Plant growth promoting components

Abstract

To provide a plant growth promoting composition that can promote plant growth more safely and inexpensively compared to conventional materials. [Solution] A plant growth promoting composition containing one or more selected from the group consisting of cocoa extract and cocoa powder as an active ingredient.

Description

Technical Field

[0001] The present invention relates to a plant growth promoting composition and a plant growth promoting method.

Background Art

[0002] Currently, due in part to the explosive population growth, producing food plants in a sufficient supply has become a global challenge. In addition, land desertification caused by reduction of green spaces has become a problem, and furthermore, the rise in surface temperature due to global warming has also become a problem, leading to an increasing number of environmental issues in plant cultivation. That is, excessive stress on plants caused by global environmental factors often poses a problem for plant growth.

[0003] Accordingly, there is a demand for technologies that promise more efficient harvests than ever before, and biostimulants are attracting attention as a new technology that mitigates plant damage caused by climate and soil conditions by controlling environmental stress on plants, thereby providing healthy plants.

[0004] Patent Document 1 discloses a plant stomatal opening promoter containing stevia, and it has been reported that artificially promoting the opening of plant stomata with stevia improves heat stress tolerance and the like. Patent Document 2 discloses a composition for activating the ethylene and / or jasmonic acid signal transduction pathway, which contains a yeast cell wall degradation product, and it has been reported that applying the yeast cell wall degradation product to plants activates the expression of plant defensin genes involved in the antibacterial properties of plant bodies and disease prevention. Patent Document 3 discloses a method for increasing the yield of cereals by inoculating a plant body for cultivating cereals with a cell wall-containing material of a methanol-assimilating bacterium.

Prior Art Documents

Patent Documents

[0005] [Patent Document 1] Patent No. 6741262 [Patent Document 2] Patent No. 4931388 [Patent Document 3] Japanese Patent Publication No. 2019-180361 [Overview of the Initiative] [Problems that the invention aims to solve]

[0006] As mentioned above, various studies have been conducted to date using different materials to improve plant tolerance to environmental stress and increase yield, but no examples have been reported using cocoa extract or cocoa powder. [Means for solving the problem]

[0007] As a result of diligent research, the inventors discovered, using a uniquely developed high-precision screening method, that one or more materials selected from the group consisting of cocoa extract and cocoa powder can be effective ingredients for promoting plant growth, thus completing the present invention.

[0008] In other words, the present invention is as follows. [1] A plant growth promoting composition containing one or more substances selected from the group consisting of cocoa extract and cocoa powder as an active ingredient. [2] The plant growth promoting composition described in [1] above, which has one or more plant growth promoting effects selected from the group consisting of elongation of stems and leaves or roots, increase in the number of leaves, promotion of flowering or fruiting, increase in the number of flowers or fruits, increase in plant weight or crop yield, greening, and promotion of tillering. [3] A plant growth promoting composition according to [1] or [2] above, which has an effect of improving environmental stress tolerance. [4] The plant growth promoting composition described in [3] above, which has an effect of improving tolerance to one or more environmental stresses selected from the group consisting of temperature stress, nutrient stress, chemical stress, light stress, drought stress, pH stress, salt stress, hypoxia stress, herbivore stress, physical stress, and disease stress. [5] The plant growth promoting composition described in [3] above, which activates genes in the stress response system. [6] A method for promoting plant growth, comprising using a plant growth promoting composition containing one or more selected from the group consisting of cocoa extract and cocoa powder as an active ingredient, on a plant, or in the soil or culture medium on which the plant grows. [7] The plant growth promoting method according to [6] above, wherein the amount of the plant growth promoting composition used in the culture medium or diluent is 0.001 to 50% by mass or 0.001 to 50% by volume. [Effects of the Invention]

[0009] By applying a composition containing one or more selected from the group consisting of cocoa extract and cocoa powder of the present invention to plants, plant growth can be promoted more safely and inexpensively compared to conventional materials. [Brief explanation of the drawing]

[0010] [Figure 1] These images show the improvement in root development of rice plants when no cocoa extract or cocoa powder was added (left) and when 3.6 mL of a plant growth promoting composition containing cocoa extract (Example 1) was added (right). [Figure 2] This graph shows the weight of rice plants when no cocoa extract or cocoa powder is added, and when 3.6 mL of a plant growth promoting composition containing cocoa extract (Example 1) is added. [Figure 3] This image shows a heatmap of the expression levels of high-temperature stress tolerance genes in rice plants when no cocoa extract or cocoa powder is added, and when 3.6 mL of a plant growth-promoting composition containing cocoa extract is added. [Figure 4] This is an image showing the expression level of the high-temperature stress tolerance gene Os03g0854500 in rice between the case where no cocoa extract or cocoa powder is added and the case where 3.6 mL of the plant growth-promoting composition containing cocoa extract is added. [Figure 5] This is an image showing the heat map of the expression level of the drought stress tolerance gene in rice between the case where no cocoa extract or cocoa powder is added and the case where 3.6 mL of the plant growth-promoting composition containing cocoa extract is added. [Figure 6] This is an image showing the expression level of the drought stress tolerance gene Os02g0115700 in rice between the case where no cocoa extract or cocoa powder is added and the case where 3.6 mL of the plant growth-promoting composition containing cocoa extract is added. [Figure 7] This is an image showing the heat map of the expression level of the low-temperature stress tolerance gene in rice between the case where no cocoa extract or cocoa powder is added and the case where 3.6 mL of the plant growth-promoting composition containing cocoa extract is added. [Figure 8] This is an image showing the expression level of the low-temperature stress tolerance gene Os03g0285800 in rice between the case where no cocoa extract or cocoa powder is added and the case where 3.6 mL of the plant growth-promoting composition containing cocoa extract is added. [Figure 9] This is an image showing the heat map of the expression level of the salt stress tolerance gene in rice between the case where no cocoa extract or cocoa powder is added and the case where 3.6 mL of the plant growth-promoting composition containing cocoa extract is added. [Figure 10] This is an image showing the expression level of the salt stress tolerance gene Os02g0543000 in rice between the case where no cocoa extract or cocoa powder is added and the case where 3.6 mL of the plant growth-promoting composition containing cocoa extract is added. [Figure 11] This is an image showing the heat map of the expression level of the osmotic stress tolerance gene in rice between the case where no cocoa extract or cocoa powder is added and the case where 3.6 mL of the plant growth-promoting composition containing cocoa extract is added. [Figure 12]This is an image showing the expression level of the rice osmotic stress tolerance gene Os02g0666200 in the case where no cocoa extract or cocoa powder is added, and in the case where 3.6 mL of the plant growth-promoting composition containing cocoa extract is added. [Figure 13] This is an image showing a heat map of the expression level of the rice high-light stress tolerance gene in the case where no cocoa extract or cocoa powder is added, and in the case where 3.6 mL of the plant growth-promoting composition containing cocoa extract is added. [Figure 14] This is an image showing the expression level of the rice high-light stress tolerance gene Os05g0592400 in the case where no cocoa extract or cocoa powder is added, and in the case where 3.6 mL of the plant growth-promoting composition containing cocoa extract is added. [Mode for Carrying Out the Invention]

[0011] Hereinafter, the mode for carrying out the present invention (hereinafter also referred to as "the present embodiment") will be described in detail. The present invention is not limited to the present embodiment, and can be implemented with various modifications within the scope of its gist.

[0012] <Plant Growth-Promoting Composition> The plant growth promoting composition in this embodiment contains one or more active ingredients selected from the group consisting of cocoa extract and cocoa powder. The plant growth promoting composition in this embodiment may contain only cocoa extract or cocoa powder, or it may contain both cocoa extract and cocoa powder. Cocoa (Theobroma cacao) is a plant of the genus Theobroma in the family Malvaceae. Specifically, Theobroma bicolor is mentioned, and more specifically, Theobroma grandiflorum is mentioned. The cacao varieties are not particularly limited; for example, Forastero, Criollo, Trinitario, Nacional, CCN51, Chuao, Arriba, Porcelana, Pentagona, Guasare, Ocumare, Almidonado, Lagarto, Uranga, Matina, Sanchez, Puerto Fino, Puerto Mare, Canoapo, Javablonde, Ameronado, Angoleta, Arriba, CATIE R-1, CATIE R-4, CATIE R-6, IMC67, Scavina6, ICS1, ICS82, ICS95, Pa30, Pa169, Pa121, and Pa169 are all acceptable. Furthermore, the origin of the cacao is not particularly limited.

[0013] In this embodiment, cocoa extract refers to specific components extracted from various parts of the cocoa plant, and is not particularly limited. Cocoa extract can be obtained, for example, by the method described in <Method for preparing plant growth promoting composition> below. Cocoa powder refers to dried and pulverized parts of the cocoa plant or cocoa extract, and is not particularly limited.

[0014] The plant growth promoting composition in this embodiment is a composition having a plant growth promoting effect, where the plant growth promoting effect includes, for example, growth promoting effects such as elongation of stems and leaves or roots, increase in the number of leaves, promotion of flowering or fruiting, increase in the number of flowers or fruits, increase in plant weight or crop yield, greening, or promotion of tillering.

[0015] More specifically, a plant growth-promoting effect is observed when, for example, the increase in actual weight (%) or dry weight (%) increases when comparing the use of the plant growth-promoting composition of this embodiment with the lack of use. The actual weight or dry weight may be the actual weight or dry weight of the entire plant, or the actual weight or dry weight of each part of the plant (preferably the roots). When the plant growth-promoting composition is used, the increase in actual weight (%) or dry weight (%) should preferably increase by 10% or more, more preferably 20%, and even more preferably 30% or more compared to the case where the plant growth-promoting composition of this embodiment is not used. A plant growth-promoting effect is observed when, in cases where no growth is observed at all without the use of the plant growth-promoting composition, growth becomes observed with the use of the plant growth-promoting composition. Note that the above values ​​are average values ​​for the entire plant to which the plant growth-promoting composition was applied.

[0016] In addition, the plant growth-promoting effect also includes the following effects that improve plant activity: 1) Improved root elongation and root survival rate 2) Increased nutrient absorption efficiency 3) Induction of flower buds, promotion of flowering, and increase of fruit set. 4) Accumulation of components such as sugars 5) Increased water-saving effect 6) Inducing effect of callus formation 7) Improvement of plant size, particularly elongation or thickening of above-ground and underground parts. 8) Wound repair In other words, the plant growth-promoting composition of this embodiment may have growth-promoting effects such as elongation of stems and leaves or roots, increase in the number of leaves, promotion of flowering or fruiting, increase in the number of flowers or fruits, increase in plant weight or crop yield, greening, or promotion of tillering, in addition to the effects described in 1) to 8) above. The effect described in 1) above may promote nutrient absorption by the roots, thereby promoting the growth of the entire plant.

[0017] The plant growth promoting composition in this embodiment may confer tolerance to various environmental stresses in general that plants experience. The plant growth promoting composition in this embodiment may also have an effect of improving environmental stress tolerance. Here, examples of environmental stresses include biological stress and abiotic stress, and specifically include temperature stress such as herbivory stress, disease stress, high temperature stress and low temperature stress, nutritional stress, chemical stress, light stress, drought stress, pH stress such as oxidative stress, salt stress such as osmotic stress, hypoxia stress, and physical stress. Nutritional stress includes, for example, nutrient deficiency stress and nutrient excess stress.

[0018] In this embodiment, "environmental stress tolerance" means the ability to grow normally or in a near-normal state even under growth conditions that subject to environmental stress, without substantially suffering undesirable effects such as inability to grow (death), poor growth (e.g., whitening or yellowing of the plant body, reduction in root length or number of leaves), reduced growth rate, or reduction in plant weight or crop yield. By improving the environmental stress tolerance of plants with the plant growth promoting composition of this embodiment, plants can be grown normally even in external environments that would normally have undesirable effects on plant growth.

[0019] The temperature stress tolerance-conforming effect of the plant growth-promoting composition in this embodiment includes, for example, the effect of promoting normal growth under high-temperature and low-temperature conditions. High-temperature conditions may be appropriately determined by those skilled in the art based on common technical knowledge in this field, but conditions of 35°C or higher are preferred, for example, conditions of 35°C to 50°C. Low-temperature conditions may be appropriately determined by those skilled in the art based on common technical knowledge in this field, but conditions of 10°C or lower are preferred, for example, conditions of 0°C to 10°C. Furthermore, the salt stress-conforming effect enables normal growth in the presence of 0.1 to 3.0% by mass of sodium chloride. The drought stress-conforming effect enables normal growth even at relative humidity of 10 to 40%. The effect of the plant growth promoting composition in this embodiment on imparting nutritional stress tolerance includes, for example, promoting normal growth in plants that are deficient in chemical substances that serve as nutrients for plants (nutrient-deficient state). Here, a nutrient-deficient state may be, for example, a state in which the amount of nutrients is less than the concentrations shown in Table 1 of the Examples, and specifically, the amount of each nutrient may be about 1 / 2 to 1 / 3, 1 / 3 to 1 / 4, 1 / 4 to 1 / 5, 1 / 5 to 1 / 6, 1 / 6 to 1 / 7, or 1 / 7 to 1 / 8 of the amount shown in Table 1 of the concentrations for the test period. The concentrations for the test period in Table 1 are concentrations that are generally used in the cultivation of crops such as rice.

[0020] In this embodiment, the environmental stress tolerance of plants can be evaluated by, for example, the following means: The presence or absence of salt stress tolerance can be confirmed by growing the target plant in a culture medium or soil to which salt has been added at an appropriate concentration, under conditions of growth temperature and growth period suitable for the plant, and evaluating its phenotype. The presence or absence of drought stress tolerance can be confirmed by growing the target plant under conditions of growth temperature, growth humidity and growth period that are dry for the plant, and evaluating its phenotype. The presence or absence of high temperature stress tolerance can be confirmed by acclimatizing the target plant to a high temperature such as 35°C to 50°C, and then growing it under conditions of growth temperature and growth period suitable for the plant, and evaluating its phenotype. The presence or absence of low temperature stress tolerance can be confirmed by acclimatizing the target plant to a low temperature that does not cause freezing, and then growing it under conditions of growth temperature and growth period suitable for the plant, and evaluating its phenotype.

[0021] If the plant growth promoting composition in this embodiment has an effect of improving environmental stress tolerance, it may activate genes in the stress response system. The genes in the stress response system are not particularly limited, and include genes that are generally known as response indicators to environmental stresses such as temperature stress, nutrient stress, chemical stress, light stress, drought stress, pH stress, salt stress, hypoxia stress, herbivory stress, physical stress, and disease stress. Genes commonly known as indicators of response to environmental stress are those disclosed in publicly available databases such as the National Center for Biotechnology Information (NCBI), Ensembl Plants, The Arabidopsis Information Resource (TAIR), and The Rice Annotation Project (RAP). Specifically, genes commonly known as indicators of response to high temperature stress include Os01g0135900, Os01g0273500, Os01g0721000, Os01g0875700, Os01g0894600, Os02g0753800, Os03g0161900, Os03g0266300, Os03g0426900, Os03g0854500, Os05g0110100, Os05g0364500, and Os05g04600. Examples include 00, Os05g0502000, Os05g0529700, Os05g0530400, Os06g0682900, Os06g0716700, Os06g0727200, Os07g0632600, Os08g0127600, Os08g0191100, Os08g0500700, Os09g0133600, Os09g0456800, Os11g0216100, Os11g0661200, Os12g0244100, etc.For example, by applying the plant growth promoting composition of this embodiment to a plant, the following values ​​are obtained in the plant acclimatized to high temperatures: Os01g0135900, Os01g0273500, Os01g0721000, Os01g0875700, Os01g0894600, Os02g0753800, Os03g0161900, Os03g0266300, Os03g0426900, Os03g0854500, Os05g0110100, Os05g0364500, Os05g0460000, O The expression levels of genes such as s05g0502000, Os05g0529700, Os05g0530400, Os06g0682900, Os06g0716700, Os06g0727200, Os07g0632600, Os08g0127600, Os08g0191100, Os08g0500700, Os09g0133600, Os09g0456800, Os11g0216100, Os11g0661200, and Os12g0244100 are elevated compared to the control group.

[0022] In addition, genes commonly known as indicators of response to drought stress include Os01g0615050, Os01g0864500, Os01g0867300, Os01g0955100, Os01g0975300, Os02g0115700, Os02g0126400, Os02g0178800, Os02g0543000, Os02g0615800, Os02g0673500, Os02g0682300, and Os02 g0731700, Os02g0753800, Os02g0766700, Os03g0140400, Os03g0177500, Os03g0225100, Os03g0285800, Os03g032150 0, Os03g0594400, Os03g0741100, Os03g0764100, Os03g0782500, Os04g0398000, Os04g0564600, Os04g0584600, Os05g 0127200, Os05g0247100, Os05g0382900, Os05g0542500, Os06g0192800, Os06g0316000, Os06g0726300, Os07g0150700 , Os07g0680400, Os07g0684000, Os08g0157600, Os08g0492500, Os08g0512400, Os09g0135700, Os09g0271100, Os09g0 Examples include 322000, Os09g0421700, Os09g0487900, Os09g0522200, Os09g0565200, Os10g0417800, Os10g0552400, Os10g0562900, Os11g0126900, Os11g0167800, Os11g0704500, Os12g0168100, Os12g0218300, Os12g0221600, Os12g0555000, etc.For example, by applying the plant growth promoting composition of this embodiment to a plant, the following values ​​were obtained in the plant grown in a dry state: Os01g0615050, Os01g0864500, Os01g0867300, Os01g0955100, Os01g0975300, Os02g0115700, Os02g0126400, Os02g0178800, Os02g0543000, Os02g0615800, Os02g0673500, Os02g0682 300, Os02g0731700, Os02g0753800, Os02g0766700, Os03g0140400, Os03g0177500, Os03g0225100, Os03g0285800, Os03g03 21500, Os03g0594400, Os03g0741100, Os03g0764100, Os03g0782500, Os04g0398000, Os04g0564600, Os04g0584600, Os05g0 127200, Os05g0247100, Os05g0382900, Os05g0542500, Os06g0192800, Os06g0316000, Os06g0726300, Os07g0150700, Os07 g0680400, Os07g0684000, Os08g0157600, Os08g0492500, Os08g0512400, Os09g0135700, Os09g0271100, Os09g0322000, Os The expression levels of genes such as 09g0421700, Os09g0487900, Os09g0522200, Os09g0565200, Os10g0417800, Os10g0552400, Os10g0562900, Os11g0126900, Os11g0167800, Os11g0704500, Os12g0168100, Os12g0218300, Os12g0221600, and Os12g0555000 are elevated compared to the control group.

[0023] In addition, genes commonly known as indicators of response to cold stress include Os01g0510100, Os01g0559600, Os01g0769900, Os02g0121100, Os02g0624300, Os03g0285800, Os03g0319400, Os03g0656900, Os03g0678800, Os04g0517100, Os04g0530900, and Os04g058460. Examples include 0, Os05g0138300, Os05g0476700, Os05g0497300, Os05g0559900, Os06g0726300, Os07g0635900, Os08g0129200, Os09g0271100, Os09g0334500, Os09g0369000, Os09g0522200, Os09g0565200, Os10g0562900, Os12g0210300, etc. For example, by applying the plant growth promoting composition of this embodiment to a plant, the following values ​​are obtained in the plant acclimatized to low temperatures: Os01g0510100, Os01g0559600, Os01g0769900, Os02g0121100, Os02g0624300, Os03g0285800, Os03g0319400, Os03g0656900, Os03g0678800, Os04g0517100, Os04g0530900, Os04g0584600, O The expression levels of genes such as s05g0138300, Os05g0476700, Os05g0497300, Os05g0559900, Os06g0726300, Os07g0635900, Os08g0129200, Os09g0271100, Os09g0334500, Os09g0369000, Os09g0522200, Os09g0565200, Os10g0562900, and Os12g0210300 are elevated compared to the control group.

[0024] In addition, genes commonly known as indicators of response to salt stress include Os01g0141000, Os01g0200700, Os01g0575200, Os01g0615100, Os01g0624500, Os01g0864500, Os01g0955100, Os01g0975300, Os02g0126400, Os02g0132300, O s02g0327000, Os02g0543000, Os02g0594700, Os02g0666200, Os03g0285700, Os03g0285800, Os03g0 364400, Os03g0594400, Os03g0678800, Os04g0304200, Os04g0401700, Os04g0473400, Os04g0493000 , Os04g0511200, Os04g0530900, Os04g0584600, Os04g0620700, Os05g0127200, Os05g0138300, Os05 g0348100, Os05g0542500, Os06g0111500, Os06g0171900, Os06g0683400, Os07g0575700, Os07g0684 Examples include 000, Os07g0694000, Os08g0157600, Os08g0492500, Os08g0512400, Os09g0322000, Os09g0344500, Os09g0522200, Os10g0370500, Os10g0431000, Os12g0512100, Os12g0518200, Os12g0555000, etc.For example, by applying the plant growth promoting composition of this embodiment to a plant, the following values ​​were obtained in the plant grown in a culture medium or soil with salt added at an appropriate concentration: Os01g0141000, Os01g0200700, Os01g0575200, Os01g0615100, Os01g0624500, Os01g0864500, Os01g0955100, Os01g0975300, Os02g012 6400, Os02g0132300, Os02g0327000, Os02g0543000, Os02g0594700, Os02g0666200, Os03g0285700, Os03g0 285800, Os03g0364400, Os03g0594400, Os03g0678800, Os04g0304200, Os04g0401700, Os04g0473400, Os04g 0493000, Os04g0511200, Os04g0530900, Os04g0584600, Os04g0620700, Os05g0127200, Os05g0138300, Os0 5g0348100, Os05g0542500, Os06g0111500, Os06g0171900, Os06g0683400, Os07g0575700, Os07g0684000, Os The expression levels of genes such as 07g0694000, Os08g0157600, Os08g0492500, Os08g0512400, Os09g0322000, Os09g0344500, Os09g0522200, Os10g0370500, Os10g0431000, Os12g0512100, Os12g0518200, and Os12g0555000 are elevated compared to the control group.

[0025] Furthermore, among salt stresses, genes generally known as response indicators to osmotic stress include Os02g0666200, Os06g0683400, Os08g0157600, Os09g0322000, and Os09g0369000. For example, by applying the plant growth promoting composition of this embodiment to plants, the expression levels of genes such as Os02g0666200, Os06g0683400, Os08g0157600, Os09g0322000, and Os09g0369000 in plants grown under an osmotic stress environment increase compared to the control.

[0026] Furthermore, genes commonly known as indicators of response to light stress include Os01g0510100, Os04g0619400, Os05g0592400, and Os11g0117400. For example, by applying the plant growth promoting composition of this embodiment to plants, the expression levels of genes such as Os01g0510100, Os04g0619400, Os05g0592400, and Os11g0117400 in plants grown under strong light increase compared to the control. Genes commonly known as indicators of response to light stress are also commonly known as indicators of response to strong light stress.

[0027] The plant growth promoting composition in this embodiment may contain one or more selected from the group consisting of cocoa extract and cocoa powder, either alone or in combination with one or more agriculturally acceptable components. Agriculturally acceptable components are not particularly limited, but examples include solvents, carriers, excipients, binders, solubilizers, stabilizers, thickeners, leavening agents, lubricants, surfactants, oily liquids, buffers, bactericides, antifreezes, defoamers, colorants, antioxidants, and further active ingredients. Preferred agriculturally acceptable carriers are agriculturally acceptable liquid carriers such as water, mineral oil fractions like kerosene or diesel oil, oils of plant or animal origin, cyclic or aromatic hydrocarbons (e.g., paraffin, tetrahydronaphthalene, alkylated naphthalenes or their derivatives, or alkylated benzenes or their derivatives), alcohols (e.g., methanol, ethanol, propanol, butanol or cyclohexanol), ketones (e.g., cyclohexanone), or amines (e.g., N-methylpyrrolidone), or mixtures thereof.

[0028] If the plant growth promoting composition in this embodiment contains one or more additional active ingredients, various compounds known in the art that have environmental stress tolerance-improving activity can be applied as the additional active ingredients. The inclusion of such one or more additional active ingredients in the plant growth promoting composition tends to further improve the environmental stress tolerance of plants.

[0029] The plant growth promoting composition in this embodiment can be used in combination with conventionally known agricultural materials such as pesticides and fertilizers. Examples of agricultural materials include pesticides, fertilizers, soil conditioners, and growing media (potting soil). Specifically, pesticides may include fungicides, insecticides, insecticide-fungicides, acaricides, nematicides, rodenticides, insecticides / repellents, attractants, spreading agents, hormones (growth regulators), herbicides, etc. Fertilizers may include organic fertilizers, chemical fertilizers, liquid fertilizers, solid fertilizers, compound fertilizers, compost, growth promoters, vitality boosters, calcium supplements, etc. Soil conditioners may be intended for physical, chemical, or biological soil improvement, and may also include oxygen supply agents, etc. Furthermore, pesticides, fertilizers, soil conditioners, and growing media may contain components derived from plants, animals, lime, minerals, or microorganisms.

[0030] The content of one or more substances selected from the group consisting of cocoa extract and cocoa powder in the plant growth promoting composition may be any value as long as one or more substances selected from the group consisting of cocoa extract and cocoa powder function as an active ingredient for promoting plant growth. For example, it may be 5, 10, 20, 30, 40, 50, 60, 70, 80, or 90% by mass (volume) or more, and 99, 95, 90, 80, 70, 60, 50, 40, 30, 20, or 10% by mass (volume) or less.

[0031] The plant growth promoting composition in this embodiment can be used as an agricultural chemical agent or pesticide to promote plant growth. The plant growth promoting composition may be in any form, such as a solid (e.g., powder or granules) or a liquid (e.g., solution or suspension), and is preferably used in the form of a liquid such as a solution or suspension. When used as a solution, it may be in liquid form or prepared as a liquid at the time of application.

[0032] The dosage form of the plant growth promoting composition of this embodiment is not particularly limited and can be formulated in dosage forms commonly used in the art, such as emulsions, wettable powders, liquids, aqueous solutions, powders, pastes, or granules.

[0033] In this embodiment, the plants to which the plant growth promoting composition can be applied are not particularly limited, and examples include angiosperms and gymnosperms. Examples of target plants include Asteraceae plants such as chrysanthemums and gerberas, Solanaceae plants such as potatoes, tomatoes and eggplants, Brassicaceae plants such as rapeseed and rapeseed, Poaceae plants such as rice, maize, wheat, sugarcane and barley, Fabaceae plants such as soybeans, Apiaceae plants such as carrots, Lamiaceae plants such as basil, mint and rosemary, Convolvulaceae plants such as morning glories, Salicaceae plants such as poplars, and Euphorbiaceae plants such as castor bean, cassava and jatropha. Examples include plants of the Convolvulaceae family such as sweet potatoes, plants of the Rutaceae family such as oranges and lemons, plants of the Rosaceae family such as cherry blossoms and roses, plants of the Orchidaceae family such as phalaenopsis orchids, plants of the Gentianaceae family such as lisianthus, plants of the Primulaceae family such as cyclamen, plants of the Violaceae family such as pansies, plants of the Liliaceae family such as lilies, plants of the Amaranthaceae family such as sugar beets, plants of the Vitaceae family such as grapes, plants of the Cupressaceae family such as cedar and cypress, plants of the Oleaceae family such as olives and osmanthus, and plants of the Pinaceae family such as Japanese red pine.

[0034] The target plants may include not only the whole plant (i.e., a complete plant body), but also plant tissues or organs (for example, cut flowers, or vegetative reproductive organs such as rhizomes, tubers, corms, or runners), cultured cells, and / or parts of the plant such as callus.

[0035] The plant growth promoting composition in this embodiment can be applied to the whole or a part of a plant at any growth stage, including before or after germination (for example, a seed, seedling, or the whole or a part of a mature plant).

[0036] <Method for preparing a plant growth promoting composition> As a method for preparing a plant growth promoting composition, for example, one or more components selected from the group consisting of cocoa extract and cocoa powder can be obtained, and then mixed with the above-mentioned agriculturally acceptable components as needed. The method for obtaining cocoa extract is not particularly limited, and general methods for extracting plant extracts can be used. Specifically, for example, it can be obtained by a method that includes the steps of: obtaining a raw material for cocoa extract by heating or crushing one or more selected from the group consisting of cocoa leaves, stems, skins, fruits, seed coats, and roots, and then removing impurities; concentrating the raw material for cocoa extract obtained in the first step to obtain a cocoa concentrate; and fractionating the cocoa concentrate obtained in the first step to obtain a cocoa extract containing specific components. Alternatively, cocoa extract can also be obtained using one or more selected from the group consisting of cocoa leaves, stems, skins, fruits, seed coats, and roots that have gone through each step in obtaining cocoa extract as a raw material. For example, a raw material for cocoa extract or a cocoa concentrate can be obtained from cocoa residue such as impurities removed in the step of obtaining the raw material for cocoa extract using the same method as described above, and then cocoa extract can be obtained from that raw material for cocoa extract or cocoa concentrate using the same method as described above. Alternatively, cocoa extract may be obtained using cocoa residue, such as impurities, obtained during the manufacturing process of any cocoa processed product. Cocoa extract raw materials or cocoa concentrate may be used as cocoa extract as is. Cocoa extract raw materials, cocoa concentrate, or cocoa extract may be heated or pressurized at or after each step. When heating, various conditions such as heating temperature and heating time can be appropriately determined depending on the variety, part, and condition of the cocoa used. The method for obtaining cocoa powder is not particularly limited. It can be obtained by drying cocoa extract raw materials, cocoa concentrate, or cocoa residue such as impurities generated when obtaining cocoa extract, or by drying fresh or frozen cocoa (one or more selected from the group consisting of cocoa leaves, stems, skins, fruits, seed coats, and roots) using methods such as hot air drying, air drying, dry heat drying, or reduced pressure drying, and then crushing it into a powder.

[0037] (Crushing process) Cocoa can be crushed using fresh or dried cocoa, for example, by a mixer. The crushed cocoa is filtered using a filter cloth, filter paper, or the like to remove impurities and obtain a cocoa extract raw material. When filtering, an extraction solvent such as an organic solvent may be added beforehand. Alternatively, impurities may be removed by obtaining the supernatant using a centrifuge instead of filtration.

[0038] (concentration process) The obtained cocoa extract raw material can be concentrated to obtain a cocoa concentrate using methods such as vacuum concentration, freeze concentration, or membrane concentration. The concentration ratio is preferably 3 to 30 times, and more preferably 4 to 20 times. When the concentration ratio is 5 times or more, the amount of plant growth promoting composition added to the plant is reduced, which improves handling and tends to reduce transportation costs. When the concentration ratio is 30 times or less, there is less precipitate in the plant growth promoting composition, which tends to make it easier to handle. Furthermore, in the preparation of the plant growth promoting composition, concentration of the cocoa extract raw material is not essential, and the obtained cocoa extract raw material may be subjected directly to the following fractionation steps.

[0039] (Fractional process) The obtained cocoa concentrate can be fractionated by solvent extraction or other methods to obtain cocoa extract containing specific components. In the fractionation process, for example, hydrophilic and hydrophobic fractions can be obtained by liquid-liquid partitioning. Further purification can be obtained by passing the fraction through a column.

[0040] The cocoa extract obtained in the fractionation process may be further purified or separated into highly active fractions as needed. Purification methods include, for example, filtration and adsorption (ion exchange resin column, activated carbon column, etc.). Separation methods for highly active fractions include gel filtration, adsorption, silica gel column chromatography, and HPLC.

[0041] <Plant growth promotion method> The plant growth promotion method in this embodiment is a method of promoting plant growth by using a composition containing one or more selected from the group consisting of cocoa extract and cocoa powder as an active ingredient on a plant, or on the soil or culture medium in which the plant grows. In this method, the plant growth promotion composition can be applied to a plant or a part thereof (e.g., a seed, seedling, or mature plant) at any growth stage, including before or after germination. Furthermore, it can be applied not only to the plant itself, but also to the soil, culture medium, or culture medium in which the plant grows. By applying the plant growth promotion composition to a plant in a growth stage or to the soil, culture medium, or culture medium in which the plant grows, plant growth can be promoted.

[0042] The plant growth promoting composition may be applied to plants once or multiple times. Similarly, if there are multiple application periods, the number of applications at each period may also be once or multiple times.

[0043] Methods for applying the plant growth-promoting composition to plants include, for example, addition, spraying, coating, and immersion. These methods may be used individually or in combination of two or more. The application of the plant growth-promoting composition to plants may be done manually or using machinery. When using machinery, there are no particular limitations, but for example, application (spraying) may be done using a drone.

[0044] The amount of plant growth promoting composition to be applied to plants is preferably 0.001 to 50% by mass, or 0.001 to 50% by volume, more preferably 0.01 to 30% by mass, or 0.01 to 30% by volume, and even more preferably 0.01 to 10% by mass, or 0.01 to 10% by volume, when applied to the culture solution in which the plants are growing, or when diluted with a diluent and applied to the field in which the plants are growing. While sufficient plant growth promoting effects tend to be obtained when the amount of plant growth promoting composition is within the above range, the amount used may be appropriately adjusted depending on the content of one or more selected from the group consisting of cocoa extract and cocoa powder in the plant growth promoting composition, the type and growth stage of the plant to which the plant growth promoting composition is applied, and the plant growth environment such as temperature, humidity, and light. The amount used may also be determined by investigating the concentration range in which the effect is obtained using known cultivation test methods. When applying a plant growth-promoting composition diluted with a diluting solvent to plants using a drone, the amount of solution that can be carried on the drone is limited by the drone's payload capacity, etc. Therefore, the amount of plant growth-promoting composition used relative to the diluting solvent is preferably 1 to 50% by mass or 1 to 50% by volume.

[0045] The components of the culture solution and diluent described above are not particularly limited as long as they do not adversely affect plant growth, and may include water, pesticides, etc.

[0046] The above usage amounts will vary depending on the concentration ratio of the cocoa extract raw material mentioned above, but the above usage amounts represent the preferred range when the concentration ratio is between 3 and 30 times. [Examples]

[0047] The present invention will be described in more detail using examples and comparative examples, but the present invention is not limited in any way by these examples.

[0048] (Method for measuring root weight) The roots were identified based on the color and shape of the rice plants, and their weight was measured in a bottle using a microbalance.

[0049] [Example 1] Cacao extract was obtained by crushing 20.0g of cacao seed husks, adding ultrapure water, extracting the raw material for cacao extract, and then concentrating the raw material for cacao extract. Rice plants were grown in the seedling stage culture solution shown in Table 1. Subsequently, the rice plants were transferred to 900 mL of the test stage culture solution shown in Table 1, and 3.6 mL (0.4 vol%) of the obtained cocoa extract was added to investigate the effect on the rice plants. As a result, the weight of the rice roots increased by 19.9% ​​compared to the case without cocoa extract, indicating an improvement in rice root development (Figures 1 and 2). Furthermore, since the concentration of nutrients during the seedling stage was about one-sixth to one-seventh of the concentration during the test stage, which is sufficient for rice growth, the rice to which cocoa extract was added was in a state of nutrient deficiency.

[0050] [Table 1]

[0051] [Example 2] We also evaluated the fluctuations in gene expression in rice.

[0052] (A) Sample preparation • Preparation of rice plants Rice seedlings, grown in the same manner as in Example 1, were transferred to 900 mL of the culture solution for the test period shown in Table 1. 3.6 mL (0.4 vol%) of the cocoa extract used in Example 3 was added to the 900 mL of culture solution, and the rice was cultivated. • RNA extraction RNA samples were obtained using the Maxwell RSC Plant RNA Kit (Promega) and Maxwell RSC Instrument (Promega) according to the instructions. • RNA quality check Measurement equipment: Agilent 5400 Bioanalyzer <5312AA> (Agilent Technologies), Reagent kit: Agilent RNA kit (15nt) <dnf-471>After confirming that the RNA samples were of sufficient quality using Agilent Technologies and ProSize analysis software, they were used for library preparation. • Library preparation A library for the next-generation sequencer DNBSEQ T7 was prepared using the NEBNext Ultra II RNA Library Prep Kit for Illumina, following the steps outlined below. 1. Purification of mRNA using oligo(dT) magnetic beads 2 Fragmentation 3. Synthesis of single-stranded and double-stranded cDNA using random primers. 4. End repair, polyadenylation, adapter bonding, size selection, and amplification are performed.

[0053] (B) Analysis using next-generation sequencers • Sequence analysis Equipment: DNBSEQ T7 (MGI) • Sequence (outline) Sequencing was performed using DNBSEQ T7 according to the following procedure. 1. Addition of sequencing reagent 2. Single-base extension reaction 3. Removal of unreacted bases 4. Uptake of fluorescent signals 5. Removal of protecting groups and fluorescence 6. Repeat the cycle (2 cycles, 3 cycles, etc.) until 300 cycles are completed.

[0054] (C) Data Analysis • Primary data analysis Operating System of the analysis computer: Ubuntu Desktop 22.04.4 LTS Analysis software: FastQC, hisat2, samtools, HTSeq, DESeq2 The analysis was performed using the software described above. 1. Checking the quality of the reeds We used FastQC to verify the quality of the bases contained in the obtained fastq file. 2. Mapping and Sorting The data was mapped to the Oryza sativa reference genome, Oryza sativa Japonica Group (assembly IRGSP-1.0), using hisat2. Subsequently, the files were sorted and converted to binary data using samtools. 3 Lead Count The number of mapped reads was counted using the htseq-count within the HTSeq. 4 Statistical analysis Using the DESeq2 package on the statistical package R, we compared gene expression levels in the control group and the group treated with cocoa extract. • Third-level data analysis We searched the Rice Annotation Project (https: / / rapdb.dna.affrc.go.jp / ) for genes that have been reported in previous studies to be involved in tolerance to various environmental stresses. We then searched for the genes found in the previous study using the gene expression list created in this experiment. • Selection of target genes for analysis and visualization of gene expression levels We collected various environmental stress tolerance genes from known databases. Subsequently, we extracted various stress tolerance genes from the list of genes with increased expression levels, and represented their expression levels as heatmaps and bar graphs. In the heatmaps of Figures 3, 5, 7, 9, 11, and 13, CTS represents the control group, and CCh-bS represents the group treated with cocoa extract.

[0055] Gene expression analysis revealed that the expression levels of several high-temperature stress tolerance genes tended to be higher in the above-ground parts of the cocoa extract-added groups compared to the control group (Figure 3). In particular, the expression level of the high-temperature stress tolerance gene Os03g0854500 was increased in the above-ground parts (Figure 4). The following has been reported about these molecules related to high-temperature stress tolerance, suggesting that they contribute to high-temperature stress tolerance. Molecules homologous to Os03g0854500 are induced by high-temperature stress and are suggested to play an important role in the survival of plants under high-temperature conditions (Reference 1). This gene and similar genes have been reported to be widely present not only in Arabidopsis thaliana and rice, but also in sunflowers, lettuce, bread wheat, etc. (Reference 2).

[0056] Gene expression analysis revealed that the expression levels of several drought stress tolerance genes tended to be higher in the above-ground parts of the cocoa extract-added groups compared to the control group (Figure 5). In particular, the expression level of the drought stress tolerance gene Os02g0115700 was increased in the above-ground parts (Figure 6). The following has been reported about these molecules related to drought stress tolerance, suggesting that they contribute to drought stress tolerance. It has been reported that molecules homologous to Os02g0115700 are induced to express by drought stress, and the activity of the protein translated from this gene increases (Reference 3). This gene and similar genes have been reported to be widely present not only in Arabidopsis thaliana and rice, but also in sunflower, cassava, bread wheat, maize, etc. (Reference 4).

[0057] Gene expression analysis revealed that the expression levels of several cold stress tolerance genes tended to be higher in the above-ground parts of the cocoa extract-added groups compared to the control group (Figure 7). In particular, the expression level of the cold stress tolerance gene Os03g0285800 was increased in the above-ground parts (Figure 8). The following has been reported about these molecules related to cold stress tolerance, suggesting that they contribute to cold stress tolerance. The MAP kinase cascade has been suggested to contribute to cold stress tolerance (Reference 5). This gene and similar genes have been reported to be widely present not only in Arabidopsis thaliana and rice, but also in soybeans, cottonweed, bread wheat, etc. (Reference 6).

[0058] Gene expression analysis revealed that the expression levels of several salt stress tolerance genes tended to be higher in the above-ground parts of the cocoa extract-added groups compared to the control group (Figure 9). In particular, the expression level of the salt stress tolerance gene Os02g0543000 was increased in the above-ground parts (Figure 10). The following has been reported about these molecules related to salt stress tolerance, suggesting that they contribute to salt stress tolerance. It has been reported that overexpression of homologous genes of Os02g0543000 increases salt stress tolerance (Reference 7). This gene and similar genes have been reported to be widely present not only in Arabidopsis thaliana and rice, but also in soybeans, sunflowers, lettuce, etc. (Reference 8).

[0059] Gene expression analysis revealed that the expression levels of several osmotic stress tolerance genes tended to be higher in the above-ground parts of the cocoa extract-added group compared to the control group (Figure 11). In particular, the expression level of the stress tolerance gene Os02g0666200 was increased in the above-ground parts (Figure 12). Regarding molecules related to osmotic stress tolerance, the following has been reported, suggesting their contribution to osmotic stress tolerance: A homologous molecule of Os02g0666200 promotes water uptake (Reference 9). This gene and similar genes have been reported to be widely present in plants other than Arabidopsis thaliana and rice, such as rapeseed, bread wheat, and maize (Reference 10).

[0060] Gene expression analysis revealed that the expression levels of several light stress tolerance genes tended to be higher in the above-ground parts of the cocoa extract-added groups compared to the control group (Figure 13). In particular, the expression level of the light stress tolerance gene Os05g0592400 was increased in the above-ground parts (Figure 14). The following has been reported about these molecules related to light stress tolerance, and therefore they are thought to contribute to light stress tolerance. Homologous genes of Os05g0592400 have been reported to be involved in the removal of UV-damaged DNA (Reference 11). This gene and similar genes have been reported to be widely present in plants other than Arabidopsis thaliana and rice, such as chili peppers, tobacco, and bread wheat (Reference 12).

[0061] From the above, it is thought that cocoa extract has the effect of improving the tolerance of one or more stresses selected from the group consisting of high temperature stress, drought stress, low temperature stress, salt stress, salt stress, osmotic stress, and osmotic stress and high light stress in many plants other than rice by stimulating high temperature stress tolerance genes, drought stress tolerance genes, low temperature stress tolerance genes, salt stress tolerance genes, osmotic stress, and high light stress, or molecules similar to these.

[0062] Reference 1: https: / / pubmed.ncbi.nlm.nih.gov / 39013704 / Reference 2: https: / / www.arabidopsis.org / locus?key=29645 Reference 3: https: / / onlinelibrary.wiley.com / doi / 10.1111 / jac.12533 Reference 4: https: / / www.arabidopsis.org / locus?key=30587 Reference 5: https: / / pubmed.ncbi.nlm.nih.gov / 29056551 / Reference 6: https: / / www.arabidopsis.org / locus?key=128489 Reference 7: https: / / onlinelibrary.wiley.com / doi / 10.1111 / j.1365-3040.2004.01251.x Reference 8: https: / / www.arabidopsis.org / locus?key=131439 Reference 9: https: / / pubmed.ncbi.nlm.nih.gov / 9681029 / Reference 10: https: / / www.arabidopsis.org / locus?key=37081 Reference 11: https: / / www.pnas.org / doi / 10.1073 / pnas.1110067109 Reference 12: https: / / www.arabidopsis.org / locus?key=128357

Claims

1. A plant growth promoting composition containing one or more substances selected from the group consisting of cocoa extract and cocoa powder as an active ingredient.

2. The plant growth promoting composition according to claim 1, having one or more plant growth promoting effects selected from the group consisting of elongation of stems and leaves or roots, increase in the number of leaves, promotion of flowering or fruiting, increase in the number of flowers or fruits, increase in plant weight or crop yield, greening, and promotion of tillering.

3. A plant growth promoting composition according to claim 1 or 2, which has an effect of improving environmental stress tolerance.

4. The plant growth promoting composition according to claim 3, which has an effect of improving tolerance to one or more environmental stresses selected from the group consisting of temperature stress, nutrient stress, chemical stress, light stress, drought stress, pH stress, salt stress, hypoxia stress, herbivore stress, physical stress, and disease stress.

5. The plant growth promoting composition according to claim 3, which activates genes in the stress response system.

6. A method for promoting plant growth, comprising using a plant growth promoting composition containing one or more selected from the group consisting of cocoa extract and cocoa powder as an active ingredient, on a plant, or in the soil or culture medium on which the plant grows.

7. The plant growth promoting method according to claim 6, wherein the amount of the plant growth promoting composition used in the culture medium or diluent is 0.001 to 50% by mass or 0.001 to 50% by volume.

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