Topical application of polynucleotide molecules to improve yield traits in plants - Patent Application 20070122967

Abstract

A composition comprising: (i) a dsRNA molecule of at least 18 consecutive nucleotides that is essentially identical to or essentially complementary to a plant gene or a transcript of the plant gene; and (ii) an introduction agent that conditions the surface of a plant for penetration of cells of the plant by the dsRNA molecule, wherein penetration of cells of the plant with the dsRNA molecule causes a transient decrease in expression of the gene, and wherein the transient decrease in expression of the gene causes a change in a yield-related trait of the plant.

Description

[Technical Field]

[0001] According to the United Nations Food and Agricultural Organization (UN FAO), the world's population is expected to exceed 9.6 billion by 2050, necessitating significant improvements in agriculture to meet the growing demand for food. At the same time, preserving supply sources, reducing the use of fertilizers, pesticides, and herbicides, and environmental sustainability are becoming increasingly important factors in how food is grown. Improved agricultural plants and farming techniques are needed that enable increased plant production using fewer resources and more environmentally sustainable inputs. [Background technology]

[0002] Yield is affected by a variety of factors, such as the number and size of plant organs, plant architecture (e.g., number of branches), seed filling, seed number, drought tolerance, shattering, flowering, and number of tillers.

[0003] Today, crop performance is primarily optimized through techniques targeting the interactions between the crop genotype (e.g., plant breeding, genetically modified (GM) crops) and its surrounding environment (e.g., fertilizers, synthetic herbicides, and pesticides). While these paradigms have helped global food production double over the past 50 years, yield growth rates have stalled for many major crops, driving an urgent need for novel solutions to crop yield improvement. Public fear of GM crops and synthetic chemistries, coupled with their long development and regulatory history, has thwarted their use in many important crops and countries, resulting in the failure of many GM traits and the exclusion of GM crops and many synthetic chemistries from several global markets. Therefore, innovative, effective, environmentally sustainable, and publicly acceptable approaches to improving yields are clearly needed. Summary of the Invention

[0004] Provided herein are compositions and methods for providing increased yield in plants by topically applying to a plant a composition comprising a polynucleotide molecule capable of hybridizing to a yield-associated gene or gene transcript and an introducing agent that conditions the surface of the plant for penetration of the plant cells by the polynucleotide molecule, thereby suppressing expression of a yield-associated gene in the plant and thereby improving yield-associated traits in the plant. Non-limiting examples of yield-associated traits in plants include increased branching in the plant, grain size, increased number of panicles, increased number of tillers, increased seed production, increased silique size in the plant, increased seed filling, increased seed number, increased heading, improved drought tolerance, reduced grain shattering, reduced abscission tissue formation, reduced petals in the plant, delayed / early flowering, shortened / longer flowering period, delayed senescence, increased oil content, oil composition, starch content, starch composition, carbohydrate content, improved carbohydrate composition, increased protein content, improved protein composition, and any combination thereof. Each possibility is a separate embodiment.

[0005] According to some embodiments, infiltration of polynucleotide molecules may cause a transient reduction in gene expression, may cause non-permanent spatial and temporal effects in the plant, and does not result in or require the integration of exogenous polynucleotides into the plant's chromosomes. This approach has several advantages. First, it avoids the need for GMO methods. Furthermore, it is a more sophisticated and efficient approach because it is dynamic and allows for user-driven application depending on real-time needs, the timing of trait improvement, and / or the environmental conditions that need to be addressed. As a non-limiting example, during drought, a farmer may decide to transiently inhibit expression of a gene / transcript to improve water stress resilience, and then discontinue the inhibition when the weather changes. As another non-limiting example, a farmer may decide to transiently inhibit a gene / transcript associated with early flowering in plants in cases of unexpected rain, temperature changes, etc.

[0006] Advantageously, this technology is applicable for use in a variety of crops, including but not limited to corn, rice, soybean, cotton, canola, rapeseed, tomato, potato, etc., and especially crops with complex genomes such as wheat, strawberries, or fruit trees.

[0007] In some embodiments, the polynucleotide molecule is provided in a composition that can be penetrated or absorbed into living plant tissue to initiate systemic gene inhibition or regulation. In certain embodiments of the invention, the polynucleotide molecule ultimately provides the plant with an RNA (e.g., dsRNA) or RNA-like molecule that, under physiological conditions in the plant cell, can hybridize to RNA transcribed from a target endogenous gene in the plant cell, thereby affecting (silencing or suppressing) expression of the target gene.

[0008] According to some embodiments, silencing / suppressing a target gene may directly improve a yield-related trait in a plant. Alternatively, silencing / suppressing a target gene may indirectly improve a yield-related trait in a plant. For example, silencing or suppressing a target gene may alter (increase or decrease) the expression of another gene that improves a yield-related trait in a plant.

[0009] As a further important practical advantage, topical application of the composition comprising an exogenous polynucleotide and an introduction agent does not require that the exogenous polynucleotide be physically bound to a particle, as is the case with biolistic introduction of polynucleotides associated with gold or tungsten particles into the interior of a plant, plant part, or plant cell.

[0010] In some embodiments, the polynucleotide molecule targets the mRNA of a plant gene. In some embodiments, the polynucleotide molecule targets a translated region of the mRNA. In some embodiments, the polynucleotide molecule targets an untranslated region of the mRNA.

[0011] According to some embodiments, a composition is provided that includes: (i) a polynucleotide molecule comprising at least 18 contiguous nucleotides that are essentially identical to or essentially complementary to a plant gene or a transcript of the plant gene; and (ii) an introduction agent that conditions a surface of a plant for penetration of cells of the plant with the polynucleotide molecule, wherein penetration of cells of the plant with the polynucleotide molecule causes a transient decrease in expression of a gene, and wherein the transient decrease in expression of the gene causes a change in a yield-related trait of the plant.

[0012] According to some embodiments, the yield-related trait of the plant is selected from the group consisting of increased grain / seed size, increased grain number, increased panicle / silique number, increased tiller number, increased branching, increased seed size, increased seed filling, increased seed number, increased heading, improved drought tolerance, reduced grain shattering, reduced abscission tissue formation, delayed flowering, earlier flowering, increased grain shattering, increased abscission tissue formation, reduced petals on the plant, increased protein content of the plant, increased carbohydrate content of the plant, increased oil content of the plant, improved oil composition, starch content, starch composition, carbohydrate content, carbohydrate composition of the plant, and any combination thereof. Each possibility is a separate embodiment.

[0013] According to some embodiments, there is provided a composition suitable for topical application to a plant, the composition comprising: a dsRNA molecule comprising at least 18 contiguous nucleotides that are essentially identical to or essentially complementary to a plant gene or a portion of a transcript of the plant gene; and a delivery 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 reduction in expression of the gene.

[0014] The transient reduction in expression of the gene causes a change in the trait of the plant.

[0015] The plant trait is selected from the group consisting of increased branching, increased grain filling, increased trehalose-6-phosphate (T6P) levels, increased panicle number, increased seed filling, increased seed number, increased seed size, reduced seed shattering, reduced abscission tissue formation, increased number of tillers, increased heading on the plant, reduced petals, increased silique size, delayed or early flowering, delayed senescence, and any combination thereof, or increased branching, increased grain filling, increased panicle number, increased seed filling, increased seed number, increased seed size, reduced shedding, reduced abscission tissue formation, increased number of tillers, increased heading on the plant, reduced petals, increased silique size, delayed or early flowering, delayed senescence, and any combination thereof. increased kernel filling, increased seed number, increased seed size, reduced grain shattering, reduced abscission tissue formation, increased number of tillers, increased heading on the plant, reduced petals, increased silique size, and any combination thereof; or selected from the group consisting of increased branching, increased kernel filling, increased number of panicles, increased seed filling, increased seed number, reduced grain shattering, reduced abscission tissue formation, increased number of tillers, reduced petals, increased silique size, and any combination thereof. Each possibility is a separate embodiment.

[0016] 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 any combination thereof. Each possibility is a separate embodiment. According to some embodiments, the plant gene is ,before The plant gene is selected from ADPG1, PTL, CKX2, BRC1, and / or any homologue thereof, or any combination thereof. Each possibility is a separate embodiment.

[0017] According to some embodiments, the plant is a canola plant and the dsRNA molecule is , distributionA dsRNA molecule comprising at least 18 contiguous nucleotides that are essentially identical to or essentially complementary to a portion of a sequence encoding any 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.

[0018] According to some embodiments, the plant is a soybean plant, and the dsRNA molecule comprises at least 18 contiguous nucleotides that are essentially identical to or essentially complementary to a portion of a sequence encoding any 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.

[0019] According to some embodiments, the plant is a soybean plant, and the dsRNA molecule comprises at least 18 contiguous nucleotides that are essentially identical to or essentially complementary to a portion of a sequence encoding any of the amino acid sequences set forth in SEQ ID NOs: 379, 517, 480, and 488. 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 NOs: 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 set forth in SEQ ID NOs: 734-741. Each possibility is a separate embodiment.

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

[0021] According to some embodiments, the plant is a rice plant, and the dsRNA molecule comprises at least 18 contiguous nucleotides that are essentially identical to or essentially complementary to a portion of a sequence encoding any of the amino acid sequences set forth in SEQ ID NOs: 407, 416, and 450. 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 NOs: 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 set forth in SEQ ID NOs: 742-747. Each possibility is a separate embodiment.

[0022] 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.

[0023] According to some embodiments, the transfer agent is N , N-dimethyldecanamide, coco amidopropyldimethyamine, siloxane polyalkylene oxide copolymer, AG-RHO® EM-30, dimethylamides of C8 / C10 fatty acids, esterified copolymers of glycerin, trisiloxane ethoxylate, or any combination thereof. Each possibility is a separate embodiment.

[0024] According to some embodiments, the transfer agent may be any of the transfer agents shown in Table 1.

[0025] According to some embodiments, there is provided a method for topically applying a composition essentially as disclosed herein to a plant surface.

[0026] According to some embodiments, the applying comprises spraying the composition onto the plant surface. According to some embodiments, the composition is sprayed onto the plant surface using a boom that extends over the crop, a boomless sprayer, an agricultural sprayer, a crop duster, a pressurized backpack sprayer, a track sprayer, or a laboratory sprayer / submerger. Each possibility is a separate embodiment.

[0027] According to some embodiments, the applying comprises providing the composition through an irrigation system.

[0028] According to some embodiments, the plant surface is a surface of one or more plant parts selected from the group consisting of hypocotyls, cotyledons, leaves, flowers, stems, tassels, meristems, pollen, ovules, and fruits, each possibility being a separate embodiment.

[0029] According to some embodiments, the method further comprises timing application of the composition at a desired developmental stage of the plant essentially as described herein, e.g., for the examples in Table 2.

[0030] Particular embodiments of the present disclosure may include some, all, or none of the above advantages. One or more technical advantages may be readily apparent to those skilled in the art from the drawings, descriptions, and claims contained herein. Furthermore, although specific advantages have been enumerated above, various embodiments may include all, some, or none of the enumerated advantages.

[0031] 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 descriptions.

[0032] In order that the present invention may be more fully understood, it will now be described in connection with specific examples and embodiments with reference to the following illustrative drawings. [Brief explanation of the drawings]

[0033] [Figure 1] Contact angles (indicating penetration) are shown as a function of time after application of the targeting agent mixture to the leaves of oilseed rape plants. [Figure 2] 1 shows illustrative photographs of flower morphology of canola plants, here Brassica napus plants, ectopically sprayed with 10 μg / ml of dsRNA set forth in SEQ ID NO: 733 targeting the sequence set forth in SEQ ID NO: 286. Control-treated plants (left) have normal petal morphology. BnPTL-dsRNA-treated plants (right) produce flowers with altered petal morphology. [Figure 3] 1 is an illustrative photograph of canola plants, here Brassica napus plants, ectopically sprayed with 10 μg / ml of dsRNA set forth in SEQ ID NO: 730 targeting the BnBRC1 sequence set forth in SEQ ID NO: 1, versus control plants (Ctrl). The total number of branches in each group is shown. [Figure 4] Shown is the average number of branches per canola plant, here Brassica napus, ectopically sprayed with 1 μg / ml or 10 μg / ml of dsRNA set forth in SEQ ID NO: 730 targeting the BnBRC1 sequence set forth in SEQ ID NO: 1, or per plant sprayed with surfactant solution alone. * indicates a significant change in treatment compared to control plants (Ctrl) (P<0.1). [Figure 5A]Figure 1 shows the average seed weight per 0.8 m2 of rapeseed plants, here Brassica napus plants, sprayed with 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 in treatment compared to control plants (Ctrl) (P<0.1). [Figure 5B] Figure 1 shows the average seed weight per 0.8 m2 of rapeseed plants, here Brassica napus plants, sprayed with 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 in treatment compared to control plants (Ctrl) (P<0.1). [Figure 6A] 1 shows the average percentage oil content of rapeseed plants, here Brassica napus plants, sprayed with 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 in treatment compared to control plants (Ctrl) (P<0.1). [Figure 6B] 1 shows the average oil content percentage of rapeseed plants, here Brassica napus plants, sprayed with 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 in treatment compared to control plants (Ctrl) (P<0.1). [Figure 7] Figure 1 shows the average number of branches per canola plant (Brassica napus plant) ectopically sprayed with 1 μg / ml or 10 μg / ml of dsRNA set forth in SEQ ID NO: 730 targeting the sequence set forth in SEQ ID NO: 1, or per plant sprayed with surfactant solution alone. * indicates a significant change in treatment compared to control plants (Ctrl) (P<0.1). [Figure 8A]Figure 1 shows the average seed weight per 0.8 m2 of rapeseed plants (Brassica napus plants) sprayed with dsRNA set forth in SEQ ID NO: 731 targeting the sequence set forth in SEQ ID NO: 158, obtained for Field 1. * indicates a significant change in treatment compared to control plants (Ctrl) (P<0.1). [Figure 8B] Figure 1 shows the average seed weight per 0.8 m2 of rapeseed plants (Brassica napus plants) sprayed with dsRNA set forth in SEQ ID NO: 731 targeting the sequence set forth in SEQ ID NO: 158, obtained for field 2. * indicates a significant change in treatment compared to control plants (Ctrl) (P<0.1). [Figure 9A] Figure 1 shows the average seed weight per 0.8 m2 of rapeseed plants (Brassica napus plants) sprayed with 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 in treatment compared to control plants (Ctrl) (P<0.1). [Figure 9B] Figure 1 shows the average seed weight per 0.8 m2 of rapeseed plants (Brassica napus plants) sprayed with 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 in treatment compared to control plants (Ctrl) (P<0.1). [Figure 10A] 1 shows the average percentage oil content of rapeseed plants (Brassica napus plants) sprayed with 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 in treatment compared to control plants (Ctrl) (P<0.1). [Figure 10B]2 shows the average percentage oil content of rapeseed plants (Brassica napus plants) sprayed with 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 in treatment compared to control plants (Ctrl) (P<0.1). [Figure 11] Figure 1 shows the average number of tillers per rice plant (Oryza sativa) treated with 1 μg / ml or 10 μg / ml of dsRNA set forth in SEQ ID NO: 742 targeting the sequence set forth in SEQ ID NO: 43, or per plant sprayed with surfactant solution alone. * indicates a significant change in treatment compared to control plants (Ctrl) (P<0.1). [Figure 12] 1 shows the average number of branches of soybean plants (Glycine max) treated with 1 μg / ml or 10 μg / ml of dsRNA set forth in SEQ ID NO: 734 targeting the sequence set forth in SEQ ID NO: 15, or of plants sprayed with surfactant solution alone. * indicates a significant change in treatment compared to control plants (Ctrl) (P<0.1). DETAILED DESCRIPTION OF THE INVENTION

[0034] In the following description, various aspects of the disclosure are described. For purposes of explanation, specific configurations and details are set forth in order to provide a thorough understanding of the different aspects of the disclosure. However, it will also be apparent to those skilled in the art that the present disclosure may be practiced without the specific details presented herein. Furthermore, well-known features may be omitted or simplified so as not to obscure the disclosure.

[0035] The following definitions and methods are provided to better define the present invention and to guide those of ordinary skill in the art in the practice of the present invention. Unless otherwise noted, terms are to be understood in accordance with conventional usage by those of ordinary skill in the relevant art.

[0036] Where terms are provided in the singular, the inventors also contemplate aspects of the invention described by the plural of that term.

[0037] As used herein, the terms "polynucleotide molecule" and "polynucleotide" may be used interchangeably and refer to any polynucleotide consisting of 18 or more nucleotides covalently linked in a chain and capable of hybridizing under physiological conditions to DNA and RNA molecules. According to some embodiments, the polynucleotide may be a synthetic and / or artificial polynucleotide molecule. According to some embodiments, the polynucleotide molecule is a biopolymer. According to some embodiments, the biopolymer is a DNA (deoxyribonucleic acid) or RNA (ribonucleic acid) molecule.

[0038] In some embodiments, the polynucleotide molecule targets the mRNA of a plant gene. In some embodiments, the polynucleotide molecule targets the translated region of the mRNA. In some embodiments, the polynucleotide molecule targets the untranslated region (UTR) of the mRNA.

[0039] As used herein, the terms "DNA," "DNA molecule," and "DNA polynucleotide molecule" refer to a polymer of deoxyribonucleotide bases or a DNA polynucleotide molecule, such as a single-stranded or double-stranded DNA molecule of genomic or synthetic origin.

[0040] As used herein, the terms "DNA sequence," "DNA nucleotide sequence," and "DNA polynucleotide sequence" refer to the nucleotide sequence of a DNA molecule.

[0041] As used herein, the term "gene" refers to any portion of a nucleic acid that provides for the expression of or encodes a transcript. Thus, a "gene" includes, but is not limited to, a promoter region, a 5' untranslated region, a transcript encoding region which may include intron regions, and a 3' untranslated region.

[0042] As used herein, the terms "RNA," "RNA molecule," and "RNA polynucleotide molecule" refer to single-stranded or double-stranded RNA molecules of genomic or synthetic origin, such as polymers of ribonucleotide bases that contain single- or double-stranded regions or any other structural element.

[0043] Unless otherwise stated, nucleotide sequences in the context of this specification are given in the 5' to 3' direction when read from left to right. The nomenclature used herein is that required by Title 37 of the United States Code of Federal Regulations § 1.822 and set forth in the tables of WIPO Standard ST.25 (1998), Appendix 2, Tables 1 and 3.

[0044] As used herein, "plant surface" refers to any outer part of a plant. Thus, plant surfaces include, but are not limited to, the surfaces of flowers, stems, tubers, fruits, anthers, pollen, leaves, roots, or seeds. A plant surface can be on a part of a plant that is attached to another part of the plant or on a part of the plant that has detached from the plant.

[0045] As used herein, the phrase "a polynucleotide that is 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 either a DNA-dependent RNA polymerase II protein or a viral RNA-dependent RNA polymerase in such a manner that the polynucleotide will be transcribed by the DNA-dependent RNA polymerase protein or viral RNA-dependent RNA polymerase. A polynucleotide that is not operably linked to a promoter can be transcribed by a plant RNA-dependent RNA polymerase.

[0046] As used herein, SEQ ID NOs: 1-364 and 729-747 are represented in the sequence listing in ssDNA form, but also encompass dsDNA equivalents, dsRNA equivalents, ssRNA equivalents, ssRNA complements, the represented ssDNA, and ssDNA complements.

[0047] As used herein, the term "transfer agent" may refer to any agent that, when applied to the surface of a plant, renders the plant susceptible to polynucleotides. According to some embodiments, the transfer agent is an agent that conditions the surface of a plant tissue, such as a seed, leaf, stem, root, flower, or fruit, for penetration of the plant cell by polynucleotide molecules. Chemical agents for conditioning or transfer include (a) wetting agents, (b) wetting agents, (c) wetting agents, (d) wetting agents, (e.g., wetting agents, (f) wetting agents, (g) wetting agents, (h) wetting agents, (i) wetting agents, (j ... b ) surfactants, ( c ) organic solvents or aqueous solutions or aqueous mixtures of organic solvents, ( d ) oxidizing agent, ( e )acid,( f )base,( g )oil,( h ) enzymes, or combinations thereof.

[0048] According to some embodiments, the introducing agent is N , It may be selected from N-dimethyldecanamide, cocoamidopropyldimethylamine, siloxane polyalkylene oxide copolymer, AG-RHO® EM-30, dimethylamides of C8 / C10 fatty acids, esterified copolymers of glycerin, trisiloxane ethoxylate, or any combination thereof.

[0049] Non-limiting examples of suitable introducing agents include organosilicon compounds.

[0050] As used herein, the phrase "organosilicone preparation" refers to a liquid containing one or more organosilicone compounds, the liquid or components of which, when combined with a polynucleotide in a composition that is topically applied to a target plant surface, can improve the penetration of the polynucleotide into plant cells. Exemplary organosilicone preparations include, but are not limited to, those commercially available under the trade names "Silwet®" or "BREAK-THRU®." In certain embodiments, the organosilicone preparation can improve the penetration of the polynucleotide into plant cells in a manner that permits polynucleotide-mediated suppression of target gene expression in the plant cells.

[0051] A non-limiting example of a specific suitable initiator is Silwet® L-77, a modified trisiloxane that combines a very low molecular weight trisiloxane with a polyether group. It is characterized by outstanding interfacial activity, which can result in dramatically reduced aqueous surface tension, excellent spreading or leveling, and stabilized foaming, all of which may be achieved using a fraction of the typical concentration levels of organic or fluorocarbon surfactants.

[0052] Another non-limiting example of a specific suitable induction agent is GENAGEN™ 4166 (Clariant, material number: 10783626892). GENAGEN™ 4166 is a dimethylamide based on naturally occurring fatty acids.

[0053] Another non-limiting example of a specific suitable delivery agent is SYNERGEN® GL5 (Clarian, material number: 20072326894). SYNERGEN® GL5 is a polyglycerol ester-based adjuvant and a TAE-free surfactant derived from renewable sources. Another non-limiting example of a specific suitable delivery agent is GENAGEN™ 4296 (Clariant, material number: 10783926892). GENAGEN™ 4296 is a dimethylamide based on naturally occurring fatty acids.

[0054] Another non-limiting example of a specific suitable introduction agent is SYNERGEN® GA (Clariant, material number: 27251626894). SYNERGEN® GA is a novel biological enhancer for salts of alkyl glucamide-based pesticides. It is a sugar-based surfactant with a renewable carbon index (RCI) of over 95% and therefore an excellent ecological profile.

[0055] Another non-limiting example of a specific suitable carrier agent is GENAGEN™ SC35 (Clariant, material number: 25923226892). GENAGEN™ SC35 is a basic surfactant blend of alkyl diglycol ether sulfate sodium salt and coconut fatty acid monoethanolamide.

[0056] Another non-limiting example of a specific suitable incorporation agent is HOSTAPHAT® 1306 (Clariant, material number: 13326826900). HOSTAPHAT® 1306 is an anionic emulsifier used for the emulsion polymerization of monomers such as pure acrylics, styrene acrylates, and vinyl acetate.

[0057] Another non-limiting example of a specific suitable loading agent is SURFECO PLUS™ (Latro), a silicone-based adjuvant used for modifying the physical properties and enhancing the biological activity of pesticides.

[0058] Further suitable transfer agents and their chemical properties are summarized in Table 1 below. [Table 1]

[0059] As used herein, the phrase "improved yield" refers to some measurable improvement in yield. In certain embodiments, improved yield in a plant or plant part can be determined in comparison to a control plant or plant part that has not been treated with a composition comprising a polynucleotide. As used in this context, a control plant is a plant that has not been treated with a polynucleotide and an introducing agent. Such control plants can include, but are not limited to, untreated plants or mock-treated plants.

[0060] Non-limiting examples of traits affected by the compositions disclosed herein include increased branching, increased seed filling, increased seed number, improved drought tolerance, reduced seed shattering, reduced abscission tissue formation, delayed / early flowering, and any combination thereof, with each possibility being a separate embodiment.

[0061] Non-limiting examples of rice plant traits affected by the compositions disclosed herein include increased grain size, increased number of panicles, increased number of tillers, increased heading, and any combination thereof, with each possibility being a separate embodiment.

[0062] Non-limiting examples of canola traits affected by the compositions disclosed herein include increased grain size, increased number of panicles, increased number of tillers, increased branching, increased seed filling, increased seed number, increased heading, improved drought tolerance, reduced grain shattering, reduced abscission tissue formation, delayed / early flowering, shorter / longer flowering period, and any combination thereof. Each possibility is a separate embodiment.

[0063] According to some embodiments, the plant may be any cultivated plant, including but not limited to, canola, rapeseed, rice, wheat, barley, soybeans, peanuts, cotton, corn, sorghum, sugarcane, sugar beets, beans, sunflowers, potatoes, sweet potatoes, alfalfa, bananas, apricots, grapes, apples, peaches, prunes, citrus fruits, dates, palm oil, peppers, tomatoes, broccoli, onions, melons, watermelons, yams, cassava, etc. Each possibility is a separate embodiment.

[0064] According to some embodiments, the plant may be a soybean plant, a rice plant, or an oilseed rape plant. Each possibility is a separate embodiment. According to some embodiments, the soybean plant may be a soybean (Glycine max) species. According to some embodiments, the rice plant may be a rice (Oryza sativa) species. According to some embodiments, the oilseed rape plant may be a rapeseed (Brassica napus) species. Each possibility is a separate embodiment.

[0065] According to some embodiments, the gene targeted by the polynucleotide molecule may be referred to by its scientific name as used in one species, for example, Arabidopsis thaliana.However, it is understood by those skilled in the art that the described scientific name also encompasses other names and homologs in other species that are called by other names (also known as / homologues).As a non-limiting example, if the gene targeted by the polynucleotide molecule is called BRC1, it encompasses the alias / homologue TB1 / FC1.

[0066] According to some embodiments, the target gene may have a nucleotide sequence selected from any of the nucleotide sequences set forth in SEQ ID NOs: 1-364. Each possibility is a separate embodiment. According to some embodiments, the target gene may encode an amino acid sequence selected from any of the amino acid sequences set forth in SEQ ID NOs: 365-728. Each possibility is a separate embodiment. According to some embodiments, the polynucleotide (i.e., dsRNA) may have a nucleotide sequence set forth in SEQ ID NOs: 729-747. Each possibility is a separate embodiment.

[0067] According to some embodiments, the polynucleotide molecule is a dsRNA having a polynucleotide sequence essentially 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 "a substantial portion thereof," when referring to a dsRNA, refers to a dsRNA that is at least 80% identical to at least 18-20 contiguous base pairs of the sequence set forth in any one of SEQ ID NOs: 729-747, a dsRNA that is at least 85% identical to at least 18-20 contiguous base pairs of the sequence set forth in any one of SEQ ID NOs: 729-747, a dsRNA that is at least 90% identical to at least 18-20 contiguous base pairs of the sequence set forth in any one of SEQ ID NOs: 729-747, a dsRNA that is at least 95% identical to at least 18-20 contiguous base pairs of the sequence set forth in any one of SEQ ID NOs: 729-747, or a dsRNA that is at least 98% identical to at least 18-20 contiguous base pairs of the sequence set forth in any one of SEQ ID NOs: 729-747. Each possibility is a separate embodiment.

[0068] As used herein, the term "essentially identical to" when referring to a dsRNA refers to a dsRNA sequence that has at least 80%, at least 90%, at least 95%, or at least 98% homology to a portion of the nucleotide sequence set forth in SEQ ID NOs: 1-364. As used herein, the term "a portion of the nucleotide sequence set forth in" refers to a portion of the nucleotide sequence targeted by the dsRNA that has essentially the same length as the dsRNA. As a non-limiting example, if a dsRNA has a length of 18 bp, the portion of the nucleotide sequence targeted by the dsRNA will have a length of approximately 18 bp. As another non-limiting example, if a dsRNA has a length of 200 bp, the portion of the nucleotide sequence targeted by the dsRNA will have a length of approximately 200 bp.

[0069] As used herein, the terms "approximately" and "about" refer to + / - 10%, or + / - 5%, or + / - 2% of the referenced range. Each possibility is a separate embodiment.

[0070] According to some embodiments, the dsRNA targets BnADPG1 (SEQ ID NO: 235) of the oilseed rape plant and has the polynucleotide sequence shown in SEQ ID NO: 729.

[0071] According to some embodiments, the dsRNA targets BnBRC1 (SEQ ID NO: 1) of the canola plant and has the polynucleotide sequence shown in SEQ ID NO: 730.

[0072] According to some embodiments, the dsRNA targets BnCKX2 of the canola plant (SEQ ID NO: 158) and has the polynucleotide sequence shown in SEQ ID NO: 731.

[0073] According to some embodiments, the dsRNA targets BnKIN10 (SEQ ID NO: 62) of oilseed rape plants and has the polynucleotide sequence shown in SEQ ID NO: 732.

[0074] According to some embodiments, the dsRNA targets BnPTL (SEQ ID NO: 286) of the canola plant and has the polynucleotide sequence shown in SEQ ID NO: 733.

[0075] According to some embodiments, the dsRNA targets GmBRC1 (SEQ ID NO: 15) of soybean plants and has the polynucleotide sequence set forth in SEQ ID NO: 734 or 735.

[0076] According to some embodiments, the dsRNA targets GmBS1 (SEQ ID NO: 116) of soybean plants and has the polynucleotide sequence set forth in SEQ ID NO: 736 or 737.

[0077] According to some embodiments, the dsRNA targets GmJAG1 (SEQ ID NO: 153) of soybean plants and has the polynucleotide sequence set forth in SEQ ID NO: 738 or 739.

[0078] According to some embodiments, the dsRNA is a GmPLD gene from a soybean plant. α 1 (SEQ ID NO: 124) and has the polynucleotide sequence shown in SEQ ID NO: 740 or 741.

[0079] According to some embodiments, the dsRNA targets OsBRC1 (SEQ ID NO: 43) of rice plants and has a polynucleotide sequence set forth in SEQ ID NO: 742 or 743.

[0080] According to some embodiments, the dsRNA targets OsPIN5b (SEQ ID NO: 86) of rice plants and has a polynucleotide sequence set forth in SEQ ID NO: 744 or 745.

[0081] According to some embodiments, the dsRNA targets OsSKIN1 (SEQ ID NO: 52) of rice plants and has a polynucleotide sequence set forth in SEQ ID NO: 746 or 747.

[0082] According to some embodiments, the compositions and methods for applying them may include timing the application of the composition to a desired plant developmental trait. As a non-limiting example, application of a composition comprising a polynucleotide configured to target the gene BS1 (or other gene involved in regulating seed filling) may be timed when the plant is at the seed filling stage (R3-R5 developmental stages). As another non-limiting example, application of a composition comprising a polynucleotide configured to target the gene BRC1 (or other gene whose reduced expression causes increased branching or tiller number) may be timed to the bolting stage in soybean plants (R1-R2 developmental stages) or the vegetative phase of axillary bud development in rice plants. As another non-limiting example, application of a composition comprising a polynucleotide configured to target the gene JAG1 (or other gene that increases seed number) may be timed when the plant is at the flowering stage (R1-R2 developmental stages). As another non-limiting example, application of a composition comprising a polynucleotide configured to target the gene SGR1 (or other target gene whose reduction increases drought tolerance) may be timed to a period 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 causes reduced grain shattering) may be timed to when the plant siliques mature. 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 controlling flowering) may be timed to when the plant is at 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 controls grain size) may be timed to when the plant is at the seed filling stage.

[0083] According to some embodiments, a composition may comprise two or more polynucleotide sequences (different sequences), such as 2, 3, 4, 5, or more polynucleotide sequences. Each possibility is a separate embodiment.

[0084] According to some embodiments, the two or more polynucleotide sequences may target the same target gene, i.e., they may be directed to different portions of the sequence of the same target gene.

[0085] According to some embodiments, the two or more polynucleotide sequences may target different target genes.

[0086] According to some embodiments, different target genes may target the same yield-related trait (e.g., reduced shattering). As a non-limiting example, the two or more polynucleotide sequences may target AGL1 and PDH1. As another non-limiting example, the two or more polynucleotide sequences may target two or more of JAG1, JAG2, CKX1, and OTU1, all of which affect seed number.

[0087] According to some embodiments, different target genes may be directed to different yield-related traits (e.g., increased drought tolerance and seed filling). As non-limiting examples, one of the two or more polynucleotide sequences may target ERA1, SGR1, SGR2, ACO2, CER9, or CytG, while a second of the two or more polynucleotide sequences may target BS1, PLD, ACO3, or PDHK.

[0088] As used herein, the phrase "reduced expression," when used in the context of a transcript or protein in a plant or plant part, refers to some measurable decrease in the level of the transcript or protein in the plant or plant part. In certain embodiments, the decrease in the level of the transcript or protein in the plant or plant part can be determined in comparison to a control plant or plant part that has not been treated with a composition comprising a polynucleotide and an introduction agent.

[0089] As used herein, the phrase "wherein the plant does not contain a transgene" refers to a plant that does not have either a DNA molecule containing a promoter operably linked to a polynucleotide or a genetically modified viral vector.

[0090] As used herein, the term "transgene" describes a segment of DNA containing a gene sequence that is isolated from one organism and introduced into the DNA of a different organism.

[0091] As used herein, the phrases "suppressing expression" or "reducing expression," when used in the context of a gene, refer to some measurable decrease in the amount and / or activity of a product encoded by the gene. Thus, gene expression can be suppressed when there is a decrease in the level of transcripts from the gene, a decrease in the level of the protein encoded by the gene, a decrease in the activity of transcripts from the gene, a decrease in the activity of the protein encoded by the gene, any one of the foregoing conditions, or any combination of the foregoing conditions. In this context, the activity of a transcript includes, but is not limited to, its ability to be translated into a protein and / or to exert any RNA-mediated biological or biochemical effect. In this context, the activity of a protein includes, but is not limited to, its ability to exert any protein-mediated biological or biochemical effect. As used in this context, a control plant or plant part is a plant or plant part that has not been treated with a polynucleotide and an introducing agent.

[0092] As used herein, the term "transient," when used in the context of reducing / suppressing the expression of a gene, refers to a time-limited decrease in the expression of a gene that lasts only as long as the polynucleotide that has penetrated the cell is not degraded, as opposed to long-term expression, typically referred to as "stable expression."

[0093] As used herein, the term "transcript" refers to any RNA produced from a gene by the process of transcription. A transcript of a gene can therefore include a primary transcript, which may contain introns, or can include a mature RNA that lacks introns.

[0094] As used herein, the term "homology," as it relates to polynucleotide molecules, refers to the degree of sequence identity or similarity (homology) between nucleotide sequences that indicates a shared ancestry. Two segments of DNA can share a common ancestry either because of a speciation event (orthologs) or a duplication event (paralogs). According to some embodiments, a homolog may refer to a polynucleotide having substantially about 70% to about 99% sequence identity, or more preferably about 80% to about 99% sequence identity, or most preferably about 90% to about 99% sequence identity, up to about 99% sequence identity, to a reference nucleotide sequence of a reference polynucleotide molecule. Each possibility is a separate embodiment.

[0095] As used herein, the terms "sequence identity," "sequence similarity," or "homology" are used to describe the sequence relationship between two or more nucleotide sequences. The percentage of "sequence identity" between two sequences is determined by comparing two optimally aligned sequences. A sequence that is identical at every position when compared to a reference sequence is said to be identical to the reference sequence, and vice versa. A first nucleotide sequence, when viewed in the 5' to 3' direction, is said to be "complementary" or complementary to a second or reference nucleotide sequence if the first nucleotide sequence exhibits complete complementarity with the second or reference sequence when viewed in the 3' to 5' direction. As used herein, nucleic acid sequence molecules are said to exhibit "complete complementarity" when every nucleotide of one of the sequences, read 5' to 3', is complementary to every nucleotide of the other sequence when read 3' to 5'. A nucleotide sequence that is complementary to a reference nucleotide sequence will exhibit a sequence identical to the reverse complement of the reference nucleotide sequence. These terms and descriptions are well defined in the art and readily understood by those of ordinary skill in the art.

[0096] According to some embodiments, the composition may further comprise a carrier. According to some embodiments, the carrier may be a liquid.

[0097] As used herein, the term "liquid" refers to both homogeneous mixtures, such as solutions, and non-homogeneous mixtures, such as suspensions, colloids, micelles, and emulsions. Each possibility is a separate embodiment.

[0098] According to some embodiments, the liquid may be an aqueous solution. According to some embodiments, the liquid may be an oil or oil mixture.

[0099] According to some embodiments, the polynucleotide may be naked. As used herein, the term "naked" refers to a polynucleotide that is not encapsulated. Naked polynucleotides may, however, be modified and / or conjugated.

[0100] According to other embodiments, the polynucleotide may be encapsulated.

[0101] According to some embodiments, polynucleotides may be delivered via and / or encapsulated in vehicles such as, but not limited to, nanoparticles, liposomes, micelles, and the like.

[0102] Provided herein are specific methods and polynucleotide compositions that can be applied to living plant cells / tissues to silence expression of a target gene and provide improved yield to plants in need thereof. Also provided herein are plants and plant parts that exhibit improved yield, as well as processed products of such plants or plant parts. The compositions may be applied topically to the surface of the plant, such as the surface of the leaves. The compositions may be applied to a variety of plants, including, but not limited to, plants of the Brassicaceae, Fabaceae, or Poaceae families, such as, but not limited to, soybean plants, rice plants, oilseed rape plants, and / or rapeseed plants. Each possibility is a separate embodiment.

[0103] As used herein, "polynucleotide" refers to a DNA or RNA molecule containing multiple nucleotides, and generally refers to both "oligonucleotides" (polynucleotide molecules 18-25 nucleotides in length) and longer polynucleotides of 26 nucleotides or greater. In embodiments of the invention, polynucleotides having lengths of 18-25 nucleotides (18-mer, 19-mer, 20-mer, 21-mer, 22-mer, 23-mer, 24-mer, or 25-mer) or intermediate-length polynucleotides having lengths of 26 nucleotides or greater (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, 110, 111, 112 , 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, about 65, about 70, about 75, about 80, about 85, about 90, about 95, about 100, about 110, about 120, about 130, about 140, about 150, about 160, about 170, about 180, about 190, about 200, about 210, about 220, about 230, about 240, about 250, about 260, about 270, about 280, about 290, or about 300 nucleotides). or a long polynucleotide having a length of more than about 300 nucleotides (e.g., a polynucleotide having a length of about 300 to about 400 nucleotides, about 400 to about 500 nucleotides, about 500 to about 600 nucleotides, about 600 to about 700 nucleotides, about 700 to about 800 nucleotides, about 800 to about 900 nucleotides, about 900 to about 1000 nucleotides, about 300 to about 500 nucleotides, about 300 to about 600 nucleotides, about 300 to about 700 nucleotides, about 300 to about 800 nucleotides, about 300 to about 900 nucleotides, or about 1000 nucleotides, or a polynucleotide longer than about 1000 nucleotides in length, for example, up to the coding or non-coding portion of the target gene, or the entire length of the target gene including both the coding and non-coding portions). When the polynucleotide is double-stranded, its length can be similarly described in terms of base pairs.

[0104] Polynucleotide compositions used in various embodiments of the present invention include compositions containing polynucleotides, including RNA, DNA, RNA / DNA hybrids, chemically modified polynucleotides, artificial polynucleotides, or mixtures thereof. In certain embodiments, the polynucleotide may be a combination of ribonucleotides and deoxyribonucleotides, such as a synthetic polynucleotide composed primarily of ribonucleotides but with one or more terminal deoxyribonucleotides, or a synthetic polynucleotide composed primarily of deoxyribonucleotides but with one or more terminal dideoxyribonucleotides. In certain embodiments, the polynucleotide comprises non-standard nucleotides, such as inosine, thiouridine, or pseudouridine. In certain embodiments, the polynucleotide comprises chemically modified nucleotides. Examples of chemically modified oligonucleotides or polynucleotides are well known in the art. Illustrative examples include, but are not limited to, the naturally occurring phosphodiester backbone of polynucleotides, which can be partially or fully modified with phosphorothioate, phosphorodithioate, or methylphosphonate internucleotide linkage modifications; modified nucleoside bases 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).

[0105] According to some embodiments, dsRNA may 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 may 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 may be modified to include a 3' overhang.

[0106] According to some embodiments, modifications can also be incorporated into the dsRNA. According to some embodiments, the modifications do not prevent the dsRNA composition from serving as a substrate for Dicer. In one embodiment, one or more modifications are made to enhance Dicer processing of the dsRNA. In a second embodiment, one or more modifications are made to result in more effective RNAi production. In a third embodiment, one or more modifications are made to support greater RNAi effects. In a fourth embodiment, one or more modifications are made to result in greater potential per dsRNA molecule to be delivered to a cell. Modifications can be introduced in the 3'-terminal region, the 5'-terminal region, both the 3'-terminal and 5'-terminal regions, or in some cases, at various positions within the sequence. With the above limitations in mind, any number and combination of modifications can be introduced into the dsRNA. When multiple modifications are present, they can be the same or different. Modifications to the base, sugar moiety, phosphate backbone, and combinations thereof are contemplated. Either 5'-terminus can be phosphorylated.

[0107] Contemplated modifications of the phosphate backbone include phosphonates, such as methylphosphonates, phosphorothioates, and phosphotriester modifications, such as alkylphosphotriesters. Contemplated modifications of the sugar moiety include 2'-alkylpyrimidines, such as 2'-O-methyl, 2'-fluoro, amino, and deoxy modifications (see, e.g., Amarzguioui et al., 2003). Contemplated modifications of the base group include abasic sugars, 2-O-alkyl-modified pyrimidines, 4-thiouracil, 5-bromouracil, 5-iodouracil, and 5-(3-aminoallyl)-uracil. Locked nucleic acids, or LNAs, can also be introduced. Many other modifications are known and can be used as long as they meet the above criteria.

[0108] Polynucleotides can be single- or double-stranded RNA, structurally characterized single- or double-stranded RNA, single- or double-stranded DNA, double-stranded DNA / RNA hybrids, and modified analogs thereof. In certain embodiments of the present invention, the polynucleotide that provides the single-stranded RNA in the plant cell may 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 hybridizing 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 may contain both ribonucleic acid and deoxyribonucleic acid residues. In certain embodiments, these polynucleotides include chemically modified nucleotides or non-standard nucleotides. In certain embodiments of the method, the polynucleotide includes double-stranded DNA formed by intramolecular hybridization, double-stranded DNA formed by intermolecular hybridization, double-stranded RNA formed by intramolecular hybridization, or double-stranded RNA formed by intermolecular hybridization. In certain embodiments, when the polynucleotide is a dsRNA, its antisense strand will contain at least 18 nucleotides that are essentially complementary to the target gene. In certain embodiments, the polynucleotide includes single-stranded DNA or single-stranded RNA that self-hybridizes to form a hairpin structure, the hairpin structure having an at least partially double-stranded structure that includes at least one segment that hybridizes to RNA transcribed from the gene targeted for suppression.Without being bound by any mechanism, it is believed that such polynucleotides will be, or will produce, single-stranded RNA having at least one segment that will hybridize to RNA transcribed from the gene targeted for repression.

[0109] The polynucleotide molecules of the present invention are designed to regulate expression by inducing control or repression of an endogenous target gene in a plant, and are designed to have a nucleotide sequence that is essentially identical to or essentially complementary to the nucleotide sequence of the endogenous target gene of the plant or the sequence of RNA transcribed from the endogenous target gene of the plant, which can be a coding sequence or a non-coding sequence.

[0110] "Essentially identical" or "essentially complementary" means that a polynucleotide (or at least one strand of a double-stranded polynucleotide) has sufficient identity or complementarity to an RNA (e.g., a transcript) transcribed from an endogenous gene or an endogenous target gene to inhibit expression of the endogenous target gene (e.g., causing a decrease in the level or activity of the gene transcript and / or encoded protein).

[0111] The polynucleotides of the methods and compositions provided herein can be used to inhibit the expression of an endogenous target gene or RNA transcribed from an endogenous target gene (i.e., transcript) to cause a decrease in the level or activity of the gene transcript or encoded protein. ) 100% identity vs. or complementarityThus, in certain embodiments, a polynucleotide or portion thereof is designed to be essentially identical to or essentially complementary to a sequence of at least 18 or 19 contiguous nucleotides in either the target gene or the messenger RNA (e.g., transcript) transcribed from that target gene. In certain embodiments, an "essentially identical" polynucleotide has 100 percent sequence identity or at least about 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99 percent sequence identity when compared to a sequence of 18 or more contiguous nucleotides in either the endogenous target gene or the RNA (e.g., transcript) transcribed from that target gene. In certain embodiments, an "essentially complementary" polynucleotide has 100 percent sequence complementarity or at least about 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99 percent sequence complementarity when compared to a sequence of 18 or more contiguous nucleotides in either the target gene or RNA transcribed from that target gene.

[0112] In certain embodiments, the polynucleotides used in the methods and compositions provided herein can be essentially identical to or essentially complementary to either i) a conserved region of a target gene in both monocotyledonous and dicotyledonous plants, ii) a conserved region of a target gene in monocotyledonous plants, or iii) a conserved region of a target gene in dicotyledonous plants. Such polynucleotides that are essentially identical to or essentially complementary to such conserved regions can be used to improve senescence delay and / or yield improvement by suppressing the expression of target genes in various dicotyledonous plants.

[0113] Certain embodiments of the compositions and methods provided herein can thus use polynucleotides containing mismatches to target genes or transcripts. In certain embodiments, a polynucleotide of 19 contiguous nucleotides that is essentially identical to or essentially complementary to an endogenous target gene or an RNA transcribed from that target gene (e.g., a transcript) can have one or two mismatches to the target gene or transcript. In certain embodiments, a polynucleotide of 20 or more nucleotides containing a contiguous 19 nucleotide span that is identical to or complementary to an endogenous target gene or an RNA transcribed from that target gene can have one or two mismatches to the target gene or transcript. In certain embodiments, a polynucleotide of 21 contiguous nucleotides that is essentially identical to or essentially complementary to an endogenous target gene or an RNA transcribed from that target gene (e.g., a transcript) can have one, two, or three mismatches to the target gene or transcript. In certain embodiments, a polynucleotide of 22 or more nucleotides containing a contiguous 21 nucleotide span having identity or complementarity to an endogenous target gene or RNA transcribed from that target gene can have one, two, or three mismatches to the target gene or transcript. When designing a polynucleotide having mismatches to an endogenous target gene or RNA transcribed from that target gene, certain types of mismatches, and in certain locations, that are more likely to be tolerated can be used.

[0114] According to some embodiments, the target gene is selected from the group consisting of SEQ ID NOs: 1-36. 4 Any of the target genes shown may be used, as well as orthologous target genes obtainable from other crops.

[0115] According to some embodiments, the plant may be a canola plant, and the polynucleotide may reduce the level of a target gene having any of the nucleotide sequences set forth 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.

[0116] According to some embodiments, the plant may be a canola plant, and the polynucleotide may reduce levels of a protein having any of the amino acid sequences set forth 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.

[0117] According to some embodiments, the plant may be a canola plant, and the polynucleotide may reduce levels 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.

[0118] According to some embodiments, the plant may be a canola plant, and the polynucleotide may reduce levels 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.

[0119] According to some embodiments, the plant may be a canola plant, and the polynucleotide may have the nucleotide sequence set forth in SEQ ID NOs: 729-733.

[0120] According to some embodiments, the plant may be a soybean plant, and the polynucleotide may reduce the level of a target gene having any of the nucleotide sequences set forth 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 a substantial portion of any of them. Each possibility is a separate embodiment.

[0121] According to some embodiments, the plant may be a soybean plant, and the polynucleotide may reduce the level of a target gene having any of the nucleotide sequences set forth 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 a substantial portion of any of them. Each possibility is a separate embodiment.

[0122] According to some embodiments, the plant may be a soybean plant, and the polynucleotide may 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 a substantial portion of any of them. Each possibility is a separate embodiment.

[0123] According to some embodiments, the plant may be a soybean plant, and the polynucleotide may reduce levels 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 a substantial portion of any of them. Each possibility is a separate embodiment.

[0124] According to some embodiments, the plant may be a soybean plant, and the polynucleotide may have a nucleotide sequence set forth in SEQ ID NOs: 734-741.

[0125] According to some embodiments, the plant may be a rice plant, and the polynucleotide may reduce the level of a target gene having any of the nucleotide sequences set forth 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 a substantial portion of any of them. Each possibility is a separate embodiment.

[0126] According to some embodiments, the plant may be a rice plant, and the polynucleotide may reduce the level of a target gene having any of the nucleotide sequences set forth 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 a substantial portion of any of them. Each possibility is a separate embodiment.

[0127] According to some embodiments, the plant may be a rice plant, and the polynucleotide may reduce the level of a target gene having any of the nucleotide sequences set forth in SEQ ID NOs: 43, 44, 52-57, 73-75, 85, 86, 225-227, 246-248, 259-264, or a substantial portion of any of them. Each possibility is a separate embodiment.

[0128] According to some embodiments, the plant may be a rice plant, and the polynucleotide may reduce levels 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 a substantial portion of any of them. Each possibility is a separate embodiment.

[0129] According to some embodiments, the plant may be a rice plant, and the polynucleotide (ie, dsRNA) may have a nucleotide sequence set forth in SEQ ID NOs: 742-747.

[0130] In certain embodiments, the polynucleotide compositions and methods provided herein typically control or regulate (e.g., suppress) gene expression over the lifespan of a treated plant over several days to several weeks or longer, and typically in a systemic manner. For example, within a few days of treating a plant leaf with a polynucleotide composition of the invention, primary and transient siRNAs can be detected in other leaves lateral to and above the treated leaf, as well as in apical tissue. In certain embodiments, a method for systemically suppressing gene expression in a plant is provided, comprising treating the plant with a composition comprising at least one polynucleotide and a transfection agent, wherein the polynucleotide comprises at least 18 or at least 19 contiguous nucleotides that are essentially identical to or essentially complementary to a gene or transcript encoding a target gene in the plant, thereby systemically suppressing gene expression in the plant or its progeny compared to a control plant not treated with the composition.

[0131] Compositions used to inhibit a target gene can include one or more polynucleotides that are essentially identical to or essentially complementary to multiple genes or to multiple segments of one or more genes. In certain embodiments, compositions used to inhibit a target gene can include one or more polynucleotides that are essentially identical to or essentially complementary to multiple contiguous segments of a target gene, multiple non-contiguous segments of a target gene, multiple alleles of a target gene, or multiple target genes from one or more species.

[0132] In certain embodiments, a polynucleotide comprises two or more copies of a nucleotide sequence (of 18 or more nucleotides) arranged in a tandem fashion. In other embodiments, a polynucleotide comprises two or more copies of a nucleotide sequence (of 18 or more nucleotides) arranged in an inverted repeat fashion (forming at least partially self-complementary strands). A polynucleotide can comprise both tandem and inverted repeat copies. Whether arranged in a tandem or inverted repeat fashion, each copy can be directly adjacent to the next copy, or pairs of copies can be separated by an optional spacer of one or more nucleotides. The optional spacer can be an unrelated sequence.

[0133] There is no upper limit to the concentration and dosage of polynucleotide molecules that may be useful in the methods and compositions provided herein, although lower effective concentrations and dosages are generally required for efficiency. Concentrations can be adjusted taking into account the volume of spray or treatment applied to the surface of plant leaves or other plant parts such as petals, stems, tubers, fruits, anthers, pollen, leaves, roots, or seeds.

[0134] Examples of agents or treatments for conditioning plants for polynucleotide infiltration include emulsions, inverse emulsions, liposomes, and other micelle-like compositions. Examples of agents or treatments for conditioning plants for polynucleotide infiltration include counterions or other molecules known to associate with nucleic acid molecules, such as inorganic ammonium ions, alkylammonium ions, lithium ions, polyamines such as spermine, spermidine, or putrescine, and other cations. Organic solvents useful for conditioning plants for polynucleotide infiltration include DMSO, DMF, pyridine, N-pyrrolidine, hexamethylphosphoramide, acetonitrile, dioxane, polypropylene glycol, and other solvents that are miscible with water or that dissolve phosphonucleotides in non-aqueous systems (such as those used in synthesis reactions). Naturally occurring or synthetic oils, such as oils of plant origin or crop oils, with or without surfactants or emulsifiers, can be used.

[0135] In certain embodiments, polynucleotide compositions can be prepared using an organosilicone formulation commercially available as Silwet® L-77 surfactant, which has CAS Number 27306-78-1 and EPA Number: CAL.REG.NO.5905-50073-AA, and is currently available from Momentive Performance Materials, Albany, N.Y. In certain embodiments, when Silwet® L-77 organosilicone formulation is used as a pre-spray treatment for plant leaves or other plant surfaces, the polynucleotide compositions can be prepared using an organosilicone formulation in the range of about 0.015 to about 2 weight percent (wt%) (e.g., about 0.01, 0.015, 0.02, 0.025, 0.03, 0.035, 0.04, 0.045, 0.05, 0.055, 0.06, 0.065, 0.07, 0.075, 0.08, 0.085, 0.095, 10 ... , 0.09, 0.095, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, and 2.5 wt% of newly prepared concentrations are effective in preparing leaves or other plant surfaces for introduction of polynucleotide molecules into plant cells from topical application to the surface. In certain embodiments of the methods and compositions provided herein, the polynucleotide molecule and Silwet® L-77 are combined in a concentration ranging from about 0.015 to about 2 weight percent (wt%) (e.g., about 0.01, 0.015, 0.02, 0.025, 0.03, 0.035, 0.04, 0.045, 0.05, 0.055, 0.06, 0.065, 0.07, and an organosilicone formulation comprising: 0.075, 0.08, 0.085, 0.09, 0.095, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.5 wt %.

[0136] According to some embodiments, polynucleotide compositions containing organosilicone formulations can include salts such as ammonium chloride, tetrabutylphosphonium bromide, and / or ammonium sulfate. The ammonium chloride, tetrabutylphosphonium bromide, and / or ammonium sulfate can be provided in the polynucleotide composition at a concentration of about 0.01% to about 5% (w / v).

[0137] According to some embodiments, other useful transfer agents or adjuvants to transfer agents that can be used in the polynucleotide compositions provided herein include surfactants and / or effective molecules contained therein. Surfactants and / or effective molecules contained therein include, but are not limited to, sodium or lithium salts of fatty acids (such as tallow or tallowamine or phospholipids) and organosilicone surfactants. In certain embodiments, polynucleotide compositions containing transfer agents are formulated with counterions or other molecules known to associate with nucleic acid molecules. Illustrative examples include tetraalkylammonium ions, trialkylammonium ions, sulfonium ions, lithium ions, and polyamines such as spermine, spermidine, or putrescine.

[0138] In certain embodiments, the polynucleotide composition further comprises glycerin. Glycerin can be provided in the composition at a concentration of about 0.1% to about 1% (w / v or v / v). Alternatively, a glycerin concentration of about 0.4% to about 0.6%, or about 0.5% (w / v or v / v) can be used in the polynucleotide composition containing the transfer agent.

[0139] In certain embodiments, the polynucleotide composition further comprises an organic solvent. Non-limiting examples of suitable organic solvents include, but are not limited to, DMSO, DMF, pyridine, N-pyrrolidine, hexamethylphosphoramide, acetonitrile, dioxane, polypropylene glycol, and other solvents that are miscible with water or will dissolve phosphonucleotides in non-aqueous systems (such as those used in synthetic reactions).

[0140] In certain embodiments, the polynucleotide composition further comprises a naturally occurring or synthetic oil, with or without a surfactant and / or emulsifier. Non-limiting examples of suitable oils include, but are not limited to, oils of plant origin, crop oils, paraffin oils, polyol fatty acid esters, or oils having short chain molecules modified with polyamines such as amides or polyethyleneimines or N-pyrrolidines.

[0141] The compositions and methods of the present invention are useful for modulating or suppressing the expression of endogenous or genetically modified target genes in plant cells or plants. In certain embodiments of the methods and compositions provided herein, expression of genes targeted by the polynucleotides disclosed herein can be completely, partially, and / or transiently suppressed, resulting in improved yield.

[0142] The target genes and plants containing those 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 use, or vi) trees in natural forests, or vii) ornamental plants. The methods and compositions provided herein can also be applied to plants produced by cutting, cloning, or grafting processes.

[0143] Compositions comprising a polynucleotide and an introduction agent provided herein can be topically applied to plants or plant parts by any convenient method, for example, by spraying or coating with a powder or with a liquid composition including an emulsion, suspension, or solution. Such topically applied sprays or coatings can be applied to all or any portion of the surface of the plant or plant part. Similarly, compositions comprising an introduction agent or other pretreatment can, in certain embodiments, be applied to plants or plant parts by any convenient method, for example, by spraying or wiping with a solution, emulsion, or suspension. Compositions comprising a polynucleotide and an introduction agent provided herein can be topically applied to plant parts, including, but not limited to, roots, flowers, stems, tubers, meristems, ovules, fruits, anthers, pollen, leaves, or seeds.

[0144] According to some embodiments, the composition may be applied by irrigation, for example, using an existing or designated irrigation system.

[0145] Specifically provided herein is the application of a composition comprising a polynucleotide and an introduction agent to seeds. The seeds can be contacted with such a composition by spraying, misting, soaking, etc. In some embodiments, progeny plants or plant parts derived from the treated seeds will exhibit improved yields due to the suppression of target gene expression.

[0146] A composition containing a polynucleotide containing an introduction agent can be topically applied to the plant surface using various methods for spraying the composition onto a plant or plant part. In the art, compositions can be applied using a boom that extends over the crop and delivers the composition to the plant surface, or using a boomless sprayer that distributes the composition over a wide area. Agricultural sprayers adapted for directional, broadcast, or band spraying can also be used in certain embodiments. Sprayers adapted for spraying specific parts of plants, including, but not limited to, leaves, leaf undersides, flowers, stems, male reproductive organs such as tassels, meristems, pollen, ovules, etc., can also be used. The composition can also be delivered by aircraft, such as a pesticide-dusting airplane. In certain embodiments, spraying can be delivered using a pressurized backpack sprayer adjusted to deliver the appropriate rate of composition.

[0147] In certain embodiments, plant parts can be sprayed either before or after harvest to provide improved yield at the plant part. The composition can be topically applied to plant parts attached to the plant by spraying as described above. The composition can be topically applied to plant parts detached from the plant by spraying as described above or by alternative methods. Alternative methods for applying the composition to the detached part include, but are not limited to, passing the plant part through a sprayer on a conveyor belt or trough, or dipping the plant part into the composition.

[0148] Compositions comprising a polynucleotide and an introduction agent can be applied to plants or plant parts at one or more developmental stages as desired and / or required. Application of the composition to seeds before germination and / or to seedlings after germination is provided in certain embodiments. Seeds are treated with the polynucleotide compositions provided herein by methods including, but not limited to, spraying, soaking, or any process that results in coating, absorption, and / or uptake of the polynucleotide composition by the seeds. Seeds can be treated with the polynucleotide composition using a seed batch treatment system or a continuous flow treatment system. Seed treatments can be applied in the laboratory or in commercial-scale treatment equipment such as a tumbler, mixer, or pan granulator. The polynucleotide composition used to treat seeds can contain one or more other desirable ingredients, including, but not limited to, a liquid diluent, a binder that acts as a base for the polynucleotide, a filler that protects the seeds during stress conditions, and a plasticizer that improves the flexibility, adhesion, and / or spreadability of the coating. Additionally, for oil-based polynucleotide compositions that contain little or no filler, a desiccant such as calcium carbonate, kaolin or bentonite clay, perlite, diatomaceous earth, or any other absorbent material can be added.

[0149] Certain embodiments provide for application of the composition during early, mid, and late vegetative stages of plant development. Certain embodiments provide for application of the composition during early, mid, and late reproductive stages. Application of the composition to plant parts at different stages of maturity is also provided.

[0150] The following examples are included to demonstrate examples of certain preferred embodiments of the invention. Those of skill in the art should understand that the techniques disclosed in the examples below represent approaches that the inventors have found to work well in carrying out the invention, and that these techniques can be considered to constitute examples of preferred modes for its practice. However, those of skill in the art should understand in light of the present disclosure that many changes can be made in the specific embodiments that are disclosed and still obtain like or similar results without departing from the spirit and scope of the invention. [Example]

[0151] Example 1 - Verification of improved yield traits in plants

[0152] The application of the composition (e.g., by irrigation or spraying) is timed according to the transcription of the target gene and the desired trait it affects. Examples of suitable timing are outlined in Table 2. Table 2 shows examples of suitable timing for applying the composition, based on the targeted gene and its expression (here for rapeseed). Depending on the timing of the expression of the specific gene, the dsRNA mixture is applied one week before the expected peak expression, at the expected peak expression, and one week after the peak expression. Following treatment, the plant is phenotyped for the targeted yield trait, for example, as outlined in Table 2. Table 2 shows examples of traits and their associated genes, for example.

[0153] Example 2 - Testing of Injection Agent Penetration

[0154] To test the systemicity of the inducers, leaves were sprayed with 0.01% to 1% or 0.1 to 10 mg / ml inducer solutions and contact angles were assessed using standard methods.

[0155] The effectiveness of the tested transfer agents is shown in Figure 1, which shows the contact angle (indicating penetration) as a function of time after application to plant leaves. As can be seen, all of the transfer agents tested significantly reduced the contact angle compared to when no transfer agent was applied.

[0156] Example 3 - Targeting BRC1 in rapeseed increases branching

[0157] A dsRNA molecule (SEQ ID NO: 730) directed against BnBRC1 (SEQ ID NO: 1) in rapeseed (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 to 10 mg / ml of a surfactant, here a siloxane polyalkylene oxide copolymer (Silwet® L-77AG), although other surfactants such as those listed in Table 1 may be used as well. BnBRC1 (SEQ ID NO: 730) dsRNA was applied from the beginning of bolting to the emergence of the second inflorescence.

[0158] Following treatment, plant branching was assessed by visual inspection. As shown in Figure 3, which shows illustrative images of Brassica napus plants following topical application of dsRNA, plant branching was significantly increased (5-6 branches per plant in mock-treated control plants (left) and 9-10 branches per plant in BnBRC1-dsRNA-treated plants with dsRNA containing the sequence set forth in SEQ ID NO: 730 (right)).

[0159] Example 4 - Ectopic application of dsRNA affects petal shape of rapeseed in the greenhouse and field

[0160] A dsRNA molecule (SEQ ID NO: 733) directed against the Brassica napus gene BnPTL (SEQ ID NO: 286) was applied using a hand spray at a dsRNA concentration of 1 ng / ml to 1 mg / ml diluted in 0.01% to 1% or 0.1 to 10 mg / ml of a surfactant, here a siloxane polyalkylene oxide copolymer (Silwet® L-77AG), although other surfactants such as those listed in Table 1 may be used as well. The dsRNA was applied when the plants reached 70% of the total stem length.

[0161] Following treatment, petal number was assessed by visual inspection. Figure 2 shows preliminary illustrative images of rapeseed plants, here Brassica napus, following topical application of BnPTL-dsRNA. Mock-treated plants (left) exhibited normal petal shape, while BnPTL-dsRNA-treated plants (right) displayed abnormal petal shape (three petals per flower or asymmetric flowers), demonstrating the ability of topically applied dsRNA to affect petal shape in rapeseed plants.

[0162] Example 5 - Ectopic application of dsRNA for yield enhancement - Brassica napus

[0163] Belinda rapeseed seeds (Brassica napus) were sown in 3-liter pots in a controlled greenhouse, one plant per pot, and watered and fertilized once daily. 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 further descriptions) and a dsRNA molecule (SEQ ID NO: 730) directed against the BnBRC1 gene (SEQ ID NO: 1) (hereinafter referred to as treatment "B"). Treatments were applied at 10 ml per plant at concentrations of 1 and 10 μg / ml of dsRNA diluted in surfactant (Silwet® L-77AG), depending on the appropriate developmental stage of the plants, as shown in Table 2 below.

[0164] Phenotypic assessments were performed one month after treatment application, and the results were compared with the control (surfactant only) for flower morphology in “A” (BnPTL-dsRNA) and branch number in “B” (BnBRC1-dsRNA).

[0165] method

[0166] 2 x 1,000 m in the Sharon region of Israel (32°10'1.55"N 34°52'33.96"E) 2 The field was divided into 12 rows of 52 m x 0.8 m, each separated by a 40 cm blank lane.

[0167] Seeds were planted using a hand-push planter, approximately 15 cm apart and 2 cm deep.

[0168] Each row was divided into 0.8m x 2m sections with approximately 70 plants per section, separated by 1m of untreated plants as spacers between adjacent different treatments.

[0169] Five types of dsRNA were tested: 1) a dsRNA molecule (SEQ ID NO: 733) directed against the Brassica napus BnPTL gene (SEQ ID NO: 286) - hereafter referred to as treatment "A"; 2) a dsRNA molecule (SEQ ID NO: 730) directed against the Brassica napus BnBRC1 gene (SEQ ID NO: 1) - hereafter referred to as treatment "B"; 3) a dsRNA molecule (SEQ ID NO: 731) directed against the Brassica napus BnCKX2 gene (SEQ ID NO: 158)—hereafter referred to as treatment “C”; 4) a dsRNA molecule (SEQ ID NO: 732) directed against the Brassica napus BnKIN10 gene (SEQ ID NO: 62)—hereafter referred to as treatment “D”; 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.”

[0170] These dsRNAs were sprayed using 200 ml of water containing the dsRNA and surfactant for each treatment. The dsRNA treatments were applied according to the expected timing of peak expression of each selected gene during the plant's "vegetative stage" (as shown in Table 2). [Table 2] TIFF0007827240000003.tif173154

[0171] For each treatment, two different dsRNA doses (1 or 10 μg / ml) and two different spray regimes (1 or 5 times in the first field, and 1 or 3 times in the second field) were applied, as shown in Table 3. The interval between subsequent treatments was one week. [Table 3]

[0172] Detergent alone (without dsRNA) was used as a control (Ctrl) at the same time.

[0173] Each treatment was replicated 10 times in different sections and sprayed using a "Solo" 2 liter hand sprayer with the smallest droplet size.

[0174] At the end of the growing season, each section was harvested by hand, dried for one week, processed by a thresher (Classic ST, Wintersteiger, Germany), and weighed for net seed weight for each section / treatment.

[0175] Data from all fields were statistically analyzed for each dsRNA treatment compared to its associated Ctrl (P<0.1).

[0176] Using a designated seed counter (Contador, Pfeuffer, Germany), 1,000 seeds were weighed in five replicates for each section and combined into a constant volume total weight. Oil content was determined using a standard seed counter (Contador, Pfeuffer, Germany) using a standard seed counter (Contador, Pfeuffer, Germany). , Ba At the Biotechnology Engineering Faculty, 「 The determination was carried out according to the Soxhlet extraction method using hexane as the solvent.

[0177] result

[0178] Process A

[0179] Flower morphology

[0180] Regarding process "A", ds Flower morphology was assessed 3 weeks after RNA application.

[0181] As can be seen in Figure 2, changes in floral morphology were observed only in dsRNA-treated plants. At least one flower in each inflorescence was missing one petal compared to the control. In addition, inflorescence development and flowering were delayed by approximately two weeks in dsRNA-treated plants compared to the control.

[0182] light transmittance

[0183] Three weeks after spray application of dsRNA (SEQ ID NO: 733), light transmittance at the base of the inflorescence was measured. Measurements were performed after peak flowering. dsRNA treatment caused a significant decrease in light transmittance for all treatments applied: 1-1 (1-1 = 1 μg / ml, 1 treatment), 10-1 (10-1 = 10 μg / ml, 1 treatment), 1-5 (1-5 = 1 μg / ml, 5 treatments), and 10-5 (10-5 = 10 μg / ml, 5 treatments) caused a 42.3%, 45.1%, 47.6%, and 50% decrease in light transmittance, respectively, compared to the control.

[0184] These results indicate that reducing BnPTL expression (SEQ ID NO: 286) by ectopic administration of dsRNA molecules targeting PTL reduces petal number, which in turn leads to increased light penetration to the lower parts of the plant and increases overall photosynthetic efficiency and yield.

[0185] Seed weight:

[0186] In field 1, treatments 1-5 and 10-5 increased seed weight by 4.9% and 9.4%, respectively (Figure 5A). In field 2, treatment 10-1 increased seed weight by 17.5% (Figure 5B).

[0187] These results indicate that reducing BnPTL expression by ectopic administration of dsRNA targeting BnPTL can increase seed weight.

[0188] Oil content:

[0189] In field 1, consistent increases in oil content of 1.4%, 1.9%, and 1.1% (for treatments 1-1, 1-5, and 10-1, respectively—FIG. 6A) were observed in dsRNA-treated plants.

[0190] In field 2, larger increases of 6%, 2.4%, 5.9%, and 2.3% (for treatments 1-1, 1-3, 10-1, and 10-3, respectively—FIG. 6B) were observed.

[0191] These results indicate that reducing BnPTL expression by ectopic administration of dsRNA targeting BnPTL can increase the oil content of rapeseed plants.

[0192] Process B

[0193] Number of branches:

[0194] For treatment "B," the number of branches was assessed 3 weeks after application of dsRNA.

[0195] As can be seen from Figure 3, dsRNA treatment (10 μg / ml) resulted in an increase in the number of branches, and as can be further seen from Figure 4, the increase in branch number was dose-dependent. Ta( P<0.1).

[0196] As can be seen from Figure 7, which shows the field results, when BnBRC1 (SEQ ID NO: 1) expression was targeted by ectopic administration of dsRNA (SEQ ID NO: 730 targeting BnBRC1), the number of branches in treatments 1-1, 1-5, and 10-5 was increased by 8.2%, 5.9, and 16.4%, respectively, compared to the control.

[0197] Process C

[0198] Seed weight:

[0199] As can be seen in Figures 8A and 8B, targeting BnCKX2 (SEQ ID NO: 158) expression by ectopic administration of CKX2-targeting dsRNA (SEQ ID NO: 731) resulted in increased seed weight in both fields tested. In field 1, treatments 1-1, 1-5, 10-1, and 10-5, increases were 2.1%, 1.3%, 1.3%, and 4%, respectively (Figure 8A). In field 2, treatments 1-1, 10-1, and 10-3 increased seed weight by 1.2%, 15.6%, and 9%, respectively (Figure 8B).

[0200] Weight and size of 1,000 seeds:

[0201] In addition, targeting BnCKX2 expression by ectopic administration of dsRNA targeting BnCKX2 resulted in increased 1,000 seed weights of 3.9%, 3.5%, and 1.6% over the control for treatments 1-1, 1-5, and 10-1, respectively, in field 1. Seed size in treatments 1-1, 1-5, and 10-5 similarly increased by 1.1%, 5.1%, and 1.2%, respectively.

[0202] In field 2, targeting BnCKX2 resulted in an increase in 1,000 seed weight of 5.8%, 1.5%, and 1.1% for treatments 1-3, 10-1, and 10-3, respectively. Seed size in treatments 1-3, 10-1, and 10-3 also increased by 4.1%, 0.7%, and 3.8%, respectively.

[0203] These results clearly demonstrate that targeting BnCKX2 expression by ectopic administration of dsRNA targeting BnCKX2 increases the weight and size of rapeseed seeds.

[0204] Processing E

[0205] Seed weight:

[0206] As shown in Figures 9A and 9B, targeting BnADPG1 (SEQ ID NO: 235) expression by ectopic administration of dsRNA (SEQ ID NO: 729) targeting BnADPG1 resulted in increased seed weight in both fields tested. In field 1, treatments 1-1, 1-5, and 10-1, increases were 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.

[0207] Weight and size of 1,000 seeds:

[0208] Targeting ADPG1 expression by ectopic administration of dsRNA targeting BnADPG1 resulted in increased 1,000 seed weights of 4.5%, 7.7%, 6%, and 7% over the control for treatments 1-1, 1-5, 10-1, and 10-5 in field 1. Seed size also increased by 6.8%, 8.2%, 1.1%, and 8.1%, respectively, in treatments 1-1, 1-5, 10-1, and 10-5.

[0209] In field 2, targeting BnADPG1 resulted in increases of 14.7%, 3%, and 13.3% per 1,000 seeds for treatments 1-3, 10-1, and 10-3, respectively. Seed size in treatments 1-1, 1-3, 10-1, and 10-3 was also increased by 2%, 13.3%, 2.2%, and 9.6%, respectively.

[0210] These results clearly demonstrate that targeting BnADPG1 expression by ectopic administration of dsRNA targeting BnADPG1 can increase rapeseed weight and size.

[0211] Oil content:

[0212] In field 1, increased oil content was observed in BnADPG1-dsRNA-treated plants (1.5%, 2.2%, and 1.5% for treatments 1-1, 10-1, and 10-5, respectively—Figure 10A). Similarly, in field 2, increased oil content was observed in treatments 1-3, 10-1, and 10-3 by 2.1%, 1.5%, and 4.1%, respectively, compared to the control (Figure 10B).

[0213] Example 6 - Ectopic application of dsRNA for yield enhancement - Rice (Oryza sativa)

[0214] Materials and Methods:

[0215] Rice (Oryza sativa spp.) seeds were sown in 3 liter pots, one seed per pot, in a greenhouse and watered and fertilized once daily.

[0216] The plants were treated with 10 μg / ml (in water and detergent) of the dsRNAs listed below. 1) a dsRNA molecule (SEQ ID NO: 742) directed against rice OsBRC1 (SEQ ID NO: 43); 2) a dsRNA molecule (SEQ ID NO: 744) directed against rice OsPIN5b (SEQ ID NO: 86); 3) A dsRNA molecule (SEQ ID NO: 746) directed against rice OsSKIN1 (SEQ ID NO: 52).

[0217] These dsRNAs were applied according to the developmental stage of the plants as shown in Table 4 below. [Table 4]

[0218] These dsRNAs were applied at a concentration of either 1 or 10 μg / ml diluted in surfactant (Silwet® L-77AG) as a single spray of 10 ml per plant, with 6 pots / plant per treatment.

[0219] These treatments were evaluated one month after dsRNA application compared with the control. For OsBRC1, the number of axillary branches was assessed, for OsPIN5b, the number of panicles was assessed, and for OsSKIN1, the seed size was assessed. Total seed weight was measured for all treatments.

[0220] result

[0221] One month after treatment, the total number of tillers in rice plants treated with dsRNA targeting OsBRC1 (SEQ ID NO: 742) was counted and compared with the control. Interestingly, targeting plants with low concentrations of OsBRC1-dsRNA caused a slight increase in the number of tillers, while high concentrations caused a decrease in the number of tillers (Figure 11). This indicates that dsRNAs targeting OsBRC1 can be used to control the number of tillers in rice.

[0222] Three months after treatment, the number of panicles in rice plants treated with dsRNAs targeting OsPIN5b (SEQ ID NOs: 744 and 745) is assessed compared to controls.

[0223] Three months after treatment, the total weight of the seeds is determined for all treatments.

[0224] Example 7 - Ectopic application of dsRNA for yield enhancement - soybean (Glycine max)

[0225] Materials and Methods:

[0226] Soybean (Glycine max, Williams 82 variety) seeds were sown in 3 liter pots, one seed per pot, in a greenhouse and watered and fertilized once daily or as needed.

[0227] Several examples of dsRNA applications (detailed below) aimed at causing phenotypic changes and increasing yield were tested. 1) SEQ ID NO: 734, directed against soybean GmBRC1 (SEQ ID NO: 15). 2) SEQ ID NO: 738, directed to soybean GmJAG1 (SEQ ID NO: 153). 3) SEQ ID NO: 736, directed against soybean GmBS1 (SEQ ID NO: 116). 4) SEQ ID NO: 740 directed to soybean GmPLDα1 (SEQ ID NO: 124).

[0228] These dsRNAs were applied according to the developmental stage of the plants as shown in Table 5 below. [Table 5]

[0229] dsRNA was applied as two sprays, the first at the predicted peak of gene expression and the second two weeks later. dsRNA was applied at a concentration of 1 μg / ml or 10 μg / ml diluted in surfactant (Silwet® L-77AG) at 10 ml for total plant surface coverage, with six pots / plant for each treatment. Each dsRNA test was performed in six replicates.

[0230] These treatments were evaluated one month after dsRNA application and compared with Ctrl. For GmBRC1 targeting, the number of axillary branches was evaluated, for GmJAG1 targeting, the number of seeds per pod was evaluated, for GmBS1 targeting, seed size was evaluated, and for GmPLDα1, seed weight / filling was evaluated. In addition, total seed weight was measured for all treatments.

[0231] result

[0232] One month after treatment, plants treated with dsRNA targeting GmBRC1 were evaluated for the total number of axillary branches. As can be seen in Figure 12, dsRNA-treated plants had a higher number of branches compared to control plants. Interestingly, the greatest increase (54.1%) was observed for lower dsRNA concentrations.

[0233] One month after dsRNA treatment targeting GmJAG1, plants are evaluated for total number of seeds per pod.

[0234] After total drying of the plant pods, plants treated with dsRNA targeting GmBS1 are assessed for seed size, and plants treated with dsRNA targeting GmPLDα1 are assessed for total seed weight.

[0235] While particular embodiments of the present invention have been illustrated and described, it will be apparent that the invention is not limited to the embodiments described herein. Numerous modifications, changes, alterations, substitutions, and equivalents will be apparent to those skilled in the art without departing from the spirit and scope of the invention, which is described by the following claims. The present invention may include the following aspects. [Aspect 1] A composition comprising: a dsRNA molecule comprising at least 18 consecutive nucleotides that are essentially identical to or essentially complementary to a portion of a plant gene or a transcript of the plant gene; and a transfer agent configured to promote 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 decrease in expression of the gene causes a change in a trait of the plant, the trait of the plant being selected from the group consisting of increased branching, increased grain filling, increased T6P levels, increased number of panicles, increased seed filling, increased seed number, increased seed size, reduced shedding tendency, reduced abscission tissue formation, increased number of tillers, increased heading of the plant, reduced petals, increased silique size, delayed or early flowering, delayed senescence, and any combination thereof. [Aspect 2] 2. The composition of aspect 1, wherein the plant gene is selected from ADPG1, PTL, CKX2, BRC1, KIN10, SKIN1, PIN5b, JAG1, BS1, PLDα1, and / or any homologue thereof or any combination thereof. Aspect 3 2. The composition of aspect 1, wherein the plant gene is selected from ADPG1, PTL, CKX2, BRC1, and / or any homologue thereof, or any combination thereof. Aspect 4 2. The composition of embodiment 1, wherein the plant is an oilseed rape plant, and the dsRNA molecule comprises at least 18 contiguous nucleotides that are essentially identical to or essentially complementary to a portion of a sequence encoding any 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. Aspect 5 2. The composition of embodiment 1, wherein the plant is a soybean plant, and the dsRNA molecule comprises at least 18 contiguous nucleotides that are essentially identical to or essentially complementary to a portion of a sequence encoding any 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. Aspect 6 6. The composition of embodiment 5, wherein the plant is a soybean plant, and the dsRNA molecule comprises at least 18 contiguous nucleotides that are essentially identical to or essentially complementary to a portion of a sequence encoding any 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. Aspect 7 2. The composition of embodiment 1, wherein the plant is a rice plant, and the dsRNA molecule comprises at least 18 contiguous nucleotides that are essentially identical to or essentially complementary to a portion of a sequence encoding any 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. Aspect 8 8. The composition of embodiment 7, wherein the plant is a rice plant, and the dsRNA molecule comprises at least 18 contiguous nucleotides that are essentially identical to or essentially complementary to a portion of a sequence encoding any of the amino acid sequences set forth in SEQ ID NO: 407, SEQ ID NO: 416, and SEQ ID NO: 450. Aspect 9 The composition of any one of aspects 1 to 8, wherein the dsRNA molecule is at least about 50 bases in length. Aspect 10 The composition of embodiment 9, wherein the dsRNA molecule is at least about 200 bases in length. Aspect 11 11. The composition of any one of aspects 1-10, wherein the introducing agent comprises N,N-dimethyldecanamide, cocoamidopropyldimethylamine, siloxane polyalkylene oxide copolymer, AG-RHO® EM-30, dimethylamide of C8 / C10 fatty acid, esterified copolymer of glycerin, trisiloxane ethoxylate, or any combination thereof. Aspect 12 A method for topically applying a composition according to any one of aspects 1 to 11 to a plant surface. Aspect 13 13. The method of embodiment 12, wherein said applying comprises spraying said composition onto the surface of the plant. Aspect 14 14. The method of embodiment 13, wherein the composition is sprayed onto the plant surfaces using a crop extension rod, a boomless sprayer, an agricultural sprayer, a crop duster airplane, a pressurized backpack sprayer, a track sprayer, or a laboratory sprayer / submersible. Aspect 15 13. The method of embodiment 12, wherein said applying comprises providing said composition through an irrigation system. Aspect 16 16. The method according to any one of Aspects 12 to 15, wherein the plant surface is a surface of one or more plant parts selected from the group consisting of hypocotyls, cotyledons, leaves, flowers, stems, tassels, meristems, pollen, ovules, and fruits. Aspect 17 17. The method of any one of aspects 12-16, further comprising timing said applying of said composition to a desired developmental stage of said plant. [Explanation of symbols]

[0236] [Figure 1] Contact angle on oilseed rape leaf: Contact angle on oilseed rape leaf Contact Angle: Contact angle Time (sec): Time (sec) No additives: No additives [Figure 2] Control: Control [Figure 3] Total number of branches: 11 (6 and 5 on each plant) Total number of branches: 19 (10 and 9 on each plant) Ctrl: Control [Figure 4] Branch Number: Number of branches Ctrl: Control [Figure 5] Seed Weight [gr]: Seed weight (g) Plot Type: Type of division Ctrls: Controls [Figure 6] 1st: 1st 2nd: 2nd Oil Content: Oil content Plot Type: Type of division Ctrls: Controls [Figure 7] Branch Number: Number of branches Plot Type: Type of division Ctrl: Control [Figures 8 and 9] 1st: 1st 2nd: 2nd Seed Weight [gr]: Seed weight (g) Plot Type: Type of division Ctrls: Controls [Figure 10] Oil Content: Oil content Plot Type: Type of division Ctrls: Controls [Figures 11 and 12] Number of Tillers: Number of tillers Ctrls: Controls

Claims

1. A composition comprising: A dsRNA molecule comprising at least 18 consecutive nucleotides that are identical to or complementary to a portion of the sequence set forth in SEQ ID NO: 43 of the rice plant gene OsBRC1; and a transfer agent configured to promote 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 the transient decrease in gene expression causes a change in a trait of the plant, the trait of the plant including an increase in the number of tillers; wherein the dsRNA molecule comprises SEQ ID NO: 742 or 743.

2. 10. The composition of claim 1, wherein the dsRNA molecule is at least 50 base pairs in length.

3. The composition of claim 2 , wherein the dsRNA molecule is at least 200 base pairs in length.

4. 10. The composition of claim 1, wherein the loading agent comprises N,N-dimethyldecanamide, cocoamidopropyldimethylamine, siloxane polyalkylene oxide copolymer, AG-RHO® EM-30, dimethylamide of C8 / C10 fatty acid, esterified copolymer of glycerin, trisiloxane ethoxylate, or any combination thereof.

5. A method for topically applying the composition of any one of claims 1 to 4 to plant surfaces.

6. 6. The method of claim 5, wherein said applying comprises spraying said composition onto the surface of the plant.

7. 7. The method of claim 6, wherein the composition is sprayed onto the surface of the plant using a crop extension rod, a boomless sprayer, an agricultural sprayer, a crop duster airplane, a pressurized backpack sprayer, a track sprayer, or a laboratory sprayer / submersible.

8. 8. The method of claim 7, wherein said applying comprises providing said composition through an irrigation system.

9. 9. The method of claim 8, wherein the plant surface is the surface of one or more plant parts selected from the group consisting of hypocotyls, cotyledons, leaves, flowers, stems, tassels, meristems, pollen, ovules, and fruits.

10. 10. The method of claim 8, further comprising timing said application of said composition to a desired developmental stage of said plant.

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