A method for increasing kernel row number, seed number per ear, and overall corn seed production

Abstract

The invention relates to methods for producing hybrid or inbred corn seed by treating corn plants with a phytosterol composition comprising at least one phytosterol and a sucrose fatty acid ester, thereby increasing kernel row number, seed number per ear, and overall seed production.

Description

[0001] TITLE OF THE INVENTION: A METHOD FOR INCREASING KERNEL ROW NUMBER, SEED NUMBER PER EAR, AND OVERALL CORN SEED PRODUCTION

[0002] FIELD OF THE INVENTION

[0003] The invention relates to a method to increase the kernel row number of corn ears which is based on the application of a phytosterol composition on the said corn at an early stage of its development. Currently mostly genetics is able to efficiently increase the row number. Hence, the invention relates to a method which uses a chemical trigger able to increase the number of rows, compared to an untreated corn.

[0004] The invention also relates to a method of producing seeds for a corn inbred plant or for a corn hybrid plant, comprising a step of applying on the said corn parent plant a phytosterol-based composition.

[0005] STATE OF THE ART

[0006] Corn (Zea mays) is a worldwide staple food, with more than one billion tons produced each year, led by the United States of America and China. The seeds are collected from corn ears which are structured as a cob with even-paired, surrounding rows and kernel lines along the length of the cob. The number of rows constitutes the number of kernels which are aligned on the circumference of a corn ear. The determination of the kernel row number starts at an early phenological stage and is generally finalized by BBCH19, up to 14 unfolded leaves, but these values largely differ from one corn variety to another. Therefore, this determination can be considered as taking place between BBCHOO and BBCH 19 up to 16 or 18 unfolded leaves.

[0007] The kernel row number is more governed by corn genetics rather than by the climate situation corn would be exposed to.

[0008] In the seed production industry, producers primarily aim to optimize both kernel row number and number of kernels per row to achieve the maximum number of seeds per ear, rather than focusing on overall yield, a goal that differs from food-oriented agricultural practices where the emphasis is placed on the total weight of the harvest (usually expressed in weight per surface of field). In other words, seed production is seeking the most elevated count of seeds after harvest, not a high weight of seeds. Such high weight per surface could even be detrimental as it can increase transport costs. Therefore, the objective here is to produce a high number of high-quality seeds characterized by an optimal kernel row number and / or number of kernels per row (thus a high number of seeds per ear), coupled with a high germination rate, ensuring better genetics for future crops. This germination rate parameter is also crucial for seed producers for two main applications. First, hybrid corn produced from cross-breeding of two inbred parent lines benefits from both parents' genetic advantages, but without departing from a reduced germination rate. Second is the propagation of corn inbred lines by selfing. The germination rate is therefore a crucial market parameter in seed production.

[0009] However, this approach presents several technical challenges, particularly in optimizing plant growth to maximize the kernel row number and / or the number of kernels per row without compromising the corn health. At the same time, producers are seeking alternative solutions to genetically-modified organisms (GMOs) or CRISPR technologies, which are often used to increase both grain production and germination rate. Gene-editing technology is thus the method of choice to increase the kernel row number and / or the number of kernels per row (US2024384280, WO2024 / 238902, US20240000031) or germination rate (WO2019 / 084342). However, achieving an increase of kernel row number and / or number of kernels per row and / or germination rate by a genetic approach is cumbersome, costly, and lengthy as it requires introgression of the relevant trait into multiple varieties. In addition, GMOs breeding is still forbidden or highly restricted in many countries worldwide to date. In this context, there is a need to identify innovative and sustainable methods to increase the kernel row number and / or number of kernels per row and / or germination rate while maintaining seed quality (notably germination rate) and avoiding reliance on GMO technology.

[0010] Surprisingly, it was found that a treatment with a phytosterol composition comprising at least one phytosterol and at least of sucrose fatty acid ester significantly increases the kernel row number and / or the number of kernels per row (thus the total number of seeds per ear) and / or the germination rate of such harvested seeds, leading to a new method of seed production.

[0011] DISCLOSURE OF THE INVENTION

[0012] Definitions As used therein, the BBCH scale is used to identify the phenological stage of plants. This scale is expressed with two digits, the first and second digits reporting main and secondary growth stages, respectively. For example, BBCH 14 refers to the principal growth stage "1": leaf development and "4" refers to 4 leaves unfolded. A BBCH monograph is available at DOI: 10.5073 / 20180906-074619. Correspondence between BBCH scale, Iowa State University scale or any other scale allowing to determine the phenological stage of corn is known by the skilled artisan.

[0013] As used therein, the expression "cross pollination" refers to a fertilization by the union of two gametes from different plants.

[0014] As used therein, the expression "hybrid variety" or simply "hybrid" refers to a substantially heterozygous hybrid line showing minor genetic modifications from the overall genetics of the hybrid line.

[0015] As used therein, the expression "inbred variety" or simply "inbred" refers to a variety developed through inbreeding or doubled haploidy that preferably comprises homozygous alleles at > 95% of its loci. An inbred can be reproduced by selfing or growing in isolation so that the plants can only pollinate with the same inbred variety.

[0016] As used therein, the terms "seed" refers to a corn kernel that is harvested from a corn ear and that is suitable for planting to produce a new corn plant. The term encompasses seeds that are sold, stored or used for propagation. In the following, the terms "kernel" and "seed" can be used interchangeably.

[0017] As used therein, the expression "early phenological stage" means a phenological stage corresponding to a BBCH scale between BBCH 00 and BBCH 19 with up to 14 unfolded leaves, that is to say between dry seed and 14 unfolded leaves.

[0018] As used therein, the expression "treated" plant or crop refers to a plant or crop which is preventively or curatively treated with the composition used in the method of the invention and which undergoes subsequent growth by being exposed to substantially the same natural phenomena (pedoclimatic context, stress) as the untreated crop.

[0019] As used herein, the expression "untreated" plant or crop refers to a plant or crop which is untreated with the composition used in the method of the invention and which undergoes growth by being exposed to the same natural phenomena (pedoclimatic context, stress) as the treated crops.

[0020] As used therein, the expression "abiotic stress" refers to a non-living stimulus on living vegetal organisms, for instance a climate hazard on a crop.

[0021] As used therein, the expression "biotic stress" refers to a damage performed on a cultivated crop by living organisms, such as bacteria, viruses, fungi, parasites, insects and weeds.

[0022] As used therein, the expressions "plant sterol" and "phytosterol" may be used indifferently and refer to any vegetally-occurring or chemically-synthesized sterol.

[0023] As used therein, the expression "sterol" refers to any vegetally-, animally-occurring, or chemically-synthesized sterol. The expression "sterol" therefore embraces the expression "plant sterol".

[0024] As used therein, "cholesterol" can be indifferently considered as a plant-derived or an animal- derived sterol, as it can be found in both certain plants and certain animals.

[0025] As used therein, the expression "phytosterol composition" refers to a composition comprising at least one phytosterol and at least one sucrose fatty acid ester.

[0026] As used therein, the expressions "combined" or "in combination with" refer to an association of at least two compounds, the two compounds being applied one prior to the other, concomitantly, or one after a step of another. In other words, these expressions encompass a sequence of application of the said at least two compounds, i.e. the first compound before the second compound, the first compound after the second compound or said at least two compounds applied concomitantly, either in the form of a single product, i.e. a mixture of said at least 2 compounds, including an extemporaneous mixture.

[0027] As used therein, the term "germination rate" refers to the percentage of germinated seeds over the total seeds used during a germination test.

[0028] As used therein, the term "seed unit" refers to a standardized seed containment being sold or provided by the seed producer. Here the seed unit is calculated as 50,000 seeds, the said seed unit being usually sold between 60 € and 120 €. As used therein, the term "cultivated corn," in contrast to a naturally existing corn, refers to corn that can be cultivated, i.e., sown, planted, and exploited by man.

[0029] As used therein, the terms "crops" and "plants" may be used indifferently and refer to all plants and plant populations such as desirable and undesirable wild plants, cultivars, and plant varieties (whether or not protectable by plant variety or plant breeder's rights). Cultivars and plant varieties can be plants obtained by conventional propagation and breeding methods which can be assisted or supplemented by one or more biotechnological methods such as by use of double haploids, protoplast fusion, random and directed mutagenesis, molecular or genetic markers or by bioengineering and genetic engineering methods.

[0030] As used therein, the term "plant" includes whole plants and parts thereof, including, but not limited to, shoot vegetative organs / structures (e.g., leaves, stems and tubers), roots, flowers and floral organs / structures (e.g., bracts, sepals, petals, stamens, carpels, anthers and ovules), seeds (including embryo, endosperm, and seed coat) and fruits (the mature ovary), plant tissues (e.g., vascular tissue, ground tissue, and the like) and cells (e.g., guard cells, egg cells, and the like), and progeny of same. "Fruit" and "plant produce" are to be understood as any plant product which is further utilized after harvesting, e.g., fruits in the proper sense, nuts, wood etc., that is anything of economic value produced by the plant.

[0031] As used therein, the term "slurry" refers to the composition of the method of the invention diluted in water or in a water-based solution and, optionally one or more active ingredients.

[0032] As used therein, the term "tank-mix" means that active ingredients are mixed in the water tank to form the slurry, which is then being sprayed on the plants.

[0033] As used therein, the term "active ingredient" refers to a substance that allows the plant to combat preferably abiotic and / or biotic stresses, or a substance aiming at improving the growing of the plant.

[0034] As used therein, the expression "foliar spray" refers to a pressurized projection of a slurry that forms numerous microdroplets that cover the upper and / or lower surfaces of the treated leaf.

[0035] As used therein, the term "seed imbibition" refers to an immersion of a seed in a solution containing the phytosterol composition. The use of "a" or "an" for a molecule does not exclude, unless otherwise stated, the presence of plurality of molecules, in such a way that the expression "one or more" can be substituted for it.

[0036] As used therein, the expression "insoluble in the aqueous phase" refers to a compound which presents the inability to form with water a homogenous, one-phase solution at the microscopic or the macroscopic level, at a given temperature and atmospheric pressure.

[0037] By contrast and as used therein, the term "solubility" refers to a compound which leads to a homogenous solution without remaining insoluble particles when it is added to a liquid, at a given temperature and atmospheric pressure.

[0038] Any percentage by weight of a molecule comprised in the composition used in the method of the invention refers to the total weight of the said composition, which means relative to the sum of all ingredients giving a hundred. Weight percent can be symbolized as wt%.

[0039] Various aspects of the invention can be presented in a range format. The description in range format is merely for convenience and conciseness and should not be construed as an inflexible limitation on the scope of the invention. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 2.7, 3, 4, 5, 5.3, and 6. This applies regardless of the breadth of the range.

[0040] Detailed description of the invention

[0041] It is an object of the present invention to provide a method of increasing the kernel row number and / or the number of kernels per row of corn ears, consisting in treating the said corn with an application of a phytosterol composition comprising at least one phytosterol and at least one sucrose fatty acid ester.

[0042] It is another object of the present invention to provide a method of producing seeds for a corn hybrid plant, the said method comprising: a. Growing a first inbred corn plant as a female and a second inbred corn plant as a male, b. Treating said first female inbred corn plant with a phytosterol composition comprising at least one phytosterol and at least one sucrose fatty acid ester, c. Detasseling said first female inbred corn plant, d. Crossing said first female inbred corn plant with said second male inbred corn plant, e. Growing said first female inbred corn plant, f. Harvesting the resultant hybrid corn seed from said first female inbred corn plant; wherein the number of seeds produced from said first female inbred corn plant is at least 5% higher than the number of seeds produced by a female inbred corn plant grown under substantially identical conditions which has not been treated with a phytosterol composition under step b.

[0043] In this object of the invention:

[0044] Step a. of the method of the invention comprises sowing inbred parent lines according to standard practice of the seed production industry, for example by alternating male rows and female rows. These parent lines can be sown at the same time or at different days, provided that the flowering stage occurs concomitantly between the male corn and the female corn. Fertilization, field and pest management, roguing, sowing density and other practice are optionally performed according to ordinary skills. Optionally the seeds of the parent lines are treated with the method of the invention according to the embodiments hereafter described.

[0045] To better ensure the efficiency of step d., step a. may further require that male and female corn plants are sown in alternating rows, according to standard practice of the seed production industry. Such alternation is, for instance, 1 male row each 4 female rows, 2 male rows each 2 female rows, or 2 male rows each 4 female rows, or more sophisticated field map patterns, in particular on the edges of the field wherein male rows are denser to create a wall against undesirable corn pollen potentially coming from other fields.

[0046] Step b. involves treating corn plants with the method of the invention. Advantageously female plants only are treated with the phytosterol composition, but optionally male plants can be treated as well, or even both female and male plants. The said treatment with the phytosterol composition is performed according to the various embodiments described below in terms of phenological stage, method of formulation, molecules comprising the formulation, applied concentration on the field and the likes.

[0047] Step c. involves detasseling the female line, according to any known process, for example using a detasseling machine, detasselers and / or using inbred male-sterile line.

[0048] Optionally, the ear of the inbred male is protected from pollination, or the inbred male line is destroyed before harvest.

[0049] Step d. involves cross-breeding of the female gametes with the male gametes. Particular care is taken to avoid genetic contamination incoming from plants with undesirable genetic characteristics, and / or self-pollinating of the female. The method is performed according to standard practice known in the seed production industry.

[0050] Step e. involves methods according to standard practice, in particular field and pest management, for example irrigation in case drought stress must be mitigated.

[0051] Step f. involves harvesting the hybrid corn seeds with any method known from the skilled artisan. At this stage or after this stage the number of hybrid corn seeds is determined.

[0052] It is also another object of the invention to provide a method of producing seeds for a corn inbred plant, the method comprising: a. Growing an inbred corn plant, b. Treating said corn inbred plant with a phytosterol composition comprising at least one phytosterol and at least one sucrose fatty acid ester, c. Self-pollinating said inbred corn plant, d. Growing said inbred corn plant, e. Harvesting the resultant inbred corn seed from said inbred corn plant; wherein the number of seeds produced from said inbred corn plant is at least 5% higher than the number of seeds produced by an inbred corn plant grown under substantially identical conditions which has not been treated with a phytosterol composition under step b.

[0053] In this second object of the invention, according to an embodiment:

[0054] The said inbred corn plant which is grown, treated and self-pollinated is an inbred plant issued from continuous self-pollination cycles over multiple generations, typically at least 2 generations, generally at least 4 generations, preferably at least 6 generations, advantageously at least 8 generations.

[0055] Step a. of the method of the invention comprises sowing inbred parent lines according to standard practice of the seed production industry for corn inbred line propagation. Fertilization, field and pest management, roguing, sowing density and other practice are optionally performed according to ordinary skills. Optionally the seeds of the parent lines are treated with the method of the invention according to the embodiments hereafter described.

[0056] Step b. involves treating inbred corn plants with the method of the invention. The said treatment with the phytosterol composition is performed according to the various embodiments described below in terms of phenological stage, method of formulation, molecules comprising the formulation, applied concentration on the field and the likes.

[0057] Step c. involves self-pollinating the said inbred corn plant. Particular care is taken to avoid genetic contamination coming from plants with undesirable genetic characteristics, so that the plants can only pollinate with the same inbred variety. Thus, to ensure selfing efficiency, step c. requires further steps to keep distance from other corn varieties. The method is performed according to standard practice known in the seed production industry.

[0058] Step d. involves methods according to standard practice, in particular field and pest management, for example irrigation in case drought stress must be mitigated.

[0059] Step e. involves harvesting the hybrid corn seeds with any method known from the skilled artisan. At this stage or after this stage the number of hybrid corn seeds is determined.

[0060] According to the invention, the phenological stage to apply the phytosterol composition comprising at least one phytosterol and at least one sucrose fatty acid ester should take place at the same time the kernel row number is under determination, or before, for instance by imbibing seeds of parent plants.

[0061] In one embodiment, the phenological stage to apply a phytosterol composition comprising at least one phytosterol and at least one sucrose fatty acid ester on corn is between BBCH 00 and BBCH 19, advantageously between BBCH 00 and BBCH 19 with up to 13 unfolded leaves, more preferably between BBCH 00 and BBCH 19 with up to 11 unfolded leaves, even more preferably between BBCH 00 and BBCH 19 with up to 10 unfolded leaves, or even between BBCH 16 and BBCH 19, advantageously between BBCH 16 and BBCH 19 with up to 13 unfolded leaves, more preferably between BBCH 16 and BBCH 19 with up to 11 unfolded leaves, even more preferably between BBCH 16 and BBCH 19 with up to 10 unfolded leaves.

[0062] This surprising effect is estimated by comparing the kernel row number of corn treated at an early phenological stage with an untreated corn, for example before or after harvest. The beneficial effect, hereafter called "increase" is expressed as a difference between the number of rows of the treated corn and the number of rows of the untreated corn. A mathematical formula can be expressed as: "increase" = (number of rows of treated corn) - (number of rows of untreated corn).

[0063] This applies as well for the number of kernels per row. In such a case, the "increase" is expressed as the difference between the number of kernels per row of the treated corn and the number of kernels per row of the untreated corn. A second mathematical formula can be expressed as: "increase" = (number of kernels per row of treated corn) - (number of kernels per row of untreated corn).

[0064] Then, the total number of seeds per ear can be calculated by multiplying the kernel row number with the number of kernels per row or any other determination method.

[0065] In one embodiment, the increase of kernel row number between the treated corn with the method of the invention and the untreated corn is more than 0.25, meaning that the kernel row number of the treated plant has in average 0.25 more rows than the untreated plant. Preferably, this increase between the treated corn with the method of the invention and the untreated corn is more than 0.30.

[0066] In another embodiment, the increase of the number of kernels per row between the corn treated with the method of the invention and the untreated corn is at least 2%, meaning that the treated corn has on average 2% more kernels per row than the untreated corn. Preferably, this increase between the treated corn with the method of the invention and the untreated corn is at least 4%.

[0067] In another embodiment, the number of seeds produced between the treated corn with the method of the invention is at least 5% higher than the number of seeds from an untreated corn grown under substantially identical condition, meaning that the treated corn has on average 5% more seeds than the untreated corn. Preferably, this increase between the treated corn with the method of the invention and the untreated corn is at least 8%, advantageously at least 10%.

[0068] Since the number of harvested seeds in increased, it means that the number of seed units per hectare is also increased, as well as the seed producer income. In one embodiment, treating corn with the method of the invention allows increasing the number of seed units per hectare by at least 5%, more advantageously by at least 8%, compared to an untreated corn which was grown under substantially the same conditions. This seed unit being sold by the seed producer between 60 and 120 €, it means that the increase in revenue gain for the seed producer is at least 4000 € / ha, advantageously at least 6000 € / ha.

[0069] In one another object of the invention, the method of the invention further allows increasing the germination rate of the harvested seeds from a corn hybrid plant or from a parent plant.

[0070] Surprisingly, it was observed that treating corn with the method of the invention does not only lead to more seeds per ear, but also to an unexpected effect on the germination rate of the harvested seeds. A beneficial effect of phytosterols transferred from the treated, parent lines to the collected seeds was not expected. This effect occurs even whether the collected seeds are obtained from hybridization or from cross-breeding of corn plants. Noteworthy is the fact that the method of the invention does not rely on treating harvested seeds, but rather on treating at least one of their parents.

[0071] The germination rate is calculated upon the percentage of seeds that is able to germinate within a given timeframe, for instance in the first 4, 5 or 7 days, the said seeds being germinated at a given temperature. Usually, seed producers are seeking seeds with a germination rate higher than 90%, but the higher, the better. Guaranteeing seeds with a very high germination rate (for instance 97% or better) leads to more expensive products, thus more expensive seed units leading to more income to the seed producer, and the security of a better emergence for the farmer who uses these seeds.

[0072] In this embodiment, the germination rate increase of the harvested seeds issued from a corn treated with the method of the invention is at least 1.0%, preferably at least 1.5%, advantageously at least 2.0% compared to an untreated corn. Both treated and untreated corn must be considered exposed to substantially the same field maintenance, pedoclimatic context and / or abiotic and / or biotic stresses possibly occurring to be adequately compared.

[0073] As these beneficial effects do not depend on the pedoclimatic context, it means that the invention is efficient in all situations, for example if corn is facing a situation of abiotic and / or biotic stress, or in a situation of no particular stress.

[0074] The phytosterol composition comprises at least one phytosterol and at least one sucrose fatty acid ester. So far, more than 250 different phytosterols have been identified. The at least one phytosterol can be selected from the group consisting of: plant sterols: beta-sitosterol, campesterol, brassicasterol, stigmasterol, lanosterol, stigmastanol, campestanol, cycloartenol, gamma-sitosterol, A5-avenasterol, A7- avenasterol, isofucosterol, 5-dehydroepisterol, clerosterol, sitostanol, stigmastadioenol, isomers and derivatives thereof, and their mixtures; zoosterols: lathosterol, cholesterol, cholestanol, dinosterol, and derivatives thereof, and their mixtures; mycosterols: ergosterol, obtusifoliol, and derivatives thereof; and their mixtures.

[0075] Sterols can be isolated from various natural sources or synthesized according to known methods in the art. Preferably, the sterols are obtained from various sources, for example from deodorizer distillates of vegetal extracts.

[0076] Preferably, the at least one phytosterol is chosen among the group of beta-sitosterol, campesterol, brassicasterol, stigmasterol and / or cholesterol, more preferably beta-sitosterol. Advantageously, beta-sitosterol represents at least 30% of the phytosterols mixture by weight, with the balance to 100% preferably containing, where appropriate, campesterol, stigmasterol and brassicasterol.

[0077] According to a specific embodiment, the at least one phytosterol represents between 0.2% and 10% by weight of the total composition, advantageously between 0.5% and 7%, and preferably between 1% and 5%. The at least one sucrose fatty acid ester is selected from the group consisting of saccharose stearate, saccharose palmitate, their polyesters, and mixtures thereof.

[0078] The concentration of the at least one sucrose fatty acid ester represents between 0.2% and 10% by weight of the total composition, advantageously between 3% and 7%.

[0079] According to a specific embodiment, the weight ratio of the at least one phytosterol to the at least one sucrose fatty acid ester is between 0.01 and 10, advantageously between 0.1 and 5, more advantageously between 0.2 and 1.5.

[0080] The phytosterol composition comprising at least one phytosterol and at least one sucrose fatty acid ester of the method of the invention can take the form of a homogenous or heterogenous aqueous solution, an emulsifiable concentrate, a concentrated suspension, an oil-in-water or a water-in-oil emulsion, an oil-in-water suspo-emulsion, an oil dispersion or any other formulation the skilled artisan may find suitable for an agricultural composition. Preferably, the phytosterol composition comprising at least one phytosterol and at least one sucrose fatty acid ester of the method of the invention is an aqueous solution, an emulsion or a suspo- emulsion.

[0081] According to a specific embodiment, the phytosterol composition is a suspo-emulsion, which comprises at least one phytosterol and at least one sucrose fatty acid ester selected from the group consisting of sucrose stearate, saccharose palmitate and their polyesters, or mixtures thereof, acting either as surfactant in an oil-in-water emulsion, or as a free surfactant which is then added to the aqueous phase of the said oil-in-water emulsion as a suspension. In one embodiment, the surfactant acting in the oil-in-water emulsion and the surfactant acting as a free surfactant are identical, e.g. sucrose stearate for both. In another embodiment, the surfactant acting in the oil-in-water emulsion and the surfactant acting as a free surfactant are different, e.g., sucrose stearate acting in the oil-in-water emulsion and saccharose palmitate as a free surfactant.

[0082] In details, the phytosterol composition is a multiphase agricultural composition in the form of a suspo-emulsion, i.e. a composition comprising lipophilic droplets containing the above- mentioned mixture of phytosterols, said lipophilic droplets being dispersed in an aqueous phase to form an oil-in-water emulsion.

[0083] The composition further comprising: at least one first sucrose fatty acid ester (SEI) located at the interface between the lipophilic droplets and the aqueous phase; and at least one second sucrose fatty acid ester (SE2) suspended in the aqueous phase of the oil-in-water emulsion, said second sucrose fatty acid ester (SE2) having the form of particles insoluble in the said emulsion.

[0084] In other words, the suspo-emulsion comprises two distinct oil and water phases, but it cannot be described as an emulsion, given that the aqueous phase also includes a third phase made of solid particles. These solid particles are composed of molecules which are insoluble in the aqueous phase, whose aim is to further improve the uptake of the sterols within the plant.

[0085] According to a first embodiment of the multiphase composition, the sucrose fatty acid ester which is located at the interface between the lipophilic droplets and the aqueous phase, and the sucrose fatty acid ester which is suspended in the aqueous phase of the oil-in-water emulsion are identical. In this case, the multiphase composition comprises only one sucrose fatty acid ester located at two different places.

[0086] Practically,

[0087] The part of the sucrose fatty acid ester which is located at the interface between the lipophilic droplets and the aqueous phase represents between 0.2 and 10 wt% of the composition, preferably between 4 and 10 wt% of the composition, more preferably between 5 and 9 wt% of the composition, in particular between 3 and 7 wt% of the composition;

[0088] The oily phase comprises the phytosterol mixture, said phytosterol mixture representing between 0.2 and 10 wt% of the composition, advantageously between 0.5 and 7 wt% of the composition, or even between 0.5 and 6 wt% of the composition, preferably between 1 and 5 wt% of the composition;

[0089] The part of the sucrose fatty acid ester which is suspended in the aqueous phase of the oil-in-water emulsion represents between 0.5 and 5 wt% of the composition, more preferably between 1 and 1.5 wt% of the composition.

[0090] According to a second embodiment of the multiphase composition, the sucrose fatty acid ester which is located at the interface between the lipophilic droplets and the aqueous phase and the sucrose fatty acid ester which is suspended in the aqueous phase of the oil-in-water emulsion are not identical. Even if not identical, they can be added to the same concentration range as mentioned above when the sucrose fatty acid esters are identical.

[0091] In both embodiments, the sucrose fatty acid ester located at the interface between the lipophilic droplets and the aqueous phase may present a preferential oil solubility, a preferential water solubility or can also present both oil and water solubility, the solubilities between oil and water being here different or identical. This last feature allows the possibility that sucrose fatty acid esters which are soluble in the aqueous phase and sucrose fatty acid esters which are soluble in the lipophilic droplets are the same molecule. If different, sucrose fatty acid esters which are soluble in the aqueous phase and sucrose fatty acid esters which are soluble in the lipophilic droplets differ from each other by the proportion of their hydrophobic and hydrophilic parts. Practically, the hydrophilic / lipophilic balance (HLB) of sucrose fatty acid esters which are soluble in the aqueous phase is higher than the one of sucrose fatty acid esters which are soluble in the lipophilic droplets. In contrast, the HLB of sucrose fatty acid esters which are soluble in the lipophilic droplets is lower than the one of sucrose fatty acid esters which are soluble in the aqueous phase.

[0092] Advantageously, the sucrose fatty acid ester is soluble in water heated at 80 °C at a concentration of at least 2 g / L.

[0093] Advantageously, the sucrose fatty acid ester is soluble in the oil phase heated at 100 °C at a concentration of at least 2 g / L.

[0094] Advantageously, the limit of solubility in water of the sucrose fatty acid ester suspended in the aqueous phase of the oil-in-water emulsion observed practically at 25 °C is less than 10 mg / L, preferably less than 5 mg / L, more preferably less than 2 mg / L.

[0095] According to the invention, the majority of the lipophilic droplets present in the multiphase composition before adding suspended sucrose fatty acid ester to the aqueous phase of the oil-in-water emulsion, advantageously at least 90% of the lipophilic droplets (also named Dv90 emulsion) have a diameter comprised between 0.01 and 70 pm, preferably between 0.1 and 50 pm, most preferably between 0.1 and 20 pm, advantageously between 0.5 to 7 pm, preferably between 2 and 6 pm as determined by laser diffraction.

[0096] Advantageously at least 90% of the particles of the multiphase composition (also named Dv90 suspo-emulsion) have a diameter comprised between 1 and 1000 pm, advantageously between 10 and 250 with a peak maximum preferably of between 10 pm and 100 pm, also determined by laser diffraction.

[0097] Sucrose fatty acid esters may be used both in water or oil phases as surfactants since they are soluble in oil and in water at high temperatures, typically more than 80 °C, with different solubility constants. Indeed, sucrose esters can become soluble in water, but at high temperature only. It concerns for example sucrose stearate which is soluble in water at around 80 °C. Sucrose fatty acid esters are also soluble in oil, but only to the condition that they undergo prior heating to their melting temperature, which can easily be determined by a person skilled in the art.

[0098] Also, these esters are solid at ambient temperature. Being naturally lipophilic compounds, they are insoluble in water at a standard temperature of 20 °C and are therefore potent candidates for the role of surfactant being suspended in the aqueous phase of the oil-in-water emulsion.

[0099] According to a specific embodiment, the sucrose fatty acid ester contains sucrose stearate or, advantageously, a mixture containing saccharose stearate and sucrose palmitate; in particular is a mixture containing: between 20% and 80% by weight, advantageously 70% saccharose stearate with a monoester content ranging between 20% and 80% by weight of saccharose stearate, advantageously 70%, with the balance being a mixture of di-, tri- and / or polyesters; and between 20% and 80% by weight, advantageously 30% saccharose palmitate with a monoester content ranging between 20% and 80% by weight of saccharose palmitate, advantageously 70%, with the balance being a mixture of di-, tri- and / or polyesters.

[0100] In another aspect of the invention, the phytosterol composition used in the method of the invention comprises at least one further molecule, in particular at least one fluidifying agent and / or at least one solubilizing agent for phytosterols (also called fatty substance) and / or at least one weting agent and / or at least one sticking agent and / or at least one preservative and / or at least one antioxidant.

[0101] According to one aspect, the invention relates to a slurry resulting from the dilution of the phytosterol composition comprising at least one phytosterol and at least one sucrose faty acid ester as previously described for use in the method of the invention. This slurry is performed by diluting the said phytosterol composition, usually in water, at a concentration ranging from 0.001% to 50%, advantageously from 0.01% to 10%, preferably from 0.1% to 5%.

[0102] According to a specific embodiment, the phytosterol composition comprising at least one phytosterol and at least one sucrose fatty acid ester is combined with at least one active ingredient. For the purpose of the invention, the term "active ingredient" refers to a product that helps the plant to fight against corn diseases and / or to grow, advantageously selected from the group comprising:

[0103] - a phytopharmaceutical product such as a plant growth regulator, a fungicide, in particular an organo-chemical fungicide traditionally used in an organo-chemical fungicide treatment sequence, a fungistatic agent, a bactericide, a bacteriostatic agent, an insecticide, an acaricide, a parasiticide, a nematicide, a mole toxicant or a herbicide; and / or

[0104] - a biocontrol product based on natural mechanisms that enables plants to combat fungal infections, bacterial infections, viral infections, pest attacks and / or competition with weeds; and / or

[0105] - a nutrient, organic or inorganic such as a micronutrient or a fertilizer.

[0106] More preferentially, the phytosterol composition according to the method of the invention can be combined with at least one fungicide, in particular an organo-chemical fungicide and / or at least one nutrient.

[0107] Organo-chemical fungicides may be selected among the group consisting of:

[0108] 1. Respiration inhibitors a. Inhibitors of complex III at Qosite (e.g., strobilurins): azoxystrobin, coumethoxystrobin, coumoxystrobin, dimoxystrobin, enestroburin, famoxadone, fenamidone, fenaminstrobin, fenoxystrobin / flufenoxystrobin, fluoxastrobin, kresoxim-methyl, metominostrobin, orysastrobin, picoxystrobin, pyraclostrobin, pyrametostrobin, pyraoxystrobin, trifloxystrobin, pyribencarb, triclopyricarb / chlorodincarb; b. inhibitors of complex III at Qi site: cyazofamid, amisulbrom, c. inhibitors of complex II (e.g. carboxamides): benodanil, bixafen, boscalid, carboxin, fenfuram, fluopyram, flutolanil, fluxapyroxad, furametpyr, isopyrazam, mepronil, oxycarboxin, penflufen, penthiopyrad, sedaxane, tecloftalam, thifluzamide, benzovindiflupyr, inpyrfluxam, isofetamid, pydiflumetofen, fluindapyr; d. complex I, uncouplers: diflumetorim; e. nitrophenyl derivates: binapacryl, dinobuton, dinocap, fluazinam, ferimzone; f. organometallic compounds: fentin acetate, fentin chloride, fentin hydroxide; g. ametoctradin, silthiofam;

[0109] 2. Sterol biosynthesis inhibitors (SBI fungicides) a. C14 demethylase inhibitors (DMI fungicides): i. triazoles: azaconazole, bitertanol, bromuconazole, cyproconazole, difenoconazole, diniconazole, diniconazole-M, epoxiconazole, fenbuconazole, fluquinconazole, flusilazole, flutriafol, hexaconazole, imibenconazole, ipconazole, mefentrifluxonazole, metconazole, myclobutanil, oxpoconazole, paclobutrazole, penconazole, propiconazole, prothioconazole, simeconazole, tebuconazole, tetraconazole, triadimefon, triadimenol, triticonazole, uniconazole; ii. imidazoles: imazalil, pefurazoate, prochloraz, triflumizol; iii. pyrimidines, pyridines and piperazines: fenarimol, nuarimol, pyrifenox, triforine; b. Deltal4-reductase inhibitors: aldimorph, dodemorph, dodemorph-acetate, fenpropimorph, tridemorph, fenpropidin, piperalin, spiroxamine; c. inhibitors of 3-keto reductase: fenhexamid;

[0110] 3. Nucleic acid synthesis inhibitors a. phenylamides or acyl amino acid fungicides: benalaxyl, benalaxyl-M, kiral-axyl, metalaxyl, ofurace, oxadixyl; b. others: hymexazole, octhilinone, oxolinic acid, bupirimate, 5-fluorocytosine;

[0111] 4. Inhibitors of cell division and cytoskeleton a. tubulin inhibitors: benzimidazoles, thiophanates: benomyl, carbendazim, fuberidazole, thiabendazole, thiophanate-methyl, triazolopyrimidines; b. cell division inhibitors: diethofencarb, ethaboxam, pencycuron, fluopicolide, zoxamide, metrafenone, pyriofenone; hibitors of amino acid and protein synthesis a. methionine synthesis inhibitors (anilino-pyrimidines): cyprodinil, mepanipyrim, pyrimethanil; b. protein synthesis inhibitors: blasticidin-S, kasugamycin, kasugamycin hydrochloridehydrate, mildiomycin, streptomycin, oxytetracyclin, polyoxine, validamycin A; nal transduction inhibitors a. MAP / histidine kinase inhibitors: fluoroimid, iprodione, procymidone, vinclozolin, fenpiclonil, fludioxonil; b. G protein inhibitors: quinoxyfen; id and membrane synthesis inhibitors a. phospholipid biosynthesis inhibitors: edifenphos, iprobenfos, pyrazophos, isoprothiolane; b. lipid peroxidation: dicloran, quintozene, tecnazene, tolclofos-methyl, biphenyl, chloroneb, etridiazole; c. phospholipid biosynthesis and cell wall deposition: dimethomorph, flumorph, mandipropamid, pyrimorph, benthiavalicarb, iprovalicarb, valifenalate; d. compounds affecting cell membrane permeability and fatty acids: propamocarb, propamocarb-hydrochloride fatty acid amide; hibitors with Multi Site Action a. thio- and dithiocarbamates: ferbam, mancozeb, maneb, metam, metiram, propineb, thiram, zineb, ziram; b. chlorinated organic compounds (e.g. phthalimides, sulfamides, chloronitriles): anilazine, chlorothalonil, captafol, captan, folpet, dichlofluanid, dichlorophen, hexachlorobenzene, pentachlorophenol and its salts, phthalide, tolylfluanid, and others: guanidine, dodine, dodine free base, guazatine, guazatine-acetate, iminoctadine, iminoctadine-triacetate, iminoctadinetris(albesilate), dithianon;

[0112] 9. Cell wall synthesis inhibitors a. inhibitors of glucan synthesis: validamycin, polyoxin B; b. melanin synthesis inhibitors: pyroquilon, tricyclazole, carpropamid, dicyclomet, fenoxanil;

[0113] 10. Plant defense inducers a. acibenzolar-S-methyl, probenazole, isotianil, tiadinil, prohexadione-calcium; b. phosphonates: fosetyl, fosetyl-aluminum;

[0114] 11. Unknown mode of action: bronopol, chinomethionat, cyflufenamid, cymoxanil, dazomet, debacarb, diclomezine, difenzoquat, difenzoquat methylsulfate, diphenylamin, fenpyrazamine, flumetover, flusulfamide, flutianil, methasulfocarb, nitrapyrin, nitrothal- isopropyl, oxine-copper, picarbutrazox, proquinazid, tebufloquin, tecloftalam, triazoxide.

[0115] According to a specific embodiment, the phytosterol composition used in the method of the invention is combined, advantageously mixed, with strobilurins, carboxamides, triazoles and / or chloronitriles.

[0116] Nutrients can be chosen among the group consisting of: nitrogen (N), phosphorus (P), potassium (K), sulfur (S), calcium (Ca), magnesium (Mg), copper (Cu), iron (Fe), manganese (Mn), zinc (Zn), boron (B), chlorine (Cl), cobalt (Co), molybdenum (Mo) and / or nickel (Ni).

[0117] According to a specific embodiment, the phytosterol composition comprising at least one phytosterol and at least one sucrose fatty acid ester is combined with at least one nutrient, preferably boron (B).

[0118] According to another specific embodiment, the phytosterol composition comprising at least one phytosterol and at least one sucrose fatty acid ester is combined with at least two nutrients, preferably with only two nutrients, preferably boron (B) as the first nutrient, and nitrogen (N) or molybdenum (Mo) as the second nutrient. Advantageously, the phytosterol composition comprising at least one phytosterol and at least one sucrose fatty acid ester is combined, advantageously mixed, with boron and nitrogen.

[0119] Unexpectedly, the Applicant has noted that when the combination of at least one phytosterol and at least one sucrose fatty acid ester of the phytosterol composition used in the method of the invention is combined with at least one active ingredient, in particular a nutrient, this phytosterol composition facilitates the diffusion and passive penetration of the active ingredient into the plant cell. The composition or the slurry described in the invention therefore allows the presence of a higher concentration or quantity of active ingredient in the plant. In addition, the composition or slurry makes it possible to decrease the dose of active ingredients used while guaranteeing the improved effectiveness of these active ingredients.

[0120] According to the invention, when added to the phytosterol composition, the at least one nutrient quantity is chosen between 0.001 and 5 wt% of the composition. When several nutrients are added, this content applies to each nutrient.

[0121] According to the invention, when added to the phytosterol composition: the boron quantity is chosen between 0.01 and 2 wt% of the composition, preferably between 0.1 and 1.8 wt% of the composition, preferably about 1.5 wt% of the composition; the molybdenum quantity is chosen between 0.002 and 1 wt% of the composition, preferably between 0.003 and 0.5 wt% of the composition, preferably about 0.25 wt% of the composition; the nitrogen quantity is chosen between 1 and 4 wt% of the composition, preferably between 1 and 3 wt% of the composition, preferably about 2.2 wt% of the composition.

[0122] Another object of the invention is also an agricultural kit containing separately the phytosterol composition comprising at least one phytosterol and at least one sucrose fatty acid ester (before dilution) and at least one active ingredient as described above.

[0123] In use, the composition of the invention may be mixed by the farmer with an effective amount of the active ingredient and then diluted in order to obtain a slurry which is applied on the plant. Another option is to dilute the composition of the invention in order to obtain the slurry and only then, to add to the slurry the at least one active ingredient.

[0124] In one embodiment, the method consists in applying to the corn plant a foliar spray of a dilution of at least one organo-chemical fungicide in combination with a phytosterol composition comprising at least one phytosterol and at least one sucrose fatty acid ester, preferably, a phytosterol composition comprising at least one phytosterol and at least one sucrose fatty acid ester and at least one nutrient, advantageously boron (B) optionally in combination with nitrogen (N) or molybdenum (Mo).

[0125] For example,

[0126] - the application of the phytosterol composition is applied concomitantly with the first application of the at least one organo-chemical fungicide, and in a subsequent stage, an application of a second at least one organo-chemical fungicide is performed; or

[0127] - the application of the at least one organo-chemical fungicide is performed before the application of the phytosterol composition which is applied concomitantly with the application of the second at least one organo-chemical fungicide; or

[0128] - the application of the first at least one organo-chemical fungicide is performed before the application of the phytosterol composition which is applied subsequently and before a further stage of application of the second at least one organo-chemical fungicide.

[0129] According to one aspect, the invention relates to a slurry resulting from the dilution of the phytosterol composition in combination with at least one organo-chemical fungicide as previously described to be applied on corn so as to increase kernel row number and / or number of kernels per row and / or improve seed quantity. This slurry is performed by diluting the said combination, usually in water, at a concentration ranging from 0.001% to 50%, advantageously from 0.01% to 10%, preferably from 0.1% to 5%.

[0130] One option is to tank-mix the phytosterol-based composition with at least one active ingredient as above described, and then to dilute the mix in water (in all possible orders) to prepare the slurry. Another option is to dilute the phytosterol composition in order to obtain the slurry and only then, to add to the slurry the said at least one active ingredient as above described. Another option is to dilute in water a pre-mix comprising at least one active ingredient as above described with the phytosterol composition. In such a case the phytosterol composition and the said at least one active ingredient as above described are sprayed on corn at the same time.

[0131] Another option is to perform the treatment of cultivated corn with the phytosterol composition used in the method of the invention combined with at least one active ingredient as above described, but the said phytosterol composition (hereafter designed as "A") is not mixed in the same tank with the said at least one active ingredient as above described (hereafter designed as "B"). This option comprises one of these situations:

[0132] - A and B are in two or more different tanks but on the same vehicle, the vehicle being a human, an animal, a flying or driving machine carrying or comprising any equipment suitable to spray a liquid,

[0133] - A and B are in two or more different tanks in two or more different vehicles, then o A and B are sprayed on the same corn plants on the same day, o A is sprayed and B is sprayed from one to three days after A has been sprayed, o B is sprayed and A is sprayed from one to three days after B has been sprayed.

[0134] In one embodiment, the method consists in applying to corn a foliar spray comprising a dilution of a phytosterol composition comprising at least one phytosterol and at least one sucrose fatty acid ester at a BBCH chosen between BBCH 16 and BBCH 19, advantageously between BBCH 16 and BBCH 19 with up to 13 unfolded leaves, more preferably between BBCH 16 and BBCH 19 with up to 11 unfolded leaves, even more preferably between BBCH 16 and BBCH 19 with up to 10 unfolded leaves. This application is performed at a total concentration of said at least one phytosterol between 2.5 g / ha to 250 g / ha, preferably between 12.5 g / ha and 125 g / ha, more preferably between 25 g / ha and 100 g / ha, the said concentration being expressed in grams of phytosterol per hectare of treated corn plants.

[0135] In another embodiment, the method consists in priming, coating, pelleting or imbibing corn seeds (BBCH 00) prior to sowing with a phytosterol composition comprising at least one phytosterol and at least one sucrose fatty acid ester. This priming or sowing step is performed either with a non-diluted or with a diluted phytosterol composition comprising at least one phytosterol and at least one sucrose fatty acid ester. In the latter case, the dilution of the phytosterol composition comprising at least one phytosterol and at least one sucrose fatty acid ester can be performed with water, giving a concentration of phytosterol in the dilution ranging from IO-7mol / L to 0.06 mol / L.

[0136] Practically, the composition can be applied by spraying, watering, adding to a hydroponic growing medium, immersing the seed and / or coating the seed.

[0137] It is possible to treat corn grown in open field, in greenhouse, in a climate chamber or in a germination chamber.

[0138] Advantageously the application of the method of the invention is recommended on corn inbred lines, but it is also possible to treat all flowering plants belonging to the genus Zea with the method of the invention.

[0139] The invention also relates to an inbred corn variety treated with a phytosterol composition comprising at least one phytosterol and at least one sucrose fatty acid ester, wherein said corn plant exhibits a phenotype of increased kernel row number and / or kernel number of rows and / or number of seeds per ear and / or improved seed germination in comparison to a plant of the same variety grown under essentially the same conditions, but not treated with the phytosterol composition. The fact that the efficiency of the product is not related to genetic improvements results in the possibility to apply this method on any variety.

[0140] Particularly, the treated corn plant exhibits at least 0.25 more rows in average, and / or at least 2% more kernels per row, and / or at least 5% more seeds per ear, and / or at least a germination rate improved by at least 1.0% in comparison to a corn plant of the same variety grown under essentially the same conditions, but not treated with the phytosterol composition.

[0141] Advantageously, the treated corn plant exhibits at least 0.35 more rows in average, and / or at least 4% more kernels per row, and / or at least 8% more seeds per ear, and / or at least a germination rate improved by at least 1.5% in comparison to a corn plant of the same variety grown under essentially the same conditions, but not treated with the phytosterol composition.

[0142] To improve seed germination, the method of the invention is applied on inbred corn lines, that is to say on substantially genetically pure lines, which are used as parents of corn hybrids when the male corn plant is crossed with the female corn, or as parents of inbred corn plants when the corn is self-pollinated.

[0143] In one embodiment, both male and female inbred lines are treated with the method of the invention. Advantageously, only the female inbred line is treated with the method of the invention.

[0144] Also, treated corn exhibits at least 1.0%, preferably at least 1.5%, advantageously at least 2.0% more germinated seeds in average in comparison to a corn plant of the same variety grown under essentially the same conditions, but not treated with the phytosterol composition.

[0145] In another embodiment, treating corn with the method of the invention allows the seed producer to gain more revenues from its production. Due to the fact that both number of seeds and germination rate are improved, the number of corn seed doses is also increased, leading to more income. Thus, treating corn with the method of the invention allows increasing the revenue by at least 4000 € / ha, advantageously at least 6000 € / ha the revenue that would be obtained from an untreated corn.

[0146] Practically, an inbred corn variety is treated with the phytosterol composition of the method of the invention comprising at least one phytosterol and at least one sucrose fatty acid ester at a phenological stage which leads to increased kernel number of rows and / or number of kernels per row and / or increased number of seeds per ear and / or improved seed germination, compared to the same corn inbred variety grown under essentially the same conditions, but not treated with the phytosterol composition. This treatment can be performed either foliar, or directly on the seeds of the said corn inbred variety.

[0147] In another embodiment, a corn seed is obtained from the method of the invention, wherein the germination rate of said corn seed is improved by at least 1.0%, preferably at least 1.5%, advantageously at least 2.0% the germination rate of a corn seed obtained from a corn plant which is not treated with the said method but grown under substantially the same conditions.

[0148] The invention and the benefits it produces are more visible in the following examples, which are given in order to illustrate the invention in a non-exhaustive fashion.

[0149] BRIEF DESCRIPTION OF THE FIGURES Figure 1: daily rainfalls (black histograms), mean daily temperature (grey curve) and temperature limit of heat stress (dashed line). Sowing is at day 0, treatment with the product is at day 29 (black arrow). The black star is the harvest (day 130).

[0150] Figure 2: daily rainfalls (black histograms), mean daily temperature (grey curve) and temperature limit of heat stress (dashed line). Sowing is at day 0, treatment with the product is at day 37 (black arrow). The black star is the harvest (day 159, estimated).

[0151] EXAMPLES OF APPLICATION OF THE INVENTION

[0152] 1. Preparation of a phytosterol composition used in the method of the invention The formulation of the phytosterol composition used in the method of the invention is a suspo-emulsion comprising the compounds of Table 1.

[0153] Table 1. Phytosterol compositions.

[0154] The various compositions according to the invention are manufactured comprising the following steps:

[0155] (i) Preparation at about 110 °C of a lipophilic phase comprising phytosterols, sucrose stearate, methyl tetradecanoate, and solvent,

[0156] (ii) Preparation at a given temperature of a hydrophilic phase comprising water and benzyl alcohol if any, (iii) Mixing the lipophilic phase of step (i) with the hydrophilic phase of step (ii) and stirring until at least 90% of the volume of lipophilic droplets with a diameter comprised between 0.1 and 20 pm are obtained, with a maximum peak between 2 and 6 pm as determined by laser diffraction,

[0157] (iv) Cooling the emulsion to ambient temperature of about 20 °C, and

[0158] (v) Adding sucrose stearate in the aqueous phase of the oil-in-water emulsion, at ambient temperature of about 20 °C and stirring until at least 90% of the particles with a diameter comprised between 10 and 250 pm are obtained and suspended in the aqueous phase, with a maximum peak between 10 pm and 1000 pm as determined by laser diffraction.

[0159] (vi) Optionally and according to formulation 2 in Table 1, adding at least one nutrient to the suspo-emulsion, at ambient temperature of about 20 °C and stirring until the at least one nutrient is fully dissolved in the aqueous phase of the said suspo- emulsion.

[0160] 2. Increase of the kernel row number of treated corn compared to an untreated corn, according to the BBCH stage of the application of the phytosterol-based composition

[0161] The objective of the trial is to demonstrate the efficiency of a phytosterol composition comprising at least one phytosterol and at least one sucrose fatty acid ester to increase the kernel row number according to the phenological stage the said treatment was applied on corn.

[0162] Numerous trials were conducted in various contexts: different corn varieties, soils, pedoclimatic conditions. All experiments included a modality wherein corn was treated with the phytosterol compositions of example 1 (formulations 1 and 2), and a modality wherein corn was not treated with the said phytosterol composition. The following Table summarizes the beneficial effects of an application of the phytosterol composition, with the increase in the kernel row number being sorted according to the BBCH stage of the application. The value of the increase is the mean value of 199 trials. Table 2. Increase in the kernel row number according to the application stage of the composition of the invention. The number of unfolded leaves is also provided. number of untreated modality.

[0163] From Table 2, the strongest difference in the kernel row number occurs when the composition of the invention is applied at a BBCH stage between 16 and 19 (with 11 unfolded leaves), more precisely at BBCH stage 19 with 10-11 unfolded leaves. The mean increase in the BBCH stage between 16 and 19 (with 11 unfolded leaves) is more than 0.15, meaning that if in one trial the kernel row number of the untreated corn is 18, then the mean kernel row number of the treated corn with the composition of the invention is 18 + 0.15 = 18.15.

[0164] 3. Efficacy of the composition of the invention to increase the kernel row number of corn exposed to drought and thermal stress

[0165] The main objective was to investigate the ability of a phytosterol-based composition to improve the kernel row number of corn while being exposed to drought and thermal stress. a. Equipment and methods i. Description of the experimental setup

[0166] The description of the experimental setup is presented in Table 3.

[0167] Table 3. Experimental setup. ii. Modalities considered The description of the modalities considered is presented in Table 4.

[0168] Table 4. Tested compositions.

[0169] A foliar spray of the slurry of the composition (formulation 1 of Table 1) was performed 29 days after sowing (BBCH 17) under these conditions: temperature 29 °C, relative humidity 65%, wind 9 km / h. b. Climate

[0170] From sowing to harvest, corn was exposed to severe abiotic stresses. The total rainfalls were approximately 390 mm. Corn suffered from 85 days of hydric stress, 16 days where the mean daily temperatures were more than 30 °C and 85 days where the maximum daily temperatures were more than 30 °C. Figure 1 shows the climatic pattern, which was obtained from Copernicus CDS, according to the GPS field coordinates (Boogaard, H. et al. Copernicus Climate Change Service (C3S) Climate Data Store (CDS). DOI: 10.24381 / cds.6c68c9bb). c. Results and conclusions

[0171] Table 5 summarizes the kernel row number of the untreated and the treated corn.

[0172] Table 5. Kernel row number for both modalities.

[0173] In conclusion, kernel row number is increased when corn is treated at BBCH17 with the composition of the invention. 4. Efficacy of the composition of the invention to increase the kernel row number, seeds per row and seeds per ear of corn exposed to drought and thermal stresses

[0174] The main objective was to investigate the ability of a phytosterol-based composition to improve the kernel row number of corn while being exposed to drought and thermal stresses. a. Equipment and methods i. Description of the experimental setup

[0175] The description of the experimental setup is presented in Table 6.

[0176] Table 6. Experimental setup. ii. Modalities considered

[0177] The description of the modalities considered is presented in Table 7.

[0178] Table 7. Tested compositions.

[0179] The treatment was performed as 0.66% slurries by diluting the formulation 2 of Table 1 in water. The foliar spray was applied 37 days after sowing (BBCH 19, 10 unfolded leaves).

[0180] Concomitantly, a fungicide treatment consisting of Metominostrobin and Tebuconazole (0.725 L / ha) was also performed at BBCH 19, followed, in a subsequent stage, by another fungicide treatment consisting of Azoxyst robin and Cyproconazole (0.40 L / ha) at pre-tasseling. b. Climate

[0181] From sowing to harvest, corn was exposed to severe abiotic stresses. The total rainfalls was approximately 410 mm. Corn suffered from 77 days of hydric stress (including 67 days of severe drought stress), with no rainfall occurrence after day 80. Added to this drought stress are 138 days with maximum temperatures above 30 °C. Figure 2 shows the climatic pattern, which was obtained from the Copernicus CDS, according to the GPS coordinates of the field. c. Results and conclusions

[0182] Table 8 summarizes the kernel row number, the kernels per row and the number of seeds per ear for the untreated and treated corn. It is seen that all values are increased when corn is treated at BBCH19 (10 unfolded leaves) with the composition of the invention.

[0183] Table 8. Mean values for both modalities.

[0184] 5. Efficacy of the method of the invention to increase the number of corn seeds and to improve the germination rate of harvested seeds

[0185] The main objective was to investigate the ability of a phytosterol-based composition to increase the number of corn seeds and to improve the germination rate of harvested seeds, in a configuration which is compliant with seed production practice. a. Experimental setup

[0186] The experimental setup is presented in Table 9. The treatment was performed by foliar- spraying a water slurry of formulation 1 of Table 1 on a seed-production suitable corn variety.

[0187] Table 9. Experimental setup. b. Climate

[0188] From sowing to harvest, corn was exposed to severe abiotic stresses. Rainfalls and irrigation were 230 mm and 216 mm, respectively, totaling 446 mm. 27 days of water stress were counted, including 2 days of severe water stress. 37 days of heat stress were also recorded. c. Results and conclusions

[0189] Table 10 summarizes the results obtained for the untreated and the treated corn. Parameters such as number of rows, number of seeds per row and seeds per ear were estimated by collecting 10 corn ears per modality a few days before harvest, whereas the germination rate, the seed units / ha value and the revenue gain were estimated after harvest.

[0190] Table 10. Mean values for both modalities.

[0191] In conclusion, it is seen that treating corn with the composition of the invention increased the total number of seeds per row (notably by increasing both number of rows and number of seeds per row) and the germination rate, which is highly desirable for seed production. This is reflected by the improvement in seed units per hectare, which means that the seed producer can sell more efficient seeds per hectare. Also, since both seed units and germination rate were improved, it is expected a revenue gain between 4300 and 10,000 € per hectare thanks to the method of the invention. 6. Increase of germination rate of seeds of hybrid corn

[0192] The main objective of this trial was to demonstrate that the method of the invention increased the germination rate of seeds obtained for a hybrid corn.

[0193] 1. Experimental setup

[0194] The description of the experimental setup is presented in Table 11. The treatment was performed at day 46 after sowing by foliar-spraying a water slurry of formulation 1 of Table 1.

[0195] Table 11. Experimental setup.

[0196] 2. Climate

[0197] From sowing to harvest, corn was not exposed to drought stress. The total rainfalls was approximately 460 mm, to which were added 210 mm of irrigation, totaling 670 mm. However, 25 days of heat stress were recorded.

[0198] 3. Results and conclusions

[0199] Table 12 summarizes the results obtained for seeds obtained from the untreated and the treated hybrid corn. It is seen that treating corn with the method of the invention increased their germination rate.

[0200] Table 12. Germination rate for both modalities.

[0201] 7. Prophetic example showing the improvement in seed count increase and germination rate from two inbred lines This first prophetic example illustrates the effect of the method of the invention when two inbred lines are hybridized. The following method can lead to an increased number of hybrid seeds per hectare, coupled with an increase in the germination rate of these hybrid seeds.

[0202] Two different inbred lines are sown either concomitantly, or at various sowing times, the most important objective being that male lines flower slightly before female lines do, which will then be fertilized by the pollen of the male lines.

[0203] The sowing scheme is performed in classical hybridization conditions, that is to say by alternating usually 2 rows of male - that optionally are destroyed before harvest - and 4 rows of female corn plants, so that the resulting hybrid results only from the cross-breeding of the inbred parents.

[0204] The total sowing density is then usually higher than hybrid seed production standards, for example around 100,000 to 110,000 plants / hectare.

[0205] The application of the phytosterol composition of the method of the invention is performed by treating the inbred female line only, with a phytosterol composition which is described as formulation 1 in Table 1 of example 1. A slurry is prepared by diluting formulation 1 at 1% in water. The slurry is sprayed on the leaves of the inbred female line at a field concentration of 100 to 600 L / ha, under suitable conditions (temperature between 5 °C and 25 °C, wind < 12 km / h, RH > 60%, no rainfalls up to 2 hours after application). This treatment is performed once, at a BBCH 16-19 (up to 13 leaves) phenological stage. Optionally the seeds of the inbred parent lines (female and eventually male line) are treated with the composition of the method of the invention prior to sowing.

[0206] For comparison purposes, a section of the field can be left untreated with the phytosterol composition of the method of the invention.

[0207] Before pollination, 99.5%+ detasseling of the female line is performed using any known process, for example using a detasseling machine, detasselers and / or using inbred male-sterile line, to avoid the female flower being fertilized with the pollen of the same line.

[0208] Optionally, the ear of the inbred male is protected from pollination, if male lines are not destroyed before harvest. Roguing is optionally performed to further enhance the gene purity of the hybrids. Other maintenance is performed on the field to mitigate pests, supply required nutrients, before sowing and during the lifecycle of the corn plant.

[0209] When water availability can negatively impact the total number of harvested seeds, and considering necessary investment to manage this production and significant water needs of corn during summer, the fields are often irrigated.

[0210] After harvest, the number of seeds per hectare is counted or extrapolated from a smaller counted surface, and it is prophesized that this number is increased by at least 5% when the composition of the method of the invention is applied according to this method, compared to an untreated corn which is grown under substantially the same conditions and same field.

[0211] Then, the germination rate of two modalities of hybrid seeds isolated from treated and nontreated parent lines is determined.

[0212] For each modality, 15 seeds are placed on water-soaked paper in a Petri dish, each modality containing at least 8 Petri dishes.

[0213] The dishes are placed in a germination chamber maintained at a temperature approximately equal to that found in the soil when sowing corn, i.e., about 10 °C, in the dark.

[0214] A daily count of the development status of the seeds is carried out: dO is the day of sowing the seeds, 1stday after sowing (dl), 2ndday after sowing (d 2), etc., until complete germination.

[0215] Several stages are distinguished:

[0216] SO: non-germinated seeds. Germinated seeds: SI (seed coat breaks), S2 (radicle appears; it must be at least 2 mm long for the seed to be considered germinated and used for subsequent measurements), and S3 (coleoptile appears).

[0217] Germination Rate Measurements: The number of germinated seeds is counted regularly throughout the trial to determine the germination rate. The germination rate is as follows:

[0218] Number of germinated seeds

[0219] 6% = - - - - - - - - x 100

[0220] Number of seeds

[0221] It is prophesized that hybrid seeds from corn plants treated with the method of the invention have an increased average germination rate of at least 1.0% compared to the germination rate of untreated parents. This improved germination rate then come in addition to the increased number of seeds to optimize the number of second generation plants issued from the parental lines hybridization.

[0222] 8. Prophetic example showing the improvement in seed count increase and germination rate from the selfing of an inbred line

[0223] This second prophetic example illustrates the effect of the method of the invention when an inbred line is pollinated with itself. The following method can lead to an increased number of inbred seeds per hectare, coupled with an increase in the germination rate of these inbred seeds.

[0224] An inbred line is sown according to standard practice of the seed production industry for corn inbred line propagation. Fertilization, field and pest management, roguing, sowing density and other practice are optionally performed according to ordinary skills. Optionally the seeds of the parent lines have been treated with the method of the invention, by seed coating or by seed imbibition for example.

[0225] The sowing density is compliant with hybrid seed production standards, for example 90,000 plants / hectare.

[0226] The application of the phytosterol composition of the method of the invention is performed by treating the corn plant with a phytosterol composition which is described as formulation 2 in Table 1 of example 1. A slurry is prepared by diluting formulation 2 at 1% in water. The slurry is sprayed on the leaves of the inbred female line at a field concentration of 100 to 600 L / ha, under suitable conditions (temperature between 5 °C and 25 °C, wind < 12 km / h, RH > 60%, no rainfalls up to 2 hours after application). In the case whether the corn seeds were not previously treated with the phytosterol composition, this treatment is performed once, at a BBCH 16-19 (up to 13 leaves) phenological stage.

[0227] For comparison purposes, a section of the field can be left untreated with the phytosterol composition of the method of the invention.

[0228] Roguing is optionally performed to further enhance the gene purity of the hybrids. This allows the parent line to be conserved by self-pollination and not degraded with undesirable genetics. Other maintenance is performed on the field to mitigate pests, supply required nutrients, before sowing and during the lifecycle of the corn plant.

[0229] When water availability can negatively impact the total number of harvested seeds, the field is irrigated.

[0230] After harvest, the number of seeds per hectare is counted or extrapolated from a smaller counted surface, and it is prophesized that this number is increased by at least 5% when the composition of the method of the invention is applied according to this method, compared to untreated corn which is grown under substantially the same conditions and same field.

[0231] Then, the germination rate of two modalities of inbred seeds isolated from treated and nontreated parent lines is determined.

[0232] For each modality, 15 seeds are placed on water-soaked paper in a Petri dish, each modality containing at least 8 Petri dishes.

[0233] The dishes are placed in a germination chamber maintained at a temperature approximately equal to that found in the soil when sowing corn, i.e., about 10 °C, in the dark.

[0234] A daily count of the development status of the seeds is carried out: dO is the day of sowing the seeds, 1stday after sowing (dl), 2ndday after sowing (d2), etc., until complete germination, with the stages and the germination rate being determined as in the prophetic example 7.

[0235] It is prophesized that hybrid seeds from corn plants treated with the method of the invention have an increased average germination rate of at least 1.0% compared to the germination rate of untreated parents. This improved germination rate then come in addition to the increased number of seeds to optimize the number of second generation plants issued from the parental lines selfing.

Claims

CLAIMS1. A method of producing seeds for a corn hybrid plant, the said method comprising: a. Growing a first inbred corn plant as a female and a second inbred corn plant as a male, b. Treating said first female inbred corn plant with a phytosterol composition comprising at least one phytosterol and at least one sucrose fatty acid ester, c. Detasseling said first female inbred corn plant, d. Crossing said first female inbred corn plant with said second male inbred corn plant, e. Growing said first female inbred corn plant, f. Harvesting the resultant hybrid corn seed from said first female inbred corn plant; wherein the number of seeds produced from said first female inbred corn plant is at least 5% higher than the number of seeds produced by a female inbred corn plant grown under substantially identical conditions which has not been treated with a phytosterol composition under step b.

2. A method of producing seeds for a corn inbred plant, the method comprising: a. Growing an inbred corn plant, b. Treating said corn inbred plant with a phytosterol composition comprising at least one phytosterol and at least one sucrose fatty acid ester, c. Self-pollinating said inbred corn plant, d. Growing said inbred corn plant, e. Harvesting the resultant inbred corn seed from said inbred corn plant; wherein the number of seeds produced from said inbred corn plant is at least 5% higher than the number of seeds produced by an inbred corn plant grown under substantially identical conditions which has not been treated with a phytosterol composition under step b.

3. The method of claim 1, wherein step a. further requires that male and female corn plants are sown in alternating rows.

4. The method of claim 2, wherein step c. further requires keeping distance from other corn varieties.

385. The method of claim 1 or 2, wherein the said method further allows increasing the germination rate of the said harvested corn seed by at least 1.0%, preferably at least 1.5%, advantageously at least 2.0% compared to an untreated corn.

6. The method of any of the previous claims, wherein the said corn is treated at an early phenological stage, said early phenological stage corresponding to a BBCH stage between BBCH 00 and BBCH 19, with up to 13 unfolded leaves.

7. The method of claim 6, wherein the said early phenological stage corresponds to a BBCH stage between BBCH 00 and BBCH 19, with up to 11 unfolded leaves.

8. The method of claim 6, wherein the said early phenological stage corresponds to a BBCH stage between BBCH 16 and BBCH 19, with up to 10 unfolded leaves.

9. The method of claim 1 or 2, wherein the said increase of the kernel row number of corn ears is more than 0.25, compared to the untreated plant.

10. The method of claim 1 or 2, wherein the said increase of the kernel row number of corn ears is more than 0.30, compared to the untreated plant.

11. The method of any of the previous claims, wherein said at least one phytosterol is selected from the group consisting of: beta-sitosterol, campesterol, brassicasterol, stigmasterol, lanosterol, stigmastanol, campestanol, cycloartenol, gamma-sitosterol, A5-avenasterol, A7-avenasterol, isofucosterol, 5-dehydroepisterol, chlerosterol, sitostanol, stigmastadioenol, cholesterol, cholestanol, dinosterol, ergosterol, obtusifoliol, and mixtures thereof.

12. The method of claim 11, wherein said at least one phytosterol is a mixture of phytosterols containing p-sitosterol .

13. The method of claim 12, wherein said at least one phytosterol is a mixture of phytosterols containing p-sitosterol, which represents at least 30% of the phytosterols mixture by weight, with the balance to 100% containing campesterol, stigmasterol and brassicasterol.

14. The method of any of the previous claims, wherein said at least one sucrose fatty acid ester is selected from the group consisting of: sucrose stearate, sucrose palmitate, their polyesters and mixtures thereof.

15. The method of any of the previous claims, wherein the mass ratio between said at least one phytosterol and said at least one sucrose fatty acid ester is between 0.01 and 10.

16. The method of any of the previous claims, wherein:said at least one phytosterol represents between 0.2% and 10% of the composition by weight; and said at least one sucrose fatty acid ester represents between 0.2% and 10% of the composition by weight.

17. The method of any of the previous claims, wherein the total concentration of said at least one phytosterol applied on cultivated corn is between 2.5 g / ha to 250 g / ha, advantageously between 12.5 g / ha and 125 g / ha, preferably between 25 g / ha and 100 g / ha.

18. The method of any of the previous claims, wherein the phytosterol composition further comprises at least one nutrient selected from the group consisting of: boron (B), nitrogen (N), molybdenum (Mo), phosphorus (P), potassium (K), sulfur (S), calcium (Ca), magnesium (Mg), copper (Cu), iron (Fe), manganese (Mn), zinc (Zn), chlorine (Cl), cobalt (Co), nickel (Ni) and their mixtures.

19. The method of claim 18, wherein the nutrient is at least boron.

20. The method of claim 18, wherein the nutrient is at least boron and nitrogen.

21. The method of claim 18, wherein the nutrient is at least boron and molybdenum.

22. The method of any of the previous claims, wherein the phytosterol composition is a suspo-emulsion.

23. The method of claim 22, wherein the suspo-emulsion is a multiphase agricultural composition, comprising: lipophilic droplets containing a mixture of phytosterols, wherein the lipophilic droplets are dispersed in an aqueous phase to form an oil-in-water emulsion, wherein the mixture of phytosterols comprises R-sitosterol present in at least 30% by weight of the mixture of phytosterols, with the balance of the mixture to 100% comprising campesterol, stigmasterol and brassicasterol; and wherein the mixture of phytosterols is between 0.2% and 10% of the composition by weight; at least one first sucrose fatty acid ester (SEI) located at the interface of the lipophilic droplets and the aqueous phase; and at least one second sucrose fatty acid ester (SE2) suspended in the water phase of the oil-in-water emulsion, wherein the said at least one second sucrose fatty acid ester (SE2) is in the form of particles insoluble in said water phase of the oil-in-water emulsion,wherein the said at least one first sucrose fatty acid ester (SEI) and the said at least one second sucrose fatty acid ester (SE2) each independently comprise sucrose stearate, or sucrose palmitate, or a mixture thereof; wherein the said at least one first sucrose fatty acid ester (SEI) is between 0.2% and 10% of the composition by weight, and the said at least one second sucrose fatty acid ester (SE2) is between 0.5% and 5% of the composition by weight.

24. A hybrid corn plant produced from a first female inbred corn plant treated with a phytosterol composition comprising at least one phytosterol and at least one sucrose fatty acid ester, wherein said hybrid corn plant exhibits a phenotype of increased kernel row number and / or number of kernels per row and / or number of seeds per ear and / or improved seed germination in comparison to a hybrid corn plant produced from a female inbred corn plant of the same variety grown under essentially the same conditions but not treated with said phytosterol composition.

25. A plant of a corn inbred variety treated with a phytosterol composition comprising at least one phytosterol and at least one sucrose fatty acid ester, wherein said corn plant exhibits a phenotype of increased kernel row number and / or kernel number of rows and / or number of seeds per ear and / or improved seed germination in comparison to a plant of the same variety grown under essentially the same conditions but not treated with said composition comprising at least one phytosterol and at least one sucrose fatty acid ester.

26. The corn plant of claim 24 or 25, wherein the treated corn plant exhibits at least 0.25 kernel rows more in comparison to a plant of the same variety grown under essentially the same conditions but not treated with said composition comprising at least one phytosterol and at least one sucrose fatty acid ester.

27. The corn plant of any of claims 24 to 26, wherein the treated corn plant exhibits at least 5% more seeds production in comparison to a plant of the same variety grown under essentially the same conditions but not treated with said composition comprising at least one phytosterol and at least one sucrose fatty acid ester.

28. The corn plant of any of claims 24 to 27, wherein the treated corn plant exhibits an improved germination rate of at least 1.0% more in comparison to a plant of the same variety grown under essentially the same conditions but not treated with saidcomposition comprising at least one phytosterol and at least one sucrose fatty acid ester.

29. A corn seed obtained from the method of claim 1 or 2, wherein the germination rate of said corn seed is improved by at least 1.0%, preferably at least 1.5%, advantageously at least 2.0% the germination rate of a corn seed obtained from a corn plant which is not treated with the said method.

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