatment, the plants were evaluated for branching by visual inspection. Figure 3 , which shows illustrative images of Brassica napus plants after surface application of a dsRNA comprising the sequence set forth in SEQ ID NO: 730, showing a significant increase in branching of the plants (5-6 branches per plant in mock-treated control plants (left panel) and 9-10 branches per plant in plants treated with BnBRC1 dsRNA (right panel), wherein the dsRNA comprises the sequence set forth in SEQ ID NO: 730).

[0179] Example 4 - Ectopic application of dsRNA affects petal architecture in Brassica napus grown in the greenhouse and in the field.

[0180] A dsRNA molecule (SEQ ID NO: 733) directed against the Brassica napus gene BnPTL (SEQ ID NO: 286) was applied using a hand sprayer at a dsRNA concentration of 1 ng / ml to 1 mg / ml diluted in a 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 may also be used, such as those listed in Table 1. dsRNA was applied when the plants reached 70% to full stem length.

[0181] After treatment, the number of petals was evaluated by visual inspection. Figure 2 Preliminary illustrative images of Brassica napus plants (here, Brassica napus) after surface application of BnPTL dsRNA are shown. In mock-treated plants (left), normal petal architecture was observed, while BnPTL dsRNA-treated plants (right) displayed abnormal petal architecture (three petals per flower or asymmetric flowers), demonstrating the ability of surface-applied dsRNA to influence petal architecture in Brassica napus plants.

[0182] Example 5 - Ectopic application of dsRNA for yield enhancement - Brassica napus

[0183] Seeds of Brassica napus cv. Belinda (Brassica napus) were sown in 3-liter pots in a controlled greenhouse, with one plant per pot, and watered and fertilized once a day. The plants were treated with a dsRNA molecule (SEQ ID NO: 733) directed against 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) directed against the BnBRC1 gene (SEQ ID NO: 1) (hereinafter referred to as treatment 'B'). The plants were treated with the dsRNA molecule (SEQ ID NO: 730) diluted in a surfactant ( Treatments were performed with dsRNA concentrations of 1 μg / ml and 10 μg / ml in L-77AG), 10 ml per plant.

[0184] Phenotypic evaluations of floral morphology for 'A' (BnPTL dsRNA) and branch number for 'B' (BnBRC1 dsRNA) were performed one month after treatment application and the results were compared with the Ctrl (surfactant only).

[0185] method

[0186] The rapeseed was planted at 2 x 1,000 m in the Sharon region of Israel (32°10′1.55″N 34°52′33.96″E). 2 The fields were sown in 12 rows, each row 52m x 0.8m, with clear lanes 40cm apart between the rows.

[0187] The seeds were sown using a push seeder at a distance of approximately 15 cm between each seed and at a depth of 2 cm.

[0188] Each row was divided into 0.8 m x 2 m plots, with 1 m untreated plants as the interval between adjacent different treatments, with approximately 70 plants per plot.

[0189] Five dsRNAs were tested, namely:

[0190] 1) a dsRNA molecule (SEQ ID NO: 733) directed against the Brassica napus BnPTL gene (SEQ ID NO: 286) - hereinafter referred to as treatment 'A',

[0191] 2) a dsRNA molecule (SEQ ID NO: 730) directed against the Brassica napus BnBRC1 gene (SEQ ID NO: 1)—hereinafter referred to as treatment 'B',

[0192] 3) a dsRNA molecule (SEQ ID NO: 731) directed against the Brassica napus BnCKX2 gene (SEQ ID NO: 158), hereinafter referred to as treatment 'C';

[0193] 4) a dsRNA molecule (SEQ ID NO: 732) directed against the Brassica napus BnKIN10 gene (SEQ ID NO: 62)—hereinafter referred to as treatment 'D';

[0194] 5) A dsRNA molecule (SEQ ID NO: 729) directed against the Brassica napus BnADPG gene (SEQ ID NO: 235) - hereafter referred to as treatment 'E'.

[0195] For each plot, 200 ml of water supplemented with dsRNA and surfactant was used to spray the dsRNA. The dsRNA treatments were applied during the plant growth stage according to the expected peak expression time of each selected gene (as listed in Table 2).

[0196] Table 2: Developmental stages of B. napus plants used for dsRNA application for each gene and their phenotypic evaluation.

[0197]

[0198]

[0199] For each treatment, two dsRNA doses (1 μg / ml or 10 μg / ml) and two spraying schedules (1 or 5 times in field 1 and 1 or 3 times in field 2) were performed as listed in Table 3. The interval between subsequent treatments was one week.

[0200] Table 3 - Field trial parameters.

[0201]

[0202]

[0203] Surfactant only (without dsRNA) at the same time was used as control (ctrl).

[0204] Each treatment was replicated 10 times on different plots and sprayed using a "Solo" 2-liter hand sprayer with minimum droplet size.

[0205] At the end of the growing season, plots were harvested manually, dried for one week, processed through a thresher (Classic ST, Wintersteiger, Germany), and the net seed weight was weighed for each plot / treatment.

[0206] Statistical analysis was performed on all field data to compare each dsRNA treatment with its associated Ctrl (P < 0.1).

[0207] The weight of 1000 seeds was measured using a designated seed counter (Contador, Pfeuffer, Germany) and five replicates per plot were combined to determine the total weight of the fixed volume. The oil content was determined by the "Soxhlet" extraction method using hexane as solvent at the Biotechnology Engineering Faculty, Ben-Gurion University, Be'er Sheva, Israel.

[0208] result

[0209] Process A

[0210] Flower morphology

[0211] For treatment 'A', flower morphology was evaluated three weeks after dsRNA application.

[0212] As from Figure 2Unlike those observed in the dsRNA-treated plants, changes in floral morphology were observed only in the dsRNA-treated plants. Compared to the controls, at least one flower in each inflorescence was missing a petal. Furthermore, inflorescence development and flowering were delayed by approximately two weeks in the dsRNA-treated plants compared to the controls.

[0213] Light penetration percentage :

[0214] Three weeks after spray application of the dsRNA (SEQ ID NO: 733), the percentage of light penetration at the base of the inflorescence was measured. Measurements were taken after peak flowering. For all treatments applied, dsRNA treatment resulted in a significant increase in light penetration compared to the control, i.e., 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) resulted in increases of 42.3%, 45.1%, 47.6%, and 50%, respectively.

[0215] These results indicate that reducing BnPTL expression (SEQ ID NO: 286) by ectopic application of a dsRNA molecule targeting PTL reduces petal number, which in turn leads to increased light penetration to the lower part of the plant and increases overall photosynthetic efficiency and yield.

[0216] Seed weight:

[0217] In field 1, treatments 1-5 and 10-5 increased seed weight by 4.9% and 9.4%, respectively ( Figure 5A In the second field, treatment 10-1 increased seed weight by 17.5% ( Figure 5B ).

[0218] These results indicate that reducing BnPTL expression by ectopic application of dsRNA targeting BnPTL can increase seed weight.

[0219] Oil content:

[0220] In field 1, consistent increases in oil content were observed in dsRNA-treated plants, 1.4%, 1.9%, and 1.1% (treatments 1-1, 1-5, and 10-1, respectively). Figure 6A ).

[0221] In the second field, greater increases were observed, 6%, 2.4%, 5.9% and 2.3% (treatments 1-1, 1-3, 10-1 and 10-3, respectively). Figure 6B ).

[0222] These results indicate that reducing BnPTL expression by ectopically applying dsRNA targeting BnPTL can increase oil content in Brassica napus plants.

[0223] Treatment B

[0224] Number of branches:

[0225] For treatment 'B', the number of branches was evaluated three weeks after dsRNA application.

[0226] As from Figure 3 It was observed that the number of branches increased as a result of dsRNA treatment (10 μg / ml), and as seen from Figure 4 It was further observed that the increase in branch number was dose-dependent (P<0.1).

[0227] As from Figure 7 Observed field results showed that targeting the expression of BnBRC1 (SEQ ID NO: 1) by ectopic application of dsRNA (SEQ ID NO: 730 targeting BnBRC1) resulted in an increase in the number of branches by 8.2%, 5.9% and 16.4% for treatments 1-1, 1-5 and 10-5, respectively, compared to the control.

[0228] Process C

[0229] Seed weight:

[0230] As from Figure 8A and Figure 8B It was observed that targeting the expression of BnCKX2 (SEQ ID NO: 158) by ectopic application of a dsRNA targeting CKX2 (SEQ ID NO: 731) resulted in an increase in seed weight in both fields tested. In field 1, the increases were 2.1%, 1.3%, 1.3%, and 4% for treatments 1-1, 1-5, 10-1, and 10-5, respectively ( Figure 8A In the second field, treatments 1-1, 10-1, and 10-3 increased seed weight by 1.2%, 15.6%, and 9%, respectively ( Figure 8B ).

[0231] 1000 seed weight and seed size:

[0232] Furthermore, targeting BnCKX2 expression by ectopically applying dsRNA targeting BnCKX2 resulted in increases in 1,000 seed weight by 3.9%, 3.5%, and 1.6% for treatments 1-1, 1-5, and 10-1 in Field 1 relative to the Ctrl. Seed size also increased by 1.1%, 5.1%, and 1.2% in treatments 1-1, 1-5, and 10-5, respectively.

[0233] In field 2, targeting BnCKX2 resulted in a 5.8%, 1.5%, and 1.1% increase in 1,000-seed weight for treatments 1-3, 10-1, and 10-3, respectively. Seed size also increased by 4.1%, 0.7%, and 3.8% in treatments 1-3, 10-1, and 10-3, respectively.

[0234] These results clearly demonstrate that targeting the expression of BnCKX2 by ectopically applying dsRNA targeting BnCKX2 increases the weight and size of B. napus seeds.

[0235] Processing E

[0236] Seed weight:

[0237] As in Figure 9A and Figure 9B As observed in the results of the present study, targeting the expression of BnADPG1 (SEQ ID NO: 235) by ectopic application of a dsRNA targeting BnADPG1 (SEQ ID NO: 729) resulted in increased seed weight in both fields tested. In field 1, treatments 1-1, 1-5, and 10-1 increased seed weight by 9.5%, 9.4%, and 12%, respectively. In field 2, treatments 1-3, 10-1, and 10-3 increased seed weight by 11.4%, 14.2%, and 10.1%, respectively.

[0238] 1000 seed weight and seed size:

[0239] Targeting ADPG1 expression by ectopically applying dsRNA targeting BnADPG1 resulted in increases in 1,000 seed weight by 4.5%, 7.7%, 6%, and 7% for treatments 1-1, 1-5, 10-1, and 10-5 in Field 1 relative to the Ctrl. Seed size also increased by 6.8%, 8.2%, 1.1%, and 8.1% in treatments 1-1, 1-5, 10-1, and 10-5, respectively.

[0240] In field 2, targeting BnADPG1 increased the weight of 1,000 seeds by 14.7%, 3%, and 13.3% for treatments 1-3, 10-1, and 10-3, respectively. Seed size also increased by 2%, 13.3%, 2.2%, and 9.6% in treatments 1-1, 1-3, 10-1, and 10-3, respectively.

[0241] These results clearly demonstrate that targeting the expression of BnADPG1 by ectopically applying dsRNA targeting BnADPG can increase the weight and size of B. napus seeds.

[0242] Oil content:

[0243] In field 1, 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 field 2, 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 ).

[0244] Example 6 - Ectopic Application of dsRNA for Yield Increase - Rice (Oryza sativa)

[0245] Materials and methods:

[0246] Rice (Oryza sativa spp.) seeds were sown in 3-liter pots in a greenhouse, one seed per pot, and watered and fertilized once a day.

[0247] Plants were treated with 10 μg / ml (in water and surfactant) of the dsRNAs listed below:

[0248] 1) a dsRNA molecule (SEQ ID NO: 742) targeting OsBRC1 (SEQ ID NO: 43) of rice,

[0249] 2) a dsRNA molecule (SEQ ID NO: 744) targeting OsPIN5b (SEQ ID NO: 86) of rice,

[0250] 3) a dsRNA molecule (SEQ ID NO: 746) targeting rice OsSKIN1 (SEQ ID NO: 52),

[0251] dsRNA was applied according to the plant development stage, as listed in Table 4 below.

[0252] Table 4: Rice (O. sativa) plant developmental stages used for specific dsRNA treatments.

[0253] dsRNA Developmental stage treated SEQ ID NO:742 Axillary buds begin to appear SEQ ID NO:744 Plants at maximum tillering number SEQ ID NO:746 Plants in the seed filling stage

[0254] dsRNA was diluted in a surfactant ( L-77AG) was applied as a single spray at a concentration of 1 μg / ml or 10 μg / ml, 10 ml per plant, with 6 pots / 6 plants per treatment.

[0255] Treatments were evaluated 1 month after dsRNA application 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. Total seed weight was measured for all treatments.

[0256] result

[0257] One month after treatment, the total number of tillers in plants treated with dsRNA targeting OsBRC1 of rice plants (SEQ ID NO: 742) was counted and compared to the control. Interestingly, treatment of plants with low concentrations of OsBRC1-dsRNA resulted in a slight increase in the number of tillers, whereas high concentrations resulted in a decrease in the number of tillers ( Figure 11 ). This indicates that dsRNA targeting OsBRC1 can be used to control tiller number in rice.

[0258] The number of panicles of rice plants treated with dsRNA targeting OsPIN5b (SEQ ID NO: 744 and SEQ ID NO: 745) was evaluated 3 months after treatment compared to controls.

[0259] Total seed weights were measured for all treatments 3 months after treatment.

[0260] Example 7 - Ectopic Application of dsRNA for Yield Enhancement - Soybean (Glycinemax)

[0261] Materials and methods:

[0262] Soybean (Glycine max, Williams 82 variety) seeds were sown in 3-liter pots in a greenhouse, one seed per pot, and watered and fertilized once a day, or as needed.

[0263] Several examples of dsRNA applications aimed at inducing phenotypic changes and increasing production were tested (as specified below):

[0264] 1) SEQ ID NO: 734 for GmBRC1 (SEQ ID NO: 15) of soybean.

[0265] 2) SEQ ID NO: 738 for GmJAG1 (SEQ ID NO: 153) of soybean.

[0266] 3) SEQ ID NO: 736 for GmBS1 (SEQ ID NO: 116) of soybean.

[0267] 4) SEQ ID NO: 740 for GmPLDα1 (SEQ ID NO: 124) from soybean.

[0268] dsRNA was applied according to the plant development stage, as listed in Table 5 below.

[0269] Table 5: Soybean (G. max) plant developmental stages used for specific dsRNA treatments.

[0270]

[0271]

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

[0273] Treatments were evaluated one month after dsRNA application and compared to the control. For GmBRC1 targeting, the number of axillary branches was evaluated; for GmJAG1 targeting, the number of seeds per pod was evaluated; for GmBS1 targeting, seed size was evaluated; and for GmPLDα1, seed weight / grain filling was evaluated. In addition, total seed weight was measured for all treatments.

[0274] result

[0275] One month after treatment, the total number of axillary branches of plants treated with dsRNA targeting GmBRC1 was evaluated. Figure 12 As observed in Figure 3, dsRNA treated plants 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 plants were evaluated for the total number of seeds per pod.

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

[0278] The present invention also provides the following items:

[0279] 1. A composition comprising:

[0280] a dsRNA molecule comprising at least 18 consecutive nucleotides that are substantially identical or substantially complementary to a plant gene or a portion of a transcript of the plant gene; and

[0281] a transfer agent configured to facilitate penetration of the dsRNA molecule into cells of the plant;

[0282] wherein penetration of the dsRNA molecule into cells of the plant causes a transient decrease in expression of the gene, and

[0283] wherein the transient reduction in gene expression causes an alteration in a trait of the plant selected from the group consisting of increased branching, increased grain filling, increased T6P levels, increased ear number, increased seed filling, increased seed number, increased seed size, reduced shattering, reduced abscission tissue formation, increased tiller number, increased heading of the plant, reduced petals, increased silique size, late flowering or early flowering, delayed senescence, and any combination thereof.

[0284] 2. The composition according to item 1, wherein the plant gene is selected from ADPG1, PTL, CKX2, BRC1, KIN10, SKIN1, PIN5b, JAG1, BS1, PLDα1 and / or any homologs or combinations thereof.

[0285] 3. The composition according to item 1, wherein the plant gene is selected from ADPG1, PTL, CKX2, BRC1 and / or any homologs or combinations thereof.

[0286] 4. The composition according to item 1, wherein the plant is a Brassica napus plant, and wherein the dsRNA molecule comprises at least 18 consecutive nucleotides that are substantially identical or substantially complementary to a portion of the following sequence, the sequence encoding any one of the amino acid sequences set forth in SEQ ID NO: 599, SEQ ID NO: 650, SEQ ID NO: 522, and SEQ ID NO: 365.

[0287] 5. The composition according to item 1, wherein the plant is a soybean plant, and wherein the dsRNA molecule comprises at least 18 consecutive nucleotides that are substantially identical or substantially complementary to a portion of the following sequence, the sequence encoding any one of the amino acid sequences set forth 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.

[0288] 6. The composition according to item 5, wherein the plant is a soybean plant, and wherein the dsRNA molecule comprises at least 18 consecutive nucleotides that are substantially identical or substantially complementary to a portion of the following sequence, the 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.

[0289] 7. The composition according to item 1, wherein the plant is a rice plant, and wherein the dsRNA molecule comprises at least 18 consecutive nucleotides that are substantially identical or substantially complementary to a portion of the following sequence, wherein the sequence encodes 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.

[0290] 8. The composition according to item 7, wherein the plant is a rice plant, and wherein the dsRNA molecule comprises at least 18 consecutive nucleotides that are substantially identical or substantially complementary to a portion of the following sequence, the sequence encoding any one of the amino acid sequences set forth in SEQ ID NO: 407, SEQ ID NO: 416, and SEQ ID NO: 450.

[0291] 9. The composition of any one of items 1-8, wherein the dsRNA molecule is at least about 50 bases in length.

[0292] 10. The composition of item 9, wherein the dsRNA molecule is at least about 200 bases in length.

[0293] 11. The composition according to any one of items 1 to 10, wherein the transfer agent comprises N,N-dimethyldecylamide, cocamidopropyl dimethylamine, silicone polyalkylene oxide copolymer, EM-30, dimethylamide of C8 / C10 fatty acids, esterified copolymer of glycerol, trisiloxane ethoxylate, or any combination thereof.

[0294] 12. A method for topically applying the composition of any one of items 1 to 11 to a plant surface.

[0295] 13. The method according to item 12, wherein the applying comprises spraying the composition onto the surface of the plant.

[0296] 14. The method of item 13, wherein the composition is sprayed onto the plant surface using a boom extended above the crop, a boomless sprayer, an agricultural sprayer, a crop spreading aircraft, a pressurized knapsack sprayer, a track sprayer, or a laboratory sprayer / immersion device.

[0297] 15. The method according to item 12, wherein said applying comprises providing said composition via an irrigation system.

[0298] 16. The method according to any one of items 12 to 15, wherein 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.

[0299] 17. The method according to any one of items 12 to 16, further comprising timing the application of the composition to a desired developmental stage of the plant.

[0300] Although certain embodiments of the present invention have been illustrated and described, it should be understood that the present invention is not limited to the embodiments described herein. Many modifications, changes, variations, substitutions and equivalents will be apparent to those skilled in the art without departing from the spirit and scope of the invention as described in the appended claims.

Claims

1. A composition comprising: a dsRNA molecule comprising at least 18 consecutive nucleotides that are substantially identical or substantially complementary to a plant gene or a portion of a transcript of the plant gene; and a transfer agent configured to facilitate penetration of the dsRNA molecule into cells of the plant; wherein penetration of the dsRNA molecule into cells of the plant causes a transient decrease in expression of the gene, and wherein the transient reduction in gene expression causes an alteration in a trait of the plant selected from the group consisting of increased branching, increased grain filling, increased T6P levels, increased ear number, increased seed filling, increased seed number, increased seed size, reduced shattering, reduced abscission tissue formation, increased tiller number, increased heading of the plant, reduced petals, increased silique size, late flowering or early flowering, delayed senescence, and any combination thereof.

2. The composition according to claim 1, wherein the plant gene is selected from ADPG1, PTL, CKX2, BRCI, KIN10, SKIN1, PIN5b, JAG1, BS1, PLDα1 and / or any homologs or combinations thereof.

3. The composition of claim 1, wherein the plant gene is selected from ADPG1, PTL, CKX2, BRC1 and / or any homologs or combinations thereof.

4. The composition of claim 1 , wherein the plant is a Brassica napus plant, and wherein the dsRNA molecule comprises at least 18 contiguous nucleotides that are substantially identical or substantially complementary to a portion of a sequence encoding any one of the amino acid sequences set forth in SEQ ID NO: 599, SEQ ID NO: 650, SEQ ID NO: 522, and SEQ ID NO:

365.

5. The composition of claim 1 , wherein the plant is a soybean plant, and wherein the dsRNA molecule comprises at least 18 contiguous nucleotides that are substantially identical or substantially complementary to a portion of a sequence encoding any one of the amino acid sequences set forth 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.

6. The composition of claim 5, wherein the plant is a soybean plant, and wherein 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 set forth in SEQ ID NO: 379, SEQ ID NO: 517, SEQ ID NO: 480, and SEQ ID NO:

488.

7. The composition of claim 1 , wherein the plant is a rice plant, and wherein 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 set forth 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.

8. The composition of claim 7, wherein the plant is a rice plant, and wherein 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 set forth in SEQ ID NO: 407, SEQ ID NO: 416, and SEQ ID NO:

450.

9. The composition of any one of claims 1-8, wherein the dsRNA molecule is at least about 50 bases in length.

10. The composition of claim 9, wherein the dsRNA molecule is at least about 200 bases in length.

11. The composition according to any one of claims 1 to 10, wherein the transfer agent comprises N,N-dimethyldecylamide, cocamidopropyl dimethylamine, silicone polyalkylene oxide copolymer, EM-30, dimethylamide of C8 / C10 fatty acids, esterified copolymer of glycerol, trisiloxane ethoxylate, or any combination thereof.

12. A method for topically applying the composition of any one of claims 1 to 11 to a plant surface.

13. The method of claim 12, wherein applying comprises spraying the composition onto a plant surface.

14. The method of claim 13, wherein the composition is sprayed onto the plant surface using a boom extended above the crop, a boomless sprayer, an agricultural sprayer, a crop spreading aircraft, a pressurized knapsack sprayer, a track sprayer, or a laboratory sprayer / immerser.

15. The method of claim 12, wherein said applying comprises providing said composition through an irrigation system.

16. The method of any one of claims 12-15, wherein 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.

17. The method of any one of claims 12-16, further comprising timing the application of the composition to a desired developmental stage of the plant.

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