Flavonoid compositions and methods of use

Abstract

The present disclosure provides an agrochemical composition having a flavonoid. In certain aspects, the present disclosure also provides methods for restoring fertility in a flavonoid dependent fertile (FDF) plant including applying an effective amount of the agrochemical to the FDF plant. In certain aspects, the present disclosure also provides methods for inducing self-pollination in an FDF plant including applying an effective amount of the agrochemical to the FDF plant.

Description

FLAVONOID COMPOSITIONS AND METHODS OF USECROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of US Provisional Application Ser. No. 63 / 644,692 filed May 9, 2024, the entire contents of which are incorporated by reference herein.TECHNICAL FIELD

[0002] The present disclosure relates to compositions having flavonoids and their methods of use, for example, restoring fertility in flavonoid dependent plants.BACKGROUND

[0003] Hybrid corn, and the production thereof, is critical to the modern economy. Hybrids are important because particular characteristics, such as plant maturity, disease resistance, grain color, food processing quality, and so on, can be controlled as desired by the grower and / or end user of the corn. Furthermore, it is well recognized that when two unrelated corn lines are crossed to form a hybrid, the resulting hybrid will have significantly higher yield and vigor than the parents (also known as “hybrid vigor”). As a result, hybrid corn accounts for about 95 percent of corn acreage in the US.

[0004] In general, hybrid corn is defined as corn which results from fertilization of one corn plant with a genetically unrelated corn plant. However, effectively obtaining hybrid corn can be challenging. In particular, hybrid production involves, e.g., usage of isolated fields; specific arrangements of plants; field management including timing of planting, elimination of off-types, preventing self pollination, and processing to maintain seed quality, etc. Each step tends to be sequential, and therefore, errors in earlier stages can compound and result in complete failure or rejection of crops. Of the various steps / controls needed for successful hybrid production, one of the most challenging is ensuring proper pollination (z.e., that pollination results in the production of a hybrid).

[0005] Traditionally, to control pollination, the tassels of “female” plants (plants designated to produce hybrid seeds) are physically removed, manually or mechanically, such that the plant does not produce pollen and cannot pollinate itself or its neighbors. Detasseling machines typically remove 60-90% of tassels in a corn field, which is less than the 99.5% necessary for the uniformity desired by farmers, and the remaining tassels are manually removed. These female plants are planted in proximity to “male” plants (plants designated to pollinate the female plants), such that pollen from the male plants reaches the female plants,thereby providing cross-pollination and hybrid seed production. Manual / mechanical removal of female tassels leaves much to be desired given the laborious and tedious nature of removal.

[0006] More recently, genetic / chemical methods have been used to prevent or reduce self- pollination. These methods include cytoplasmic male sterility (CMS); roundup hybridized systems (RHS); and flavonol dependent fertility (FDF). Despite these new systems, manual / mechanical detasseling remains prevalent.

[0007] One challenge associated with genetic / chemical systems lies in the production of inbred lines used to produce hybrids. Inbred lines are plants which essentially have a fixed or uniform genetic composition which results from repeated self-pollination over many generations of plants. While inbreds allow for better control of properties / traits, inbred lines are usually smaller, less vigorous, and lower-yielding than open pollinated maize plants due to “inbreeding depression.” Furthermore, genetic traits which can facilitate pollination control can make production of the underlying inbred line more difficult.

[0008] Another challenge associated with genetic / chemical systems relates to the need to bulk inbreds of different maturities, which requires distinct production fields and variable application timings.

[0009] Accordingly, Applicant desires to improve genetic / chemical methods, and more specifically applicant desires formulations and related methods that improve FDF plant systems used to produce the inbred lines necessary for hybrid seed production. Further still, Applicant desires concentrated compositions that can be used to create stable dilutions suitable for use in bulking inbred plants of different maturities.SUMMARY

[0010] Embodiments of the disclosure relate to a sprayable agrochemical composition. The composition can include a flavonol, where the flavonol is configured as an active ingredient in the composition and is capable of restoring fertility of a flavonoid dependent fertility (FDF) plant. The composition can further include at least one flavonol enhancer.

[0011] Compositions of the disclosure can be applied to an FDF plant.

[0012] Methods of the disclosure further include methods of preparing hybrid seeds and / or bulking an inbred female FDF line, and seeds produced by such methods.BRIEF DESCRIPTION OF THE DRAWINGS

[0013] FIG. 1 shows a graph of surface tensions versus surface age.DETAILED DESCRIPTION

[0014] The present disclosure may be understood more readily by reference to the following detailed description of desired embodiments and the examples included therein.

[0015] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. In case of conflict, the present document, including definitions, will control. Preferred methods and materials are described below, although methods and materials similar or equivalent to those described herein can be used in practice or testing. All publications, patent applications, patents and other references mentioned herein are incorporated by reference in their entirety. The materials, methods, and examples disclosed herein are illustrative only and not intended to be limiting.

[0016] The singular forms “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise.

[0017] As used in the specification and in the claims, the term "comprising" can include the embodiments "consisting of' and "consisting essentially of.” The terms “comprise(s),” “include(s),” “having,” “has,” “can,” “contain(s),” and variants thereof, as used herein, are intended to be open-ended transitional phrases, terms, or words that require the presence of the named ingredients / steps and permit the presence of other ingredients / steps. However, such description should be construed as also describing compositions or processes as "consisting of' and "consisting essentially of' the enumerated ingredients / steps, which allows the presence of only the named ingredients / steps, along with any impurities that might result therefrom, and excludes other ingredients / steps.

[0018] As used herein, the terms “about” and “at or about” mean that the amount or value in question can be the value designated or some other value approximately or about the same. It is generally understood, as used herein, that it is the nominal value indicated ±10% variation unless otherwise indicated or inferred. The term is intended to convey that similar values promote equivalent results or effects recited in the claims. That is, it is understood that amounts, sizes, formulations, parameters, and other quantities and characteristics are not and need not be exact, but can be approximate and / or larger or smaller, as desired, reflecting tolerances, conversion factors, rounding off, measurement error and the like, and other factors known to those of skill in the art. In general, an amount, size, formulation, parameter or other quantity or characteristic is “about” or “approximate” whether or not expressly stated to besuch. It is understood that where “about” is used before a quantitative value, the parameter also includes the specific quantitative value itself, unless specifically stated otherwise.

[0019] Unless indicated to the contrary, the numerical values should be understood to include numerical values which are the same when reduced to the same number of significant figures and numerical values which differ from the stated value by less than the experimental error of conventional measurement technique of the type described in the present application to determine the value.

[0020] All ranges disclosed herein are inclusive of the recited endpoint and independently of the endpoints. The endpoints of the ranges and any values disclosed herein are not limited to the precise range or value and include values approximating these ranges and / or values.

[0021] As used herein, approximating language can be applied to modify any quantitative representation that can vary without resulting in a change in the basic function to which it is related. Accordingly, a value modified by a term or terms, such as “about” and “substantially,” may not be limited to the precise value specified, in some cases. In at least some instances, the approximating language can correspond to the precision of an instrument for measuring the value. The modifier “about” should also be considered as disclosing the range defined by the absolute values of the two endpoints. For example, the expression “from about 2 to about 4” also discloses the range “from 2 to 4.” The term “about” can refer to plus or minus 10% of the indicated number. For example, “about 10%” can indicate a range of 9% to 11%, and “about 1” can mean from 0.9-1.1. Other meanings of “about” can be apparent from the context, such as rounding off, so, for example “about 1” can also mean from 0.5 to 1.4. Further, the term “comprising” should be understood as having its open- ended meaning of “including,” but the term also includes the closed meaning of the term “consisting.” For example, a composition that comprises components A and B can be a composition that includes A, B, and other components, but can also be a composition made of A and B only.

[0022] The term “locus” as used herein means fields in or on which plants are growing, or where seeds of cultivated plants are sown, or where seed will be placed into the soil. It includes soil, seeds, and seedlings, as well as established vegetation.

[0023] The term “plants” refers to all physical parts of a plant, including seeds, seedlings, saplings, roots, tubers, stems, stalks, foliage, and fruits.

[0024] The term “plant propagation material” is understood to denote generative parts of the plant, such as seeds, which can be used for the multiplication of the latter, and vegetative material, such as cuttings or tubers, for example potatoes. There can be mentioned for example seeds (in the strict sense), roots, fruits, tubers, bulbs, rhizomes and parts of plants. Germinated plants and young plants which are to be transplanted after germination or after emergence from the soil, may also be mentioned. These young plants can be protected before transplantation by a total or partial treatment by immersion. Preferably “plant propagation material” is understood to denote seeds.

[0025] The term “flavonol enhancer” refers to component which enhances the ability of a flavonol to restore fertility in an FDF plant as compared to a composition without the component.

[0026] Flavonol enhancers, and compositions of the disclosure, can improve seed production / be configured to reach at least 3,750 (1000X) viable kernels per acre (9270 (1000X) viable kernels per hectare), preferably 5,000 (1000X) viable kernels per acre (12360 (1000X) viable kernels per hectare), and most preferably 5,500 (1000X) viable kernels per acre (13590 (1000X) viable kernels per hectare).

[0027] The term “agrochemical” or equivalent terms include compounds or ingredients used to achieve a desired response, directly or indirectly, from a plant or crop. In general, agrochemical active ingredients include compounds listed in: The Pesticide Manual, 12th edition, 2001, British Crop Protection Council. Agrochemicals include, but are not limited to herbicides, fungicides, other insecticides, bactericides, insect growth regulators, plant growth regulators, nematicides, molluscicides or mixtures of several of these preparations.

[0028] The term “agrochemically effective” or equivalent terms generally refer to rates of application of an agrochemical. In general, an agrochemically effective amount is an amount which elicits a desired response, for example, an amount which improves yield while avoiding undesirable levels phytotoxicity. An agrochemically effective amount is generally determined by the specific agrochemical and target (crop and effect). In specific embodiments, the term agrochemically effective amount refers to an amount of agrochemical which restores fertility of a sterile plant.

[0029] The rates of application of agrochemicals may vary within wide limits and depend on the nature of the soil, the method of application, the crop plant, the prevailing climaticconditions, and other factors governed by the method of application, the time of application and the target crop.

[0030] Agrochemical compositions of the disclosure can have a sole active ingredient or can be admixed with one or more additional active ingredients. An additional active ingredient may, in some cases, result in unexpected synergistic activities. In preferred embodiments, the composition contains a flavonol as the sole active ingredient.

[0031] The compositions of the disclosure may be employed in any conventional form, for example in the form of a twin pack (e.g., pack 1 = agrochemical / flavonol; pack 2 = flavonol enhancer), an emulsion concentrate (EC), a suspension concentrate (SC), a suspension concentrate for direct application (SD), a suspo-emulsion (SE), a capsule suspension (CS), a water dispersible granule (WG), an emulsifiable granule (EG), an emulsion, water in oil (EO), an emulsion, oil in water (EW), a micro -emulsion (ME), an oil dispersion (OD), an oil miscible flowable (OF), an oil miscible liquid (OL), a soluble concentrate (SL), an ultra-low volume suspension (SU), an ultra-low volume liquid (UL), a technical concentrate (TK), a dispersible concentrate (DC), a wettable powder (WP) or any technically feasible formulation in combination with agriculturally acceptable adjuvants. In general, such compositions are concentrates and diluted prior to use, however, composition can be provided in ready-to-use form (i.e., no dilution required). In preferred embodiments, the composition is a wettable powder (WP).

[0032] In general, the concentrate compositions include from 0.01 to 90% by weight of active agent, from 0 to 20% agriculturally acceptable surfactant and 10 to 99.99% solid or liquid formulation inerts and adjuvant(s). Concentrated forms of compositions generally contain between about 2 and 80%, preferably between about 5 and 70% by weight of active agent.

[0033] Application forms of compositions can, for example, contain from 0.001 to 20% by weight, or preferably from 0.002 to 5% by weight, active agent.

[0034] In specific embodiments, the composition of the disclosure can include pollen from an FDF plant, however, the composition preferably does not contain pollen from an FDF plant. WO2022271502A1 and W02023230039A1, both incorporated by reference in their entirety, provide techniques for storage and application of pollen.

[0035] In embodiments comprising pollen, the ratio (w / w) of pollen to flavonol (e.g., quercetin) can be from 1 : 1 to 1000: 1; preferably 20:1 to 200:1; or most preferably 50: 1 to100: 1. In embodiments where pollen is not included in the composition, the plant (e.g., corn) being treated will retain its tassel to allow self-pollination or be within pollination proximity of a second plant configured to pollinate the plant. Generally, the plant or the second plant will be able to pollinate itself and other plants within pollination proximately. Typical planting densities include from 30,000 to 150,000 plants / hectare; preferably 75,000 to 125,000 plants / hectare; or most preferably 90,000 to 110,000 plants / hectare.

[0036] In certain embodiments, the composition for application can include one or more buffers to control pH. In specific embodiments, the composition can have a pH between 1 and 10; preferably between 6 and 7; or about 6 or about 7.

[0037] As used herein, the term “adjuvant” refers to a substance, or a combination of compounds or substances, that is used to increase the efficacy or potency of certain active ingredients. An adjuvant can be a flavonol enhancer if the adjuvant meets the definition of flavonol enhancer, as used herein. In certain embodiments, the adjuvant can have a boiling point between about 150-275°C; preferably about 218-257°C; or most preferably about 227- 254°C.

[0038] As used herein, adjuvants include surfactants. Surfactants can increase surface contact, reduce runoff, and increase leaf penetration. Surfactants include nonionic surfactants (NIS) and anionic surfactants. Typical amounts of surfactants can be 1-2 pints per 100 gallons of spray solution or 0.25 to 0.5% volume per volume (v / v).

[0039] In general, there are three oil-based adjuvants: crop oil concentrates, crop oils, and vegetable oils concentrates. Crop oil concentrates (COCs) are normally formed as emulsifiable petroleum-based oils (83 to 85%) with a minor percentage of a nonionic surfactant. Standard application rates are 1-2 quarts per 100 gallons (or 1 to 2.5% v / v). COCs are often known as penetrating agents. Vegetable oil concentrates (VOCs) include crop oils such as cotton, linseed or soybean oil and a small percentage of a non-ionic surfactant.Methylated seed oils (MSOs) are vegetable oils that have been modified through a process of esterification. MSOs are typically recommended at 0.25 to 1.0% v / v of spray solution. Crop oils are more than 95 percent paraffin or naptha-based petroleum oil with 1 to 2 percent nonionic surfactant.

[0040] The crop oil adjuvant can include a synthetic isoparaffinic solution, or a hydrotreated light distillate, such as those defined in CAS 64742-47-8. The isoparaffinic solution canpreferably include a mixture of C11-C16 alkanes, in more preferred embodiments, the isoparaffinic solution is composed mostly (greater than 50%) of C13-C14 alkanes.

[0041] In some embodiments, the synthetic isoparaffinic solution can include an ISOPAR™ M solution. ISOPAR™ M can be 0.5-15% w / w; preferably 5-20% w / w; and most preferably 8-10 % w / w of the composition. In some embodiments the crop oil adjuvant is at least partially an ISOPAR™ M solution. In some embodiments, the crop oil adjuvant is 100% an ISOPAR™ M solution. ISOPAR™ M contains about the following amounts of alkanes

[0042] In some embodiments, the synthetic isoparaffinic solution that is used as crop oil adjuvant in this formulation has a lower viscosity and lower boiling temperature than other oil adjuvants. The lower boiling temperature decreases the time the synthetic isoparaffinic solution remains on the plant.

[0043] Flavonoids are phytochemical compounds which are a class of polyphenolic secondary metabolites. In general flavonoids have a 15-carbon skeleton, which consists of two phenyl rings (A and B) and a heterocyclic ring (C, a ring containing an embedded oxygen). Flavonoids can be classified into different groups, such as anthocyanidins, flavonols, flavanones, flavan-3-ols, flavanonols, flavones, and isoflavones. In addition, flavonoids can be found as both glycoside-bound and free aglycone forms.

[0044] Flavonols are a class of flavonoids. Flavonols have a 3 -hydroxyflavone backbone. Table A provides a list of flavonol glycosides and their aglycones.Table A

[0045] In preferred embodiments, the flavonol is an aglycone. Examples of flavonols includes 3-hydroxyflavone, azaleatin, fisetin, galangin, gossypetin, kaempferide, kaempferol, isorhamnetin, morin, myricetin, natsudaidain, pachypodol, quercetin, rhamnazin, and rhamnetin. In preferred embodiments, the flavonols include compounds having the following formula:

[0046] wherein Ri, R2, R3, R4, Rs, R7, and Rs, are hydrogen, hydroxyl or alkoxy having from 1 to 3 carbon atoms. More preferably, not more than two of Ri -Rs are hydroxyl or methoxy and the remaining Ri -Rs are hydrogen, and R7 and Rs are hydrogen, hydroxyl or methoxy.

[0047] In more preferred embodiments, the flavonol is quercetin. In most preferred embodiments, the flavonol is quercetin dihydrate.

[0048] Flavonols useful in the practice of the disclosure may be readily determined using an in vitro pollen rescue assay. Examples of such assays are discussed in U.S. Patent 5,733,759, incorporated by reference in its entirety. U.S. Patent 5,733,759, further discusses flavonols and methods useful in the present disclosure.

[0049] In certain embodiments, where the flavonol is formulated with liquid components when applied to the plant, the flavonol can have a target particle size distribution that maximizes formulation efficacy. For example, a Dx [4,3] of 2.39 pm to 4.03 pm; Dx (50) of 1.49 pm to 2.54 pm; Dx (95) of 7.24 pm to 12.6 pm; Dx (90) of at least 5.53 pm to 8.93 pm. Typical ranges for flavonol particle size can be a Dx [4,3] of 2.39 pm to 12 pm; preferably 2.39 pm to 4.03 pm; and most preferably 2.39 pm.

[0050] Application rates of flavonols can be 23 mg to 705 mg per plant; preferably 23 mg to 235 mg; and most preferably 23 mg, however, the amount may vary depending on the specific flavonol and formulation components therein. Application rates in the context of a field of crops can be 0.70 to 105.73 kg per hectare; preferably 0.70 to 35.24 kg per hectare; or most preferably 0.70 to 3.52 kg per hectare. The application rate of the flavonol can also be based on algorithmic estimates which take into account the type of crop or cultivar (including the amount of pollen typically produced), the specific application equipment, and environmental conditions.

[0051] As used herein, the term flavonoid deficient or flavonoid dependent fertility (FDF) plants, refers to plants which are male sterile based on impaired flavonol activity in the plant. These plants are conditionally male fertile (CMF) plants. FDF plants can be produced using techniques known in the art. For example, FDF plants may have chaicone synthase (CHS), a flavanone-3 -hydroxylase (F3H), or a flavonol synthase (FLS) activity impaired, naturally, transgenically, or through genome editing, to disrupt the natural production of flavonoids in the plant. F3H activity may be impaired by blocking F3H production in the plants, directly or indirectly, for example, by inactivating F3H naturally produced by the plants or by impairing the activity of a precursor enzyme, such as chaicone synthase (CHS) in the flavonol biosynthetic pathway. A flavonoid-deficient, conditionally male fertile plants will typically be pigment deficient, resulting in a white or pale coloration, and will typically be self sterile. Disclosure related to FDF plants is disclosed in, e.g., U.S. Patent 6,005,167 and W02024030797A2, incorporated by reference in their entirety.

[0052] The sterility is conditional as male fertility can be rescued or restored by providing fertility restoring flavonols to the pollen. Although viable pollen is produced by flavonol deficient plants, pollen germination and tube growth are severely reduced, resulting in plants which are self sterile. However, contacting pollen of the plant with an amount of a fertilityrestoring flavonol is effective to enhance germination of the pollen and normal pollen tube growth may be restored.

[0053] Plants which can be relevant include perennial and annual crops, such as berry plants for example blackberries, blueberries, cranberries, raspberries and strawberries; cereals for example barley, maize (corn), millet, oats, rice, rye, sorghum triticale and wheat; fibre plants for example cotton, flax, hemp, jute and sisal; field crops for example sugar and fodder beet, coffee, hops, mustard, oilseed rape (canola), poppy, sugar cane, sunflower, tea and tobacco; fruit trees for example apple, apricot, avocado, banana, cherry, citrus, nectarine, peach, pear and plum; grasses for example Bermuda grass, bluegrass, bentgrass, centipede grass, fescue, ryegrass, St. Augustine grass and Zoysia grass; herbs such as basil, borage, chives, coriander, lavender, lovage, mint, oregano, parsley, rosemary, sage and thyme; legumes for example beans, lentils, peas and soya beans; nuts for example almond, cashew, ground nut, hazelnut, peanut, pecan, pistachio and walnut; palms for example oil palm; ornamentals for example flowers, shrubs and trees; other trees, for example cacao, coconut, olive and rubber; vegetables for example asparagus, aubergine, broccoli, cabbage, carrot, cucumber, garlic, lettuce, marrow, melon, okra, onion, pepper, potato, pumpkin, rhubarb, spinach and tomato; and vines for example grapes. However, preferred crops / plants are corn plants.

[0054] Crops are to be understood as being those which are naturally occurring, obtained by conventional methods of breeding, transgenically, or through genome editing. They include crops which contain so-called output traits (e.g. improved storage stability, higher nutritional value and improved flavour).

[0055] Crops are to be understood as also including those crops which have been rendered tolerant to herbicides or classes of herbicides (e.g. ALS-, GS-, EPSPS-, PPO-, ACCase- and HPPD-inhibitors) by conventional methods of breeding or by genetic engineering. An example of a crop that has been rendered tolerant to imidazolinones, e.g. imazamox, by conventional methods of breeding is Clearfield® summer rape (canola). Examples of crops that have been rendered tolerant to herbicides by genetic engineering methods include e.g. glyphosate- and glufosinate-resistant maize varieties commercially available under the trade names RoundupReady® and LibertyLink®.

[0056] Crop plants can also be tolerant to protoporphyrinogen oxidase (PPO) inhibitors. Such plants can be obtained, for example, by transforming crop plants with nucleic acids which encode a suitable protoporphyrinogen oxidase, which may contain a mutation in order tomake it more resistant to the PPO inhibitor. Examples of such nucleic acids and crop plants are disclosed in WO95 / 34659, WO97 / 32011, W02007 / 024739, W02012 / 080975, WO2013 / 189984, WO2015 / 022636, WO2015 / 022640, WO2015 / 092706, WO2016 / 099153, WO20 17 / 023778, WO2017 / 039969, WO2017 / 217793, WO2017 / 217794, WO2018 / 114759, WO20 19 / 117578, WO2019 / 117579 and WO2019 / 118726, all incorporated by reference in their entirety herein.

[0057] Crops are also to be understood as being those which have been rendered resistant to harmful insects by genetic engineering methods, for example Bt maize (resistant to European corn borer), Bt cotton (resistant to cotton boll weevil) and also Bt potatoes (resistant to Colorado beetle). Examples of Bt maize are the Bt 176 maize hybrids of NK® (Syngenta Seeds). The Bt toxin is a protein that is formed naturally by Bacillus thuringiensis soil bacteria. Examples of toxins, or transgenic plants able to synthesise such toxins, are described in EP-A-451 878, EP-A-374 753, WO 93 / 07278, WO 95 / 34656, WO 03 / 052073 and EP-A- 427 529. Examples of transgenic plants comprising one or more genes that code for an insecticidal resistance and express one or more toxins are KnockOut® (maize), Yield Gard® (maize), NuCOTIN33B® (cotton), Bollgard® (cotton), NewLeaf® (potatoes), NatureGard® and Protexcta®. Plant crops or seed material thereof can be both resistant to herbicides and, at the same time, resistant to insect feeding ("stacked" transgenic events). For example, seed can have the ability to express an insecticidal Cry3 protein while at the same time being tolerant to glyphosate.

[0058] Further examples of such crops include: RR1, RR2 XTEND, XtendFlex, LibertyLink, Enlist E3, GTS 40-3-2, MON89788, A2704-12, GT27, LLGT27, or GMB 151. In preferred embodiments, the seeds are HT4 soybean (MON94313) or HT4 corn (MON87429). As used herein, the term transgenic “event” refers to a recombinant plant produced by transformation and regeneration of a single plant cell with heterologous DNA, for example, an expression cassette that includes a gene of interest. The term “event” refers to the original transformant and / or progeny of the transformant that include the heterologous DNA. The term “event” also refers to progeny produced by a sexual outcross between the transformant and another corn line. Even after repeated backcrossing to a recurrent parent, the inserted DNA and the flanking DNA from the transformed parent is present in the progeny of the cross at the same chromosomal location. Normally, transformation of plant tissue produces multiple events, each of which represent insertion of a DNA construct into a different location in the genomeof a plant cell. Based on the expression of the transgene or other desirable characteristics, a particular event is selected. Thus, for example, “event 3272”, “3272” or “3272 event” as used herein means the original 3272 transformant and / or progeny of the 3272 transformant and / or plants derived in any way from the original 3272 transformant. For 3272, see WO06 / 098952.

[0059] Other examples of transgenic events include, but are not limited to, MIR162 (See W007142840), Btl l (See US6114608 (construct) and WO8705629 (gene)), GA21 (See W09704103 (gene) WO9844140 (cassette)), MIR604 (See WO05103301), MZIR098 (See WO18231890), 5307 (See W010077816), DAS40278 (See US8598413), TC1507 (See WO04099447), DAS-59122-7 (See WO06 / 039376), NK603 (See US6825400), MON810 (See US6713259), MON863 (See US7705216), MON89034 (See W007140256), MON88017 (See W005059103), DP-4114 (See WO11084621), and MON87411 (See WO13 169923).

[0060] In specific embodiments, the present disclosure provides agrochemical compositions which are wettable powders. The wettable powder composition can include one or more active ingredients and one or more surfactants / adjuvants. The adjuvant can serve to increase the penetration of the active ingredient upon application, including, for example, spray application. The wettable powder composition includes one or more active ingredients including, for example, one or more flavonoid compounds. The one or more flavonoid components can include, for example, quercetin. The active ingredient can include a concentration of at least about 10 g / L. The active ingredient can include a concentration in the range of from about 10 g / L to about 30 g / L.

[0061] In certain embodiments, the wettable powder includes one or more surfactants / adjuvants. The one or more surfactants can include one or more anionic surfactants.

[0062] The wettable powder can further include a wettable powder base such as ATLOX™ BS-50. ATLOX™ BS-50 is a powder developed by CRODA that incorporates a combination of wetting agents, dispersants, and fillers or inerts. In general, the wetting agents and dispersants in a wettable powder are configured to disperse the active ingredient of the wettable powder throughout a carrier liquid (e.g. water) in a tank prior to application. The amounts of wetting agents, dispersants, and fillers can be selected based on the active ingredient.

[0063] The active ingredient in a wettable powder may be present in an amount of 10 to 80% w / w, preferably 30 to 50% w / w, and most preferably 30 to 35% w / w.

[0064] Examples of wetting agents include: Mowet® D-425 (a sodium salt of naphthalene sulfonate condensate), Agrilan® 700 (a hydrophobically modified polyacrylate copolymer), Enviomet® WT (e.g., ENVIOMET® WT 4510 (Sodium lauryl sulfate); ENVIOMET® WT 4950 (Alpha olefin sulfonate sodium salt); ENVIOMET® WT 7175 (Sodium methyl cocoyl taurate)), Lissapol® D powder (sulphate alcohols based composition). Wetting agents can be present in a range of 2 to 5% w / w.

[0065] Examples of dispersants include: Atlox metasperse® 550S (modified styrene acrylic polymer), REAX®280 (sodium sulfite lignosulfonate), Krafstperse® DW-5 (kraft lignin). Dispersants can be present in a range of 2 to 5% w / w.

[0066] Examples of fillers or inerts include: kaolin, bentonite, talc, and attapulgite. Fillers can be present in a range of 30 to 70% w / w.

[0067] In sum, compositions of the disclosure include a composition having: a flavonol at 0.1 to 80% w / w, preferably 1 to 50% w / w, and most preferably 1 to 15% w / w; a flavonol enhancer at 0.5 to 40% w / w, preferably 10 to 20% w / w, and most preferably 5 to 10 % w / w; a wettable powder base or mixture of wetting agents, dispersants, and fillers at 10 to 70 % w / w, preferably 30 to 70% w / w, and most preferably 50 to 70% w / w; and a liquid carrier (preferably water) at 10 to 95% w / w; preferably 50 to 95% w / w; and most preferably 80 to 95 % w / w.

[0068] Further compositions of the disclosure can include a composition having: a flavonol at 10 to 80 % w / w, preferably 30 to 50 % w / w, and most preferably 30 to 35 % w / w; and a wettable powder base or mixture of wetting agents, dispersants, and fillers at 10 to 90% w / w, preferably 30 to 80 % w / w, and most preferably 50 to 70 % w / w.

[0069] In embodiments having a flavonol and wettable powder base, the flavonol and wettable powder base can be mixed with flavonol enhancer prior to application which can also include an carrier (preferably water). In such embodiments, the flavonol enhancer is mixed with the flavanol containing component and applied such that the flavonol is not degraded. As understood by a person of ordinary skill in the art, application of thecomposition can be configured such that the application (or batch thereof) is completed prior to degradation of the flavanol. Application windows for the combination of flavonol and flavonol enhancer is preferably less than 2-3 days from mixing; preferably the application is same day from mixing.

[0070] The wettable powders of the disclosure can further be incorporated into a dissolvable pouch to facilitate storage and metering prior to application.

[0071] In certain embodiments, the wettable powder can be subjected to high sheer mixing prior to or during application. As used herein high sheer mixing refers to mixing sufficient to create an isotropic composition. The force required to create an isotropic composition will vary based on the components of the composition. However, the high sheer force required to form an isotropic composition can be between 3000 and 6000 ft / min and preferably 3000 to 4000ft / min. In general, the high shear strength disperses the wettable powder composition into the Isopar® M liquid.

[0072] In certain aspects, the agrochemical compositions as disclosed herein are sprayable. In some embodiments, the agrochemical compositions as disclosed herein can be prepared into a liquid form.

[0073] In general, the composition applied to the plant, or tassel thereof, can be configured to provide a minima film thickness to ensure gas exchange is maintained between the tassel and surrounding environment. The minima film thickness is a thickness which does not result in suffocation or death of the tassel.

[0074] In some embodiments, the agrochemical compositions of the present disclosure include one or more additives. The one or more additives can include one or more thickeners, stabilizers, biocides, surfactants, stabilizers, pigments / dyes, or one or more combinations thereof. Embodiments of the disclosure can be configured such that the flavonol, per se, generates an optically detectable composition thereby negating the need of pigments / dyes.

[0075] In certain aspects, the present disclosure provides methods for restoring fertility in a FDF organism / plant. The FDF plant can include any plant as understood in the art including, for example, one or more agricultural crop plants. Embodiments of the methods include administering to the FDF plant an effective amount of the agrochemical composition as disclosure herein. The agrochemical composition can include a composition in a liquid form.In certain embodiments, the methods can include administering the agrochemical composition using one or more suitable techniques including, for example, spraying.

[0076] The effective amount of the agrochemical composition can include an amount sufficient to restore fertility in the FDF plant. The methods can include administering the agrochemical composition a sufficient number of times in order to sufficiently restore fertility in the FDF plant. In some embodiments, the methods include administering the agrochemical to the FDF plant in a single application.

[0077] Specific methods of the disclosure further include methods of preparing hybrid seed by, inter alia, bulking an inbred female FDF line, where the inbred female line is bulked by planting a plurality of inbred female FDF seeds in an inbreed production field, and applying an effective amount of flavonol composition to the plurality of inbred female FDF plants grown from the seed; harvesting the bulked inbred female FDF seed; planting the bulked inbred female FDF seed in a hybrid seed production field; planting a plurality of inbred male seed in the hybrid seed production field, where the inbred male seed and the bulked inbred female FDF seed are planted in at least one of the following ratios 1 : 1, 1 :2, 1:3, 1 :4, 1 :5, 1 :6, 1 :7, 1 :8, 1 :9 and 1 : 10 (or ranges thereof); and harvesting the hybrid seed.

[0078] In specific embodiments the method can include specifically harvesting the cob. The method can also include conditioning the hybrid seed, where conditioning includes at least one of shelling the hybrid seed, cleaning the hybrid seed, sizing the hybrid seed, and treating the hybrid seed with a fungicide. Additional steps can include bagging the hybrid seed and / or destroying a plurality of the plants grown from the inbred male seed prior to harvesting the hybrid seed.

[0079] More general methods of the disclosure include applying an effective amount of a flavonol composition to a tassel of an inbred female FDF plant between the start of emergence from the leaf whorl and prior to the start of anthesis (anther extrusion and pollen shed).

[0080] The disclosure further includes both FDF seeds and hybrid seeds produced by the above methods.

[0081] Although only a few exemplary embodiments of this disclosure have been described in detail above, those skilled in the art will readily appreciate that many modifications are possible in the exemplary embodiments without materially departing from the novelteachings and advantages of this disclosure. Accordingly, all such modifications are intended to be included within the scope of this invention as defined in the following claims.

[0082] Following the protocol generally outlined in Mo & Taylor, Proc. Natl. Acad. Sci. USA Vol. 89, pp. 7213-7217, August 1992 (incorporated by reference herein), an initial formulation was developed by focusing on direct pollen complementation. The protocol used for development entailed collecting FDF maize pollen from a native mutant line, mixing the pollen with powdered flavonol (quercetin, kaempferol, or a 1:1 mix of quercetin to kaempferol) at a 20: 1 pollen to flavonol powder ratio. Testing mixed maize pollen and flavonol powders by weight (w / w) to increase consistency across experiments. One form of kaempferol and three forms of quercetin were sourced for this testing. These are described in Table B.Table B

[0083] Three commonly available forms of quercetin were sourced in order to test for differences in performance during direct pollen complementation; laboratory grade quercetin anhydrous, laboratory grade quercetin dihydrate, and dietary supplement grade quercetin dihydrate. The laboratory grade quercetin anhydrous was tested in the first experiment.Table C

[0084] The results demonstrated that kaempferol was not required for fertility restoration in FDF maize pollen, and surprisingly, seed set was higher in plants treated with quercetin alone. However, both were below the theoretical maximum kernel count of about 400 kernelsper ear for this line. It was observed that both treatments decreased pollen tube germination when pollen mixed with flavonols was placed onto pollen tube germination media.Accordingly, further treatments focused on optimizing pollen tube germination and seed set performance.

[0085] It was hypothesized that the reduced rate of pollen tube germination was due to small quercetin particles excessively coating FDF pollen grains and inhibiting the direct pollen to silk or pollen to germination media contact required to initiate pollen tube germination. Based on this theory, additional experiments were conducted to test the three commonly available forms of quercetin and whether the quercetin dosage could be reduced to reduce inhibition of pollen tube germination.

[0086] Pollen tube germination media was used to assess the interaction between samples using the ordinal categorical rating scale described in Table D.Table D

[0087] Results from testing the three forms of quercetin and dosage effect are summarized in Table E.Table ENA= Not Applicable

[0088] Germination plate testing validated that there were negative interactions between fine powdered quercetin anhydrous and maize pollen. The germination rating of FDF maize pollen alone was the maximum score of 5, while the germination rating after mixing withquercetin anhydrous at a 20: 1 ratio w / w was the lowest score of 1. The same negative interactions and reduction to pollen tube germination to a score of 1 was observed when FDF pollen was mixed at a 20: 1 ratio w / w with a 1 : 1 mixture of quercetin anhydrous and kaempferol anhydrous. Pollen tube germination plate testing demonstrated an immediate improvement in pollen tube germination when the dietary supplement grade quercetin dihydrate was substituted into the original protocol. Anhydrous quercetin and anhydrous kaempferol reduced pollen tube germination on media to zero or low levels. Reducing the pollen to quercetin anhydrous ratio from 20: 1 to 50: 1 by weight showed a slight improvement to pollen tube germination, suggesting that titrating the quercetin dose would yield further improvement. Accordingly, further titration testing was conducted focusing on the dietary supplement grade quercetin dihydrate.

[0089] In the titration testing, non-FDF pollen was used as a positive check for seed set. Pollen from wild-type plants was not mixed with quercetin prior to pollination. FDF pollen that was not mixed with any quercetin prior to pollination was used as a negative check. The results are shown in Table F.Table FNA= Not Applicable

[0090] The titration experiment demonstrated that reducing the pollen to quercetin dihydrate ratio from 20: 1 to 100: 1 could yield improved seed set while using less flavonol per pollination. Reducing the pollen to quercetin ratio below 100: 1 showed a dramatic reduction in seed set per pollination.

[0091] Following the direct pollen complementation discussed above, a series of range finding experiments were conducted to explore applying quercetin directly to tassels as an active ingredient suspended in a liquid carrier. It was hypothesized that directly applying quercetin to tassels would restore fertility in FDF plants by one of two methods: 1) quercetin would enter the anther tapetai cell layer in a bioavailable form where it would be recognizedby endogenous enzymes and loaded onto the mature pollen coat direct as quercetin or as a quercetin or glycoside, or 2) the quercetin would directly coat the pollen grains within the anther tapetai cell layer and be present on the mature pollen coat at the time of anthesis. Novec™ 7500, a hydrofluoroether based engineered fluid, was selected for testing as a positive check based on known capacity to carry insoluble molecules into plant cells. 5-25% DMSO solutions were tested based on their common use to deliver molecules into plant cells. Paraffin and sunflower oils were selected as carriers for quercetin based on quercetin being effectively insoluble in aqueous solutions and the known compatibility of mature maize pollen with these oils during pollination. Tested ranges of quercetin concentrations in suspension provided an initial assessment of required dosage. Tassels were treated at various timepoints in development to assess when in FDF pollen maturation quercetin could be applied and result in restored pollen fertility. For treatments applied up to seven days prior anthesis (start of anther extrusion and pollen shed), 10 ml of liquid suspension was applied directly to the emerged maize tassels. For formulations applied greater than seven days prior to anthesis, 10 ml of liquid suspension was applied by pouring the liquid down the leaf whorl to reach the developing tassels prior to emergence. Negative check treatments were accomplished both by leaving some tassels completely untreated. An additional check for the baseline effect of Novec™ 7500 on FDF maize pollen was accomplished by treating selected tassels with Novec™ 7500 that did not contain any quercetin. The results of these range finding experiments are reported in Table G.Table GAl = active ingredient

[0092] These range finding experiments provided a number of key learnings to inform formulation development. Novec™ 7500 successfully delivered quercetin anhydrous and quercetin dihydrate to the FDF maize pollen and resulted in restored fertility. The restored male fertility was able to achieve complete seed set in some test pollinations. The highest seed set was observed when 20 mg / ml quercetin anhydrous suspended in Novec™ 7500 suspension was applied two days prior to the start of pollen shed. Quercetin anhydrous suspensions in Novec™ 7500 restored fertility when applied up to ten days prior to the start of pollen shed. The highest seed sets were obtained when the quercetin anhydrous suspended in Novec™ 7500 was applied up to seven days prior to the start of pollen shed. Applying quercetin anhydrous suspended in Novec™ 7500 at 28 and 32 days prior to the start of pollen shed did not result in fertility restoration and seed set. These results indicated that the formulation had to be applied directly to the tassel to result in restored male fertility. This direct application was possible when the maize tassel emerges seven days prior to the start of pollen shed. Direct application is also possible up to 10 days prior to the start of pollen shed when the maize tassel is visible within the leaf whorl and liquid poured down the leaf whorl will directly coat the pre-emerged tassel. Applying quercetin anhydrous suspended in Novec™ 7500 resulted in higher seed set than applying quercetin dihydrate suspended in Novec™ 7500 when the applications occurred four days prior to the start of pollen shed. Negative check treatments including untreated tassels and tassels treated with only Novec™ 7500 resulted in zero or near-zero seed set, indicating that male sterility was stable and that airborne pollen contamination in the crossing environment was negligible.

[0093] Pure oil suspensions completely coated maize tassels to the point of blocking gas exchange, resulting in suffocation and death of the entire tassel prior to anthesis. These results demonstrated that liquid carrier oils applied to maize tassels must leave behind minima film thickness to ensure gas exchange is maintained between the rapidly developing tassel and the surrounding environment. It was noted that suspending insoluble compounds in DMSO solutions did not successfully deliver quercetin to the developing pollen.

[0094] Additional testing performed in relation to this disclosure included study of the alternative flavonoid active ingredients. The flavanone naringenin, the flavanonol taxifolin (dihydroquercetin), and flavonol morin, the flavonoid glycosides naringin and rutin, and the flavones apigenin and luteolin were tested singly or in combination by suspending the powders in Novec™ 7500 and applying the suspension to FDF maize tassels several days prior to the start of pollen shed. The flavonol kaempferol suspended in Novec™ 7500 was also included to study efficacy in a liquid formulation. As a test of partial efficacy, pollen collected from treated tassels was suspended in a strong base solution, IM potassium hydroxide (KOH, pH=14). This strong base solution converted flavonoids on the pollen grain surface into highly charged forms, making these molecules water soluble and able to dissolve into the IM KOH solution. This resulted a visible solution color change to yellow or orange depending on the flavonoid and concentration in solution. The color change was used to verify that the active ingredient tested reached the pollen during Novec™ 7500 application. The flavonol isorhamnetin was tested by direct pollen complementation due to high costs and limited quantity available. A summary of the testing is provided in Table H.Table H

[0095] These studies indicated that the flavonols morin and isorhamnetin are viable active ingredients that can restore FDF maize pollen fertility and seed set from pollination. These results also confirmed that kaempferol alone can restore FDF maize pollen fertility and that kaempferol can be delivered to maturing maize pollen grains when applied to maize tassels in a liquid suspension. For all Novec™ 7500 suspension tassel treatments, the pollen shed from treated tassels produced a positive IM KOH solution color change from clear to yellow or orange. This indicated that the active ingredients were reaching the maize pollen grains. The other active ingredients tested did not restore FDF maize pollen fertility.

[0096] In order to verify that liquid formulations were delivering bioavailable quercetin to developing pollen within the anthers on maize tassels prior to emergence and pollen shed, pollen samples were collected and analyzed by mass spectrometry for the presence of quercetin, quercetin-3 -glucoside (isoquercetin), and rutin. It was hypothesized that if the formulation was delivering bioavailable quercetin into the anther tapetai cell layer, and that if quercetin was recognized by enzymes (e.g., flavonol 3-O-glucosyltransferase) that conjugate the quercetin into quercetin glycosides, and if transporters within the anther tapetai cells could export those quercetin glycosides to the pollen exine, then quercetin glycosides should be detected on shed pollen grains. Alternatively, it was hypothesized that if the liquid formulation was simply delivering raw quercetin directly to the pollen exine without recognition by native enzymes, then only quercetin would be detected on the shed pollen. This hypothesis was tested using Novec™ 7500 wettable powder formulations applied to FDF and non-FDF maize tassels. Lab reagent quercetin anhydrous (Flavonol Powder 1-2 in Table B) was selected for use in this experiment due to high purity (>95% HPLC) that would minimize the presence of exogenous flavonoids other than quercetin in treated samples.

[0097] Concentration of quercetin, quercetin-3 -glucoside (isoquercetin), and rutin in maize pollen samples was analyzed by mass spectrometry. Wild type pollen was sourced from isogenic check plants that did not contain FDF alleles. Negative check FDF plants did not receive any formulation treatments prior to pollen shed. Tassels on Novec™ check FDF plants were treated with Novec™ 7500 five days prior to the start of pollen shed. The Novec™ fluid did not contain any flavonols in this treatment. Tassels on Novec™ quercetin formulation FDF plants were treated with Novec™ 7500 with suspended anhydrous quercetin five days prior to the start of pollen shed. Two concentrations of anhydrous quercetin were included in this experiment. Pollen samples were free of anthers and other maize tissues priorto cryogenic freezing in preparation for analysis. The results of the mass spectrometry testing are summarized in Table I. Flavonoid concentrations are expressed as nanograms of compound per gram of pollen fresh weight.Table INA= Not ApplicableND = Not Detected

[0098] The data demonstrated that the liquid formulations were delivering bioavailable quercetin to the anther tapetai cell layer, as evidenced by the detection of high quantities of quercetin-3 -glucoside and rutin in pollen samples treated with two different concentrations of quercetin suspended in Novec™ 7500. Levels of quercetin-3 -glucoside and rutin in treated FDF pollen were more similar to WT pollen than that of untreated FDS pollen or pollen treated with Novec™ 7500 alone.

[0099] Surprisingly, the data confirmed that raw quercetin is present within mature pollen grains from both non-FDF and FDF maize plants. Raw quercetin concentrations were higher in FDF pollen samples treated with Novec™ 7500 containing suspended quercetin than in untreated FDF pollen or pollen from isogenic non-FDF plants. Without being bound by theory, this higher concentration of raw quercetin may be the result of Novec™ 7500 delivering excessive quercetin directly to the pollen exine, the tapetai cell layer depositingexcess quercetin delivered by Novec™ 7500 directly onto the exine, raw quercetin deposited by Novec™ 7500 on the tassel surface flaking off during pollen sample collection, or a combination of all three hypothesized modes of action.

[0100] While highly effective for laboratory proof of concept, Novec™ 7500 and similar hydrofluoroethers were not considered a scalable option for use in seed production. 20 mg / ml quercetin anhydrous suspended in Novec™ 7500 was retained as a positive check treatment for ongoing formulation development.

[0101] Efforts to develop a stable quercetin liquid formulation suitable for commercial use began by examining quercetin particle size. Quercetin anhydrous flavonol powder 1-2 (Quercetin, >95% (HPLC), Sigma- Aldrich Q4951) and quercetin dihydrate flavonol powder 1-4 (Quercetin dihydrate, BulkSupplements.com) were carried forward from direct pollen complementation testing. Different formulations of quercetin were milled to reduce the particle size. Particle size of the formulations was measured before and after milling using a Malvern Mastersizer 3000. The particle size of the formulation was tested after diluting them in water.Table J

[0102] An additional range finding experiment was conducted to assess whether FDF maize silks could be coated in a formulation that would deposit a fine layer of quercetin over the silks. It was hypothesized that this fine layer of quercetin would then interact with FDF maize pollen that lands on the silks in a similar manner to the protocol in which FDF maize pollen is mixed with flavonol powders. This interaction would restore male fertility and result in seed set. Ethylene glycol and polypropylene glycol were used as solvents for quercetin in this experiment. Quercetin loading in solution ranged from 5 mg / ml to 20 mg / ml. These formulations were applied using a nebulizer to ensure that a fine layer of droplets was deposited over the entire surface of the emerged silks and to avoid large droplets depositingan excessively thick coating of quercetin that may inhibit pollen tube germination. A positive check treatment was conducted by mixing FDF maize pollen with quercetin powder form 1-4 and applying the mixture to FDF maize silks that had not been treated with a formulation. Negative check treatment plants did not receive any silk treatment and FDF maize pollen was applied with no added quercetin powder.Table KNA = Not Applicable

[0103] The 50% polyethylene glycol with 20 mg / ml quercetin anhydrous solution demonstrated that formulation application to FDF maize silks prior to pollination with FDF pollen can result in male fertility restoration. Overall, nebulizer treatments to silks resulted in zero or low seed set. Positive check treatments reinforced the efficacy of the established method of mixing FDF maize pollen with quercetin powder prior to pollination. Negative check treatments demonstrated that FDF male sterility was stable in the absence of quercetin and that the pollination environment was free of airborne pollen contamination. This method of testing through application to silks was discontinued due to relatively low performance compared to Novec™ 7500 target.

[0104] Formulation testing transitioned back to quercetin formulations applied to maize tassels. The quercetin formulations were tested by direct application to emerged tassels from flavonol dependent fertile maize plants. FDF plants used during this testing produced normal quantities of pollen that was completely sterile. This completely sterile pollen would not produce fertilization and seed set when applied to the stigma (silks) of an FDF maize plant orthe stigma on a non-FDF maize plant. The sterile pollen could be restored to completely fertile if exogenous flavonols were applied to the surface of the pollen.

[0105] For all formulations tested, the formulations were mixed or diluted into their final targeted concentration on the same day as application to tassels. For wettable powder formulations, the required quantity of powdered flavonol was measured by weight prior to being suspended in the required quantity of liquid formulation components. Liquid formulation components were measured by weight or volume based on the handling instructions specific to each formulation. In some instances, the flavonol powder was suspended into the liquid formulation by shaking. In other instances, the flavonol powder was suspended into the liquid formulation using an immersion blender to accomplish high-sheer mixing.

[0106] For concentrate formulations in which the flavonol powder was already dissolved in solution or suspended in liquid, the concentrate was dispensed by volume into a target volume of reverse osmosis water. The concentrate and water were thoroughly mixed by shaking prior to application. For ready to use liquid formulations in which the flavonol powder was already dissolved in solution or suspended in liquid, the formulations were thoroughly mixed by shaking prior to measuring out application quantity by volume. In general, formulations were prepared for application less than four hours prior to application.

[0107] Formulation applications targeted maize tassels between the start of emergence from the leaf whorl and prior to the start of anther extrusion and pollen shed. Maize tassels were left uncovered in the growing environment prior to the start of pollen shed. For initial formulation testing, 10 milliliters of formulation were applied to each targeted tassel in the respective treatment group. The 10 milliliters of formulation per tassel were thoroughly dispersed over the entire tassel to ensure complete coverage. Emerged silks on all maize plants in the testing environment were covered in wax paper bags prior to, during, and after formulation application to avoid off-target application of flavonols to the silk tissue. Formulation over spray or droplets falling from the tassels that reached maize plant leaves, stem tissue, or the soil were not considered as a variable due to the lack of precedent for flavonoid mobility within the plant.

[0108] Maize tassels treated with flavonol formulations were left uncovered for a minimum of twenty-four hours following flavonol formulation application. At the start of pollen shed, tassels were covered with wax paper pollination bags in order to collect pollen for testpollinations and to avoid contamination between treatments. Collected pollen was applied to silks on the same FDF maize plant from which the pollen was collected, silks on other FDF maize plants present in the same trial, or silks on non-FDF maize plants. Collected pollen samples were observed for the presence of unbound flavonoid powders that may have deposited, dried, and flaked off of green tissue surfaces of the tassel during application. Unbound flavonoid powder mixed with pollen could restore fertility in a small number of pollen grains and result in low (<10 kernel per ear or per plant) seed set that was clearly distinguishable from the complete pollen fertility restoration observed from high formulation efficacy.

[0109] A battery of quercetin formulations was prepared to assess three criteria; 1) type of formulation and general formulation compositions capable of delivering quercetin to maturing FDF maize pollen grains within the anthers during tassel application, 2) differences in ability for formulations to deliver different forms of quercetin to maturing pollen grains, and 3) any components phytotoxic to maize tassels and pollen.

[0110] With respect to the first criteria, a series of suspension concentrates (SC), suspension concentrates for direct application (SD), emulsion concentrates (EC), oil in water emulsions (EW), and oil dispersion (OD) were prepared for testing. Criteria two involved testing three forms of quercetin powder described in Table J, milled quercetin anhydrous (2-1), quercetin anhydrous (2-2), and quercetin dihydrate (2-4). The third criteria of phytotoxicity to tassels and pollen was a known challenge due to plant reproductive tissues being more sensitive to chemical agents than waxy green tissues. To address this criteria, a diversity of solvents, surfactants, and inerts / fillers were tested, at various concentrations, to rule out phytotoxic components and prioritize components for further testing. The results from testing are described in Table L.Table L

[0111] In general, SD formulations of ethylene glycol, TEHP -penetrant, and quercetin anhydrous demonstrated chemistries for potential delivery quercetin to maturing FDF pollen through direct application to tassels. The quercetin could restore male fertility in the FDF maize pollen and resulted in seed set. Initial testing of quercetin dihydrate did not result in efficacy. Several components including methyl esters and higher concentrations of tris(2- ethylhexyl) phosphate (TEHP) proved phytotoxic to maize tassels at tested concentrations. This phytotoxicity was characterized by tassel desiccation in the days following formulation application. Tassel desiccation resulted in dead anthers and dead FDF pollen prior to the start of pollen, thus eliminating the potential for restored fertility in FDF pollen. Interestingly, SD formulations of ethylene glycol, TEHP -penetrant, and quercetin anhydrous resulted in efficacious restoration of FDF maize pollen fertility in some preparations and phytotoxicity in other preparations. It was hypothesized that this was a result of application technique (volume applied to tassel) and the developmental stage at which the formulation was applied. Higher potential for phytotoxicity was associated with application to earlier tassel maturation stages.

[0112] No options demonstrated the required high efficacy and seed set. It was hypothesized that quercetin was unstable as an active ingredient in formulation due to the quercetin degrading through oxidation or the quercetin crystallizing into larger particles that were unable to penetrate the target plant tissues. To test this hypothesis, flavonol powders were suspended in SD formulations immediately prior to application. In some iterations, the SD formulation already contained quercetin. In other iterations, new blank SD formulations were prepared that had the same composition as formulations reported in Table L. These blank formulations did not contain any form of quercetin as an active ingredient. In all iterations, the quercetin powder was suspended in SD formulation by shaking to form an enriched SDformulation. These enriched SD formulations were applied to tassels within an hour of preparation. The results of this enriched SD formulation testing are reported in Table M.Table M

[0113] These SD formulation enrichment experiments largely confirmed the hypothesis that quercetin was losing efficacy as an active ingredient in formulation. SD formulations of ethylene glycol and TEHP -penetrant in which quercetin powder was added into suspension immediately prior to application resulted in higher efficacy than any previously tested SD formulation. Seed set was inconsistent across enriched SD formulations tested. SD formulation enrichment with quercetin dihydrate resulted in zero or low seed set across combinations tested. Further optimization was needed to achieve consistent dispersion of quercetin in formulation and consistent efficacy when applied to tassels.

[0114] In parallel to the SD formulation enrichment experiments described above, a new series of suspension concentrates for direct application (SD), micro emulsion (ME), and oil dispersion (OD) formulations were developed based on Isopar™ V. It hypothesized that the reduced surface tension of these formulations would improve delivery of the quercetin active ingredient through the complex floral tissues of individual florets on the developing tassels,thus allowing the quercetin to reach the anthers and maturing FDF maize pollen grains.Quercetin anhydrous form 2-2 and milled form 2-1 were selected as active ingredients based on efficacy in the larger battery of SD formulation testing. The results of testing Isopar™ V formulations are reported in Table N, all of which used Quercetin Anhydrous.Table N

[0115] It was observed that Isopar™ V provided advantage in formulation penetrance into the individual florets on maize tassels during application of Isopar™ V formulations. This partially confirmed the hypothesis that reduced formulation surface tension was a favorable route to improve quercetin delivering to maturing maize pollen. An evaluation of formulation efficacy was inhibited by the high phototoxicity of the tested Isopar™ V formulations to the developing maize tassels. Tassels treated with formulations that were not completely phytotoxic showed signs of stress and partial desiccation of tassel branch tips, indicating that mild phytotoxicity may have prevented formulation efficacy.

[0116] Based on observed success with reduced formulation surface tension, elements from the formulation enrichment testing were combined in a search of less phytotoxic synthetic isoparaffinic hydrocarbon fluids. Isopar™ M was identified based on tested compatibility with mature maize pollen and low surface tension. This reduced surface tension is demonstrated in FIG. 1, where a 10% Isopar™ M + 5% Atlox™ BS-50 SD formulation is compared to distilled water. For testing on plants, quercetin powder was suspended in 100% Isopar™ M and 10% Isopar™ M by shaking immediately prior to application to maize tassels. The results of this experiment are reported in Table O.Table O

[0117] Application of Isopar™ M suspensions to tassels demonstrated the desired penetration of the liquid into individual florets on the maize tassels. Quercetin anhydrous powder suspended in 10% Isopar™ M immediately showed potential for improved efficacy and seed set performance. 100% Isopar™ M proved phytotoxic to the single maize tassel tested. This experiment further demonstrated that quercetin dihydrate was not achieving the same efficacy as quercetin anhydrous. Further optimization of the flavonol powder suspended in 10% Isopar™ M strategy into a stable formulation was expected to deliver increased efficacy.

[0118] A third set of experiments was conducted which focused on testing oil dispersion (OD) and technical concentrate (TK) formulations. Quercetin anhydrous forms 2-1 and 2-2 were the focus of testing due to prior demonstrations of efficacy. The synthetic isoparaffinic hydrocarbon fluids Isopar™ V, Isopar™ M, and Norpar™ 13 were the focus of testing.Formulations were diluted over a series to reduce the potential for phytotoxicity. The results of this testing are reported in Table P.Table P

[0119] These experiments demonstrated that the instability of quercetin efficacy as an active ingredient in formulation continued to limit the viability of a single container combined formulation. These formulations were observed to demonstrate the desired penetrance into individual florets on maize tassels during application and absence of phytotoxicity, indicating that this line of synthetic isoparaffinic hydrocarbon fluid was still a favorable strategy for formulation development.

[0120] The synthetic isoparaffinic hydrocarbon fluid formulation development was combined with the quercetin powder formulation enrichment strategy to assess whether a wettable powder formulation strategy was needed. Two of the previously tested Isopar™ M oil dispersion (OD) formulations and a new pair of Isopar™ M technical concentrates (TK) were tested with and without quercetin anhydrous form 2-2 suspended in the formulation immediately prior to application. These experiments included additional suspension concentrates for direct application (SD) and one suspo-emulsion (SE) in an attempt to stabilize quercetin as an active ingredient. The non-formulated 10% Isopar™ M treatment was included as a positive check. The results of these experiments are described in Table Q.Table QRTU = Ready to UseNA = Not Applicable

[0121] These experiments confirmed that quercetin must be added into suspension shortly prior to application to retain efficacy as an active ingredient that can restore FDF maize pollen fertility and result in seed set. Isopar™ M technical concentrate formulations containing quercetin demonstrated zero or near zero efficacy when applied alone, while those same formulations with freshly suspended quercetin anhydrous demonstrated efficacy of >80 kernels per plant average seed set. The non-formulated 10% Isopar™ M check demonstrated high efficacy of >200 kernels per plant on average, further informing that Isopar™ M was a reliable basis for the liquid component of a future wettable powder formulation. Interestingly, the Isopar™ M oil dispersion did not demonstrate efficacy with formulated quercetin or withfreshly suspended quercetin anhydrous, indicating that the oil dispersion formulation liquid component strategy was not a favorable path moving forward.

[0122] Next, quercetin aqueous suspensions were used to study the impact of dispersion medium pH on fertility restoration efficacy. It was hypothesized that formulation pH may be a criteriato quercetin degradation as an active ingredient in formulation, thus optimizing pH prior to further formulation development would increase success. 0.01M buffers were prepared and adjusted to the target pH using either hydrochloric acid or potassium hydroxide. Quercetin anhydrous form 2-2 was suspended into the dispersion medium at a rate of 20 mg / ml and applied single to maize tassels within one hour of mixing. The results are shown below in Table R.Table R*pH was outside of buffer range.

[0123] These data indicate that quercetin may be applied in an aqueous suspension to tassels on flavonol dependent fertile maize plants in a dispersion medium at a wide range of pH values and result in pollen fertility restoration. Interestingly, the use of 0.01M aqueous buffers enabled the liquid suspensions to penetrate into individual florets on maize tassels. This is in contrast to how unbuffered water beads on the maize tassel exterior surfaces and did not penetrate into the florets. The presence of efficacy and seed set across all pH values tested suggested that quercetin is bioavailable to flavonol dependent fertile plants when delivered in a cationic, neutral, or anionic form. The highest seed set was observed at mildly acidic to neutral pH values (pH=6-7) where the first anionic and neutral forms of quercetin are predominant. Seed set decreases when the dispersion medium was greater than pH 8, indicating that the increasingly charged anionic forms may be less efficacious in restoring male fertility. Similar seed set was observed between pH 1 and pH 5 dispersion media,suggesting that there was not a significant difference in efficacy between the cationic and neutral forms of quercetin. It was concluded that formulation pH was not a key factor in quercetin stability during prior formulation testing and that further formulations could continue to be developed at mildly acidic or neutral pH values.

[0124] Based on the above discoveries, an initial wettable powder (WP) formulation was developed. This formulation combined the 10% Isopar™ M liquid carrier base with a powder consisting of 30% milled quercetin anhydrous form 2-1 and 70% Atlox™ BS-50. The liquid and solid components of the WP were mixed together at a ratio of 4.6:0.4, yielding a final quercetin concentration of 24 mg / ml. Three additional suspo-emulsion (SE) formulations and two additional suspension concentrates for direct application (SD) formulations were included in testing as an attempt to optimize single bottle liquid formulations in view of the maximum observed efficacy at mildly acidic dispersion mediums. The results of this testing are described in Table S.Table SRTU = Ready to UseNA = Not Applicable

[0125] The wettable powder (WP) formulation consisting of a 10% Isopar™ M liquid dispersion media base combined with a milled quercetin anhydrous and Atlox™ BS-50 powder demonstrated efficacy and seed set higher than other formulation types in which the efficacy of quercetin as an active ingredient was inhibited by degradation after being formulated into a liquid. Notably, one of the suspension concentrates for direct application (SD) formulations demonstrated some efficacy and delivered over 30 kernels per plant on average. Inconsistency was observed in the stability and dwell time of solid components in the wettable powder. A second batch that was prepared and tested which showed similarinconsistency between experiments. It was hypothesized that differences in mixing techniques were leading to inconsistent powder suspension into the dispersion media, resulting in differences in efficacy. Lab bench testing using high-sheer blender mixing was observed to result in superior suspension to the mixing by shaking that is common during greenhouse and field trialing.

[0001] A trial was then conducted comparing the wettable powder (WP) mixed using high sheer immersion blender mixing and the WP mixed through shaking. As a measure of formulation efficacy, these two preparation methods for the WP formulation were compared to non-formulated 10% Isopar™ M with suspended quercetin powders. A treatment consisting of 10% Isopar™ M with no suspended quercetin powder was included as a check measuring the efficacy of Isopar™ M in isolation. Quercetin powders suspended into RO water with no added buffers were to compare the tissue penetrance of the WP formulation and 10% Isopar™ M suspensions against the high surface tension of water alone. Novec™ 7500 with suspended quercetin powder was included as a positive check and measure of maximum potential seed set. The results of this trial are reported in Table T.Table T

[0126] This experiment demonstrated that high sheer mixing was a criteria to wettable powder formulation efficacy. The 10% Isopar™ M liquid carrier base with a powder consisting of 30% milled quercetin anhydrous form 2-1 and 70% Atlox™ BS-50 was efficacious in restored FDF maize pollen fertility and delivered over 300 kernels per plant seed set on average. This was complete seed set to the point that silks had emerged from the husk leaves and was comparable to the complete seed set achieved by quercetin powder suspended in Novec™ 7500. The same wettable powder mixed by shaking showed the expected severe reduction in efficacy due to poor mixing of the active ingredient in the dispersion medium. Testing multiple forms of quercetin suspended in 10% Isopar™ M using high sheer mixing demonstrated the critical role of Atlox™ BS-50 in the formulation, with these treatments returning much lower seed set than the WP. Treating FDF maize tassels with 10% Isopar™ M alone with no suspended quercetin powder demonstrated that Isopar™ M does not mobilize or alter endogenous flavonols and cannot restore FDF maize pollen fertility without the presence of the active ingredient. Negative check treatments using water demonstrated the critical capacity for Isopar™ M to modify formulation surface tension and ensure the quercetin penetrates into the individual maize florets where it can reach the anthers and restore fertility to the FDF maize pollen. Untreated tassels demonstrated that the trialing took place in an environment free of airborne pollen contamination.

[0127] In parallel to formulation development, the compositions of the disclosure were compared to the commercial flavonoid biostimulant product, CropBioLife, for the capacity to restore male fertility in FDF maize. CropBioLife is a flavonoid composition containing bitter orange extract CAS 72968-50-4 (flavonoid component); Glycerin CAS 56-81-5 (20%); Yucca Extract CAS 90147-57-2 (5%); an organic acid blend, and having a pH of 3.5 (neat). CropBioLife was applied to FDF maize tassels and test pollinations were conducted in the same manner as other disclosed compositions. CropBioLife was applied at the labeled rate of 0.2% as an initial evaluation. Additional testing was conducted by suspending quercetinpowders in CropBioLife. For quercetin powder suspensions, the CropBioLife was tested at0.1%, 0.2%, and 0.5% concentrations.Table UNA = Not Applicable

[0128] These experiments indicated that the bitter orange extract (CAS 72968-50-4) in CropBioLife does not restore male fertility in FDF maize tassels. The CropBioLife formulation can act as a liquid carrier for suspended quercetin powders due to the reduced surface tension of the final dilution compared to water alone. Seed set performance of these liquid suspensions is similar to that obtained from 0.0 IM citric acid and potassium phosphate buffers. The concentration of CropBioLife in the liquid suspension did not appear to influence efficacy of the suspended quercetin powders. Quercetin anhydrous continued to demonstrate higher efficacy and seed set performance than quercetin dihydrate. It was concluded that flavonoid biostimulant products are not capable of restoring male fertility in FDF maize without the addition of the quercetin active ingredients in this disclosure.

Claims

What is Claimed:

1. An agrochemical composition, comprising: a flavonol, wherein the flavonol is configured as an active ingredient in the composition and capable of restoring fertility of a flavonoid dependent fertility (FDF) plant; and at least one flavonol enhancer.

2. The composition of claim 1, wherein the flavonol is present in an agrochemically effective amount which restores fertility of a sterile plant.

3. The composition of claim 1, wherein the at least one flavonol enhancer is a crop oil adjuvant.

4. The composition of claim 3, wherein the flavonol is selected from: 3 -hydroxyflavone, azaleatin, fisetin, galangin, gossypetin, kaempferide, kaempferol, isorhamnetin, morin, myricetin, natsudaidain, pachypodol, quercetin, rhamnazin, and rhamnetin.

5. The composition of claim 4, wherein the flavonol is quercetin.

6. The composition of claim 1, further comprising water, and wherein the composition is a liquid.

7. The composition of claim 1, wherein the composition is a wettable powder (WP).

8. The composition of claim 1, wherein the composition is configured as a concentrate for dilution with a carrier, wherein the carrier is preferably water.

9. The composition of claim 1, further comprising an adjuvant, wherein the adjuvant preferably increases the penetration of the flavonoid to a plant.

10. The composition of claim 9, wherein the adjuvant is a crop oil concentrate adjuvant, wherein the adjuvant is optionally present in an amount of about 0.1-30% w / w and more preferably 1-20% w / w.

11. The composition of claim 1, wherein the composition comprises: about 10-30 g / L of quercetin.

12. The composition of claim 1, further comprising Atlox® BS-50.

13. The composition of claim 1, further comprising Isopar® M (synthetic isoparaffinic) (CAS: 64742-47-8).

14. A method, comprising: applying the composition of any one of the proceeding claims to a flavanoid dependent fertile (FDF) plant.

15. A method of preparing hybrid seed, comprising: bulking an inbred female FDF line, wherein the inbred female line is bulked by planting a plurality of inbred female FDF seeds in an inbreed production field, and applying an effective amount of a composition of any one of claims 1-13 to the plurality of inbred female FDF plants grown from the seed; harvesting the bulked inbred female FDF seed; planting the bulked inbred female FDF seed in a hybrid seed production field; planting a plurality of inbred male seed in the hybrid seed production field, wherein the inbred male seed and the bulked inbred female FDF seed are planted in at least one of the following ratios 1 : 1, 1 :2, 1 :3, 1 :4, 1 :5, 1 :6, 1 :7, 1:8, 1 :9 and 1 :10; and harvesting the hybrid seed.

16. The method of claim 15, wherein harvesting includes harvesting the cob.

17. The method of claim 15, further comprising conditioning the hybrid seed, wherein conditioning comprises at least one of shelling the hybrid seed, cleaning the hybrid seed, sizing the hybrid seed, and treating the hybrid seed with a fungicide.

18. The method of claim 15, further comprising bagging the hybrid seed.

19. The method of claim 15, further comprising destroying a plurality of the plants grown from the inbred male seed prior to harvesting the hybrid seed.

20. A method of bulking an inbred female FDF line, comprising: applying an effective amount of a composition of any one of claims 1-13 to a tassel of an inbred female FDF plant between the start of emergence from the leaf whorl and prior to the start of anther extrusion and pollen shed.

21. An FDF seed produced by the method of claim 21.

22. A composition, comprising: a flavonol; and a wettable powder base; wherein the composition is configured as a wettable powder.

23. The composition of claim 22, wherein the wettable powder base comprises a wetting agent, a dispersant, and a filler or inert.

24. The composition of claim 22, wherein the flavonol is present in an amount of 10 to 80% w / w.

25. The composition of claim 22, wherein the wettable powder base is present at 10 to 90% w / w.

26. The composition of claim 22, wherein the wettable powder base comprises a filler or inert, and the filler or inert is present 30 to 70% w / w of the composition.

27. The composition of claim 5, wherein the filler or inert includes at least one of kaolin, bentonite, and attapulgite.

28. The composition of claim 22, wherein the wettable powder base comprises a dispersant, and the dispersant is present at 2 to 5% w / w of the composition.

29. The composition of claim 22, wherein the wettable powder base comprises a wetting agent, and the wetting agent is present at 2 to 5% w / w of the composition.

30. A kit for restoring fertility of a flavonoid dependent fertility (FDF) plant, comprising: the composition of claim 22 in a first package; and at least one flavonol enhancer in a second package.

31. The kit of claim 30, wherein the at least one flavonol enhancer is a crop oil adjuvant.

32. The kit of claim 30, wherein the at least one flavonol enhancer is an isoparaffinic solution.

33. The kit of claim 30, wherein the at least one flavonol enhancer comprises greater than 50% C13-C14 alkanes.

34. The kit of claim 30, wherein the at least one flavonol enhancer has a boiling point between 150-275°C.

35. The kit according to claim 30, wherein the w / w ratio of the flavonol to the at least one flavonol enhancer is at least one of:10 to 80 : 0.5 to 40;10 to 80 : 10 to 20;10 to 80 : 5 to 10;30 to 50 : 0.5 to 40;30 to 50 : 10 to 20;30 to 50 : 5 to 10;30 to 35 : 0.5 to 40;30 to 35 : 10 to 20; or30 to 35 : 5 to 10.

36. A method of preparing a composition for restoring fertility of a flavonoid dependent fertility (FDF) plant, comprising: mixing a flavonol and at least one flavonol enhancer in a liquid carrier to form an agrochemically effective composition effective amount to restore fertility of a sterile plant.

37. The method of claim 36, wherein the mixing utilizes a high sheer force.

38. The method of claim 37, wherein the high sheer force is between 3000 and 6000 ft / min.

39. A composition, comprising: a flavonol present in an agrochemically effective amount which restores fertility of a sterile plant; at least one flavonol enhancer; and a liquid carrier; wherein the composition is configured as a liquid sprayable composition.

40. The composition of claim 39, wherein the flavonol is present at 10 to 80% w / w.

41. The composition of claim 39, wherein the at least one flavonol enhancer is present at0.5 to 40% w / w.

42. The composition of claim 39, wherein the liquid carrier is present at 10 to 95% w / w.

43. The composition of claim 39, wherein the composition further comprises at least one wetting agent or dispersant.

44. The composition of claim 39, wherein the liquid carrier is water.

45. The composition of claim 39, wherein the flavonol is solid and has a particle size distribution of at least one of a Dx [4,3] of 2.39 pm to 4.03 pm; Dx (50) of 1.49 pm to 2.54 pm; Dx (95) of 7.24 pm to 12.6 pm; or Dx (90) of at least 5.53 pm to 8.93 pm.

46. A method of restoring fertility of a flavonoid dependent fertility (FDF) plant, comprising applying the composition of claim 39 to a tassel of the FDF plant.

47. The method of claim 46, wherein the applying results in a production of at least 3,750 (1000X) viable kernels per acre or 9270 (1000X) viable kernels per hectare.

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