Plant growth promoting components

Abstract

To provide a plant growth-promoting composition capable of promoting plant growth more safely and inexpensively than conventional materials. [Solution] A plant growth-promoting composition containing one or more active ingredients selected from the group consisting of cocoa extract and cocoa powder.

Description

[Technical Field]

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

[0002] With the current explosive growth in the human population, producing a sufficient supply of plants for food has become a global challenge. Furthermore, desertification due to the reduction in green space has become a problem, and rising earth temperatures due to global warming have also become a problem, resulting in an increasing number of environmental problems affecting plant growth. In other words, excessive stress on plants caused by global environmental factors often causes problems for plant growth.

[0003] Therefore, there is a demand for technologies that promise more efficient harvesting than conventional technologies, and biostimulants are attracting attention as a new technology that reduces damage to plants 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 reports that artificially promoting the opening of plant stomatal pores with stevia improves heat stress resistance. Patent Document 2 discloses a composition for activating genes that activate the ethylene and / or jasmonic acid signaling system, which contains a yeast cell wall decomposition product, and reports that applying the yeast cell wall decomposition product to plants activates the expression of plant defensin genes that are involved in the antibacterial properties and disease prevention of the plant body. Patent Document 3 discloses a method for increasing the yield of grains by inoculating a cell wall-containing substance of a methanol-utilizing bacterium into a plant for cultivating grains. [Prior art documents] [Patent documents]

[0005] [Patent Document 1] Patent No. 6741262 [Patent Document 2] Patent No. 4931388 [Patent Document 3] Patent Publication No. 2019-180361 Summary of the Invention [Problem to be solved by the invention]

[0006] As mentioned above, research has been conducted on improving environmental stress tolerance and increasing yields in plants using various materials, but no examples have been reported using cocoa extract or cocoa powder. [Means for solving the problem]

[0007] As a result of extensive research, the inventors have discovered that, by using a highly accurate screening method that they independently developed, one or more substances selected from the group consisting of cocoa extract and cocoa powder, among various other substances, can be used as an active ingredient for promoting plant growth, thereby completing the present invention.

[0008] That is, the present invention is as follows. [1] A plant growth-promoting composition comprising, as an active ingredient, one or more members selected from the group consisting of cocoa extract and cocoa powder. [2] The plant growth-promoting composition described in [1] above has one or more plant growth-promoting effects selected from the group consisting of elongation of stems, leaves or roots, increased leaf number, promotion of flowering or fruiting, increased number of flowers or fruits, increased plant weight or crop yield, greening, and promotion of tillering. [3] The plant growth-promoting composition according to [1] or [2] above, which has the effect of improving environmental stress tolerance. [4] The plant growth-promoting composition described in [3] above has the effect of improving environmental stress tolerance against one or more stresses selected from the group consisting of temperature stress, nutritional stress, chemical stress, light stress, drought stress, pH stress, salt stress, hypoxic stress, herbicide stress, physical stress, and disease stress. [5] The plant growth-promoting composition according to [3] above, which activates genes in the stress response system. [6] A method for promoting plant growth, comprising applying a plant growth-promoting composition containing one or more active ingredients selected from the group consisting of cocoa extract and cocoa powder to a plant or to soil or culture solution in which the plant grows. [7] The plant growth-promoting method according to [6] above, wherein the amount of the plant growth-promoting composition used is 0.001 to 50% by mass or 0.001 to 50% by volume relative to the culture solution or dilution solvent. [Effects of the Invention]

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

[0010] [Figure 1] These images show the improvement in root growth 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] 1 is a graph showing the weight of rice plants when no cocoa extract or cocoa powder was added and when 3.6 mL (Example 1) of a plant growth-promoting composition containing a cocoa extract was added. [Figure 3] 1 is an image showing a heat map of the expression levels of high temperature stress tolerance genes in rice 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] 10 is an image showing the expression level of the rice high temperature stress tolerance gene Os03g0854500 when no cocoa extract or cocoa powder was added and when 3.6 mL of a plant growth-promoting composition containing cocoa extract was added. [Figure 5] 1 is an image showing a heat map of expression levels of drought stress tolerance genes in rice 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 6] 10 is an image showing the expression level of the rice drought stress tolerance gene Os02g0115700 when no cocoa extract or cocoa powder was added and when 3.6 mL of a plant growth-promoting composition containing cocoa extract was added. [Figure 7] 1 is an image showing a heat map of expression levels of cold stress tolerance genes in rice 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 8] 10 is an image showing the expression level of the rice cold stress tolerance gene Os03g0285800 when no cocoa extract or cocoa powder was added and when 3.6 mL of a plant growth-promoting composition containing cocoa extract was added. [Figure 9] 1 is an image showing a heat map of expression levels of salt stress tolerance genes in rice 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 10] 10 is an image showing the expression level of the rice salt stress tolerance gene Os02g0543000 when no cocoa extract or cocoa powder was added and when 3.6 mL of a plant growth-promoting composition containing cocoa extract was added. [Figure 11] 1 is an image showing a heat map of the expression levels of osmotic stress tolerance genes in rice 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 12]10 is an image showing the expression level of the rice osmotic stress tolerance gene Os02g0666200 when no cocoa extract or cocoa powder was added and when 3.6 mL of a plant growth-promoting composition containing cocoa extract was added. [Figure 13] 1 is an image showing a heat map of the expression levels of genes resistant to high light stress in rice when no cocoa extract or cocoa powder was added and when 3.6 mL of a plant growth-promoting composition containing cocoa extract was added. [Figure 14] 10 is an image showing the expression level of the rice high light stress tolerance gene Os05g0592400 when no cocoa extract or cocoa powder was added and when 3.6 mL of a plant growth-promoting composition containing cocoa extract was added. DETAILED DESCRIPTION OF THE INVENTION

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

[0012] <Plant growth promoting composition> The plant growth-promoting composition of this embodiment contains one or more active ingredients selected from the group consisting of cocoa extract and cocoa powder. The plant growth-promoting composition of this embodiment may contain only either cocoa extract or cocoa powder, or may contain both cocoa extract and cocoa powder. Cocoa (Theobroma cacao) is a plant of the genus Theobroma in the family Malvaceae. Specific examples include Theobroma bicolor, and more specific examples include Theobroma grandiflorum. The cacao variety is not particularly limited, and examples thereof include Forastero, Criollo, Trinitario, Nacional, CCN51, Chuao, Arriba, Porcellana, Pentagona, Guasare, Ocmare, Almidonado, Lagarto, Uranga, Matina, Sanchez, Puerto Fino, Puerto Mare, Canoapo, Java Blonde, Ameronado, Angoleta, Arriva, CATIE R-1, CATIE R-4, CATIE R-6, IMC67, Scavina6, ICS1, ICS82, ICS95, Pa30, Pa169, Pa121, and Pa169. The cacao production area is also not particularly limited.

[0013] In this embodiment, the term "cacao extract" refers to an extract of specific components from various parts of the cacao plant, and is not particularly limited. The cacao extract can be obtained, for example, by the method described in "Method for preparing a plant growth-promoting composition" below. The term "cacao powder" refers to a dried and pulverized cacao part or cacao extract, and is not particularly limited.

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

[0015] More specifically, a case where a plant growth-promoting effect is observed refers to, for example, an increase in actual weight gain (%) or dry weight gain (%) when comparing the use of the plant growth-promoting composition of the present embodiment with the use of the composition without the use of the composition. 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 actual weight gain (%) or dry weight gain (%) is preferably increased 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 the present embodiment is not used. A plant growth-promoting effect includes a case where no growth is observed without the use of the plant growth-promoting composition, but growth is observed after 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 is applied.

[0016] In addition, the plant growth promoting effect also includes the effect of improving the activity of plants as follows. 1) Root elongation and improved root establishment 2) Increased efficiency of nutrient absorption 3) Induction of flower buds, promotion of flowering, and increase in fruit production 4) Accumulation of sugar and other substances 5) Increased water-saving effect 6) Callus induction effect 7) Increase in plant size, especially elongation or thickening of above-ground and underground parts 8) Wound repair That is, the plant growth-promoting composition of the present embodiment may have the above-mentioned actions 1) to 8) in addition to growth-promoting effects such as elongation of stems, leaves, or roots, increased leaf number, promotion of flowering or fruiting, increased number of flowers or fruits, increased plant weight or crop yield, greening, or promotion of tillering. By exerting the above-mentioned action 1), nutrient absorption by roots may be promoted, and the growth of the entire plant may be promoted.

[0017] The plant growth-promoting composition of this embodiment may confer tolerance to a variety of environmental stresses to which a plant is exposed. The plant growth-promoting composition of this embodiment may have the effect of improving environmental stress tolerance. Here, examples of environmental stress include biotic stress and abiotic stress, and specific examples include herbicide stress, disease stress, temperature stress such as 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, hypoxic stress, and physical stress. Nutritional stress includes, for example, nutrient deficiency stress and nutrient excess stress.

[0018] In this embodiment, "environmental stress tolerance" refers to a trait that allows a plant to grow normally or nearly normally, even under growth conditions that cause environmental stress, without substantially suffering from undesirable effects such as inability to grow (withering), poor growth (e.g., bleaching or yellowing of the plant, reduced root length or reduced leaf number), reduced growth rate, or reduced plant weight or crop yield. By improving the plant's environmental stress tolerance with the plant growth-promoting composition of this embodiment, the plant can grow normally even in external environments that cause undesirable effects on the growth of ordinary plants.

[0019] The temperature stress tolerance-imparting effect of the plant growth-promoting composition of 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 general knowledge in the technical field, but are preferably 35°C or higher, for example, 35°C to 50°C. Low-temperature conditions may be appropriately determined by those skilled in the art based on common general knowledge in the technical field, but are preferably 10°C or lower, for example, 0°C to 10°C. Furthermore, the salt stress-imparting effect allows normal growth in the presence of 0.1 to 3.0% by mass of sodium chloride. The drought stress-imparting effect allows normal growth even at a relative humidity of 10 to 40%. The effect of the plant growth-promoting composition of this embodiment in imparting nutritional stress tolerance is, for example, the effect of promoting normal growth in plants that are deficient in chemical substances that serve as nutrient sources for the plant (nutrient-deficient state). Here, the nutrient-deficient state may be, for example, a state in which the amount of each nutrient is less than the amount indicated by the concentration during the test period in Table 1 of the Examples. Specifically, the amount of each nutrient may be approximately one-half to one-third, one-third to one-quarter, one-quarter to one-fifth, one-fifth to one-sixth, one-sixth to one-seventh, or one-seventh to one-eighth of the amount indicated by the concentration during the test period in Table 1. The concentrations during the test period in Table 1 are concentrations commonly used in the cultivation of crops such as rice.

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

[0021] When the plant growth-promoting composition of the present embodiment has an effect of improving environmental stress tolerance, it may activate a gene in a stress response system. Examples of the gene in a stress response system include, but are not limited to, genes generally known as indicators of response to environmental stresses such as temperature stress, nutritional stress, chemical stress, light stress, drought stress, pH stress, salt stress, hypoxic stress, herbicide stress, physical stress, and disease stress. Genes generally known as indicators of response to environmental stress are genes disclosed in publicly known databases such as the National Center for Biotechnology Information (NCBI), Ensembl Plants, The Arabidopsis Information Resource (TAIR), The Rice Annotation Project (RAP), etc. Specifically, genes generally known as indicators of response to high temperature stress include Os01g0135900, Os01g0273500, Os01g0721000, Os01g0875700, Os01g0894600, Os02g0753800, Os03g0161900, Os03g0266300, Os03g0426900, Os03g0854500, Os05g0110100, Os05g0364500, and Os05g04600. 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 compounds can be effectively inhibited in the plant acclimatized to high temperatures: Os01g0135900, Os01g0273500, Os01g0721000, Os01g0875700, Os01g0894600, Os02g0753800, Os03g0161900, Os03g0266300, Os03g0426900, Os03g0854500, Os05g0110100, O05g0364500, 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 increased compared to the control.

[0022] In addition, genes commonly known as response indicators to drought stress include Os01g0615050, Os01g0864500, Os01g0867300, Os01g0955100, Os01g0975300, Os02g0115700, Os02g0126400, Os02g0178800, Os02g0543000, Os02g0615800, Os02g0673500, Os02g0682300, and Os02g06900. 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 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 compounds can be effectively inhibited in the plants grown under dry conditions: 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 increased compared to the control.

[0023] In addition, genes generally known as indicators of response to low temperature stress include Os01g0510100, Os01g0559600, Os01g0769900, Os02g0121100, Os02g0624300, Os03g0285800, Os03g0319400, Os03g0656900, Os03g0678800, Os04g0517100, O4g0530900, and Os04g058460. 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 compounds can be used to promote growth of plants acclimated to low temperatures: Os01g0510100, Os01g0559600, Os01g0769900, Os02g0121100, Os02g0624300, Os03g0285800, Os03g0319400, Os03g0656900, Os03g0678800, Os04g0517100, O04g0530900, Os04g0584600, Os04g0517100, Os ... The expression levels of genes such as s05g0138300, Os05g0476700, Os05g0497300, Os05g0559900, Os06g0726300, Os07g0635900, Os08g0129200, Os09g0271100, Os09g0334500, Os09g0369000, Os09g0522200, Os09g0565200, Os10g0562900, and Os12g0210300 are increased compared to the control.

[0024] In addition, genes generally known as indicators of response to salt stress include Os01g0141000, Os01g0200700, Os01g0575200, Os01g0615100, Os01g0624500, Os01g0864500, Os01g0955100, Os01g0975300, Os02g0126400, Os02g0132300, Os02g01410 ... 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 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 plant can be grown in a medium or soil containing salt at an appropriate concentration, and the following compounds can be grown: 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 increased compared to the control.

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

[0026] Furthermore, genes generally known as indicators of a response to light stress include Os01g0510100, Os04g0619400, Os05g0592400, Os11g0117400, etc. For example, by applying the plant growth-promoting composition of this embodiment to a plant, the expression levels of genes such as Os01g0510100, Os04g0619400, Os05g0592400, Os11g0117400 in the plant grown under strong light are increased compared to controls. Genes generally known as indicators of a response to light stress are also genes generally known as indicators of a response to strong light stress.

[0027] The plant growth-promoting composition of 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 ingredients. Agriculturally acceptable ingredients include, but are not limited to, solvents, carriers, excipients, binders, solubilizers, stabilizers, thickeners, leavening agents, lubricants, surfactants, oily liquids, buffers, bactericides, antifreeze agents, antifoaming agents, colorants, antioxidants, and additional active ingredients. Preferred agriculturally acceptable carriers include water, mineral oil fractions such as kerosene or diesel oil, vegetable or animal oils, 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), amines (e.g., N-methylpyrrolidone), and mixtures thereof.

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

[0029] The plant growth-promoting composition of this embodiment can be used in combination with conventional agricultural materials such as pesticides and fertilizers. Examples of agricultural materials include pesticides, fertilizers, soil conditioners, and potting soil (culture soil). Specifically, the pesticides may be fungicides, insecticides, insecticides, fungicides, miticides, nematicides, rodenticides, insect repellents, attractants, spreaders, hormones (growth regulators), herbicides, and the like. The fertilizers may be organic fertilizers, chemical fertilizers, liquid fertilizers, solid fertilizers, compound fertilizers, compost, growth promoters, stimulants, calcium supplements, and the like. The soil conditioners may be those intended for physical, chemical, or biological soil improvement, and may also be oxygen suppliers, and the like. Furthermore, the pesticides, fertilizers, soil conditioners, and potting soil may contain components derived from plants, animals, lime, ores, or microorganisms.

[0030] The content of one or more selected from the group consisting of cocoa extract and cocoa powder in the plant growth-promoting composition relative to the entire plant growth-promoting composition may be any value as long as the one or more selected from the group consisting of cocoa extract and cocoa powder functions as an active ingredient for promoting plant growth, but may be, for example, 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 of this embodiment can be used as an agricultural chemical formulation 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., a 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 a liquid state, or may be used as a liquid prepared just before use.

[0032] The formulation of the plant growth-promoting composition of this embodiment is not particularly limited, and can be formulated into formulations commonly used in the art, such as emulsions, wettable powders, liquids, water-soluble powders, dusts, powders, pastes, or granules.

[0033] In this embodiment, the plant to which the plant growth-promoting composition is applied is not particularly limited, and examples thereof 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, corn, wheat, sugarcane, and barley, Legumes such as soybeans, Apiaceae plants such as carrots, Lamiaceae plants such as basil, mint, and rosemary, Convolvulaceae plants such as morning glory, Salicaceae plants such as poplars, and Euphorbiaceae plants such as castor beans, cassava, and jatropha. Convolvulaceae plants such as sweet potato, Rutaceae plants such as oranges and lemons, Rosaceae plants such as cherry blossoms and roses, Orchidaceae plants such as moth orchids, Gentianaceae plants such as bellflowers, Primulaces such as cyclamen, Violets such as pansies, Liliaceae plants such as lilies, Amaranthaceae plants such as sugar beets, Vitaceae plants such as grapes, Cupressaceae plants such as cedars and cypresses, Oleaceae plants such as olives and Osmanthus, and Pinaceae plants such as red pine.

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

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

[0036] <Method for preparing plant growth-promoting composition> The plant growth-promoting composition can be prepared, for example, by obtaining one or more selected from the group consisting of cocoa extract and cocoa powder, and then mixing it with the agriculturally acceptable ingredients described above, if necessary. The method for obtaining cocoa extract is not particularly limited, and general methods for extracting plant extracts can be used. Specifically, for example, cocoa extract can be obtained by a method comprising the steps of 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 to obtain a cocoa extract raw material; concentrating the cocoa extract raw material obtained in the above step to obtain a cocoa concentrate; and fractionating the cocoa concentrate obtained in the above step to obtain a cocoa extract containing specific components. Alternatively, cocoa extract can be obtained from one or more selected from the group consisting of cocoa leaves, stems, skins, fruits, seed coats, and roots that have undergone each step in obtaining the cocoa extract. For example, a cocoa extract raw material or a cocoa concentrate can be obtained from the cocoa residue, such as impurities removed in the step of obtaining the cocoa extract raw material, by a method similar to that described above, and then a cocoa extract can be obtained from the cocoa extract raw material or the cocoa concentrate by a method similar to that described above. Alternatively, the cocoa extract may be obtained from cocoa residue, such as impurities, obtained during the production of any processed cocoa product. The cocoa extract raw material or cocoa concentrate may be used as is as the cocoa extract. The cocoa extract raw material, cocoa concentrate, or cocoa extract may be heated or pressurized during or after each step. When heating, the heating conditions, such as the heating temperature and heating time, can be determined appropriately depending on the variety, part, and condition of the cocoa used. The method for obtaining cocoa powder is not particularly limited, and the powder can be obtained by drying fresh or frozen cocoa residues such as cocoa extract raw materials, cocoa concentrates, or impurities generated when obtaining cocoa extract, cocoa extract raw materials, cocoa concentrates, cocoa extract, or cocoa (one or more selected from the group consisting of cocoa leaves, stems, skins, fruits, seed coats, and roots) using a method such as hot air drying, air drying, dry heat drying, or vacuum drying, and then crushing the dried product into powder.

[0037] (Crushing process) Cocoa can be crushed using fresh or dried cocoa, for example, in a mixer. The crushed cocoa is filtered using a filter cloth, filter paper, or the like to obtain a cocoa extract raw material from which impurities have been removed. An extraction solvent such as an organic solvent may be added before filtration. Alternatively, impurities may be removed by collecting the supernatant using a centrifuge instead of filtration.

[0038] (concentration process) The obtained cocoa extract raw material can be concentrated using a method such as vacuum concentration, freeze concentration, or membrane concentration to obtain a cocoa concentrate. The concentration ratio is preferably 3 to 30 times, and more preferably 4 to 20 times. A concentration ratio of 5 times or more tends to improve handling and reduce transportation costs because the amount of plant growth-promoting composition added to plants is reduced, while a concentration ratio of 30 times or less tends to reduce sediment in the plant growth-promoting composition, making it easier to handle. In preparing the plant growth-promoting composition, it is not necessary to concentrate the cocoa extract raw material, and the obtained cocoa extract raw material may be subjected to the following fractionation step as is.

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

[0040] The cocoa extract obtained in the fractionation step may be further purified or subjected to separation of highly active fractions, if necessary. Examples of purification include filtration and adsorption (ion exchange resin column, activated carbon column, etc.). Examples of separation of highly active fractions include gel filtration, adsorption, silica gel column chromatography, HPLC, etc.

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

[0042] The plant growth-promoting composition may be applied to a plant once or multiple times. Furthermore, when the plant growth-promoting composition is applied at multiple application periods, the plant growth-promoting composition may be applied once or multiple times at each application period.

[0043] Examples of means for applying the plant growth-promoting composition to plants include addition, spraying, coating, and immersion. These may be performed alone or in combination of two or more. The plant growth-promoting composition may be applied to plants manually or by machine. When using a machine, there are no particular limitations, but it may be applied (sprayed) by, for example, a drone.

[0044] The amount of plant growth-promoting composition applied to plants, whether applied to a culture solution in which plants grow or diluted with a diluent and applied to a field or other area where plants grow, 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. While sufficient plant growth-promoting effects tend to be achieved when the plant growth-promoting composition is used in an amount within the above ranges, the amount used may be adjusted appropriately 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 state of the plant to which the plant growth-promoting composition is applied, and the plant's growth environment, such as temperature, humidity, and light. The amount used may be determined by investigating the concentration range in which an effect is obtained using a known cultivation test method. When a plant growth-promoting composition diluted with a diluting solvent is applied to a plant using a drone, the amount of solution that can be carried on the drone is limited by the drone's carrying capacity, etc., so the amount of plant growth-promoting composition used relative to the diluting solvent is preferably 1 to 50 mass % or 1 to 50 volume %.

[0045] The components of the culture medium and dilution solvent are not particularly limited as long as they do not adversely affect the growth of the plant, and may contain water, pesticides, etc.

[0046] The preferred range of the amount used varies depending on the concentration of the cacao extract raw material, but the preferred range is when the concentration is 3 to 30 times. [Example]

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

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

[0049] [Example 1] 20.0 g of cacao seed coat was crushed and then ultrapure water was added to extract the cacao extract raw material, which was then concentrated to obtain the cacao extract. Rice was grown in the seedling culture solution shown in Table 1. The rice was then transferred to 900 mL of the test culture solution shown in Table 1, and 3.6 mL (0.4% by volume) of the obtained cocoa extract was added to investigate the effect on the rice. As a result, the weight of the rice roots increased by 19.9% ​​compared to when the cocoa extract was not added, demonstrating an improved effect on the root establishment of rice (Figures 1 and 2). Furthermore, the concentration of nutrients during the seedling stage was approximately one-sixth to one-seventh of the concentration during the test stage, which is sufficient for rice growth, so the rice plants to which the cocoa extract had been added were in a state of nutritional deficiency.

[0050] [Table 1]

[0051] [Example 2] Variation in gene expression in rice was also assessed.

[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 culture medium for the test period shown in Table 1, and 3.6 mL (0.4% by volume) of the cocoa extract used in Example 3 was added to the 900 mL culture medium, and the rice was cultivated. RNA extraction RNA samples were obtained using the Maxwell RSC Plant RNA Kit (Promega) and the Maxwell RSC Instrument (Promega) according to the manufacturer's instructions. RNA quality check Measurement equipment: Agilent 5400 Bioanalyzer <5312AA> (Agilent Technologies), Reagent kit: Agilent RNA kit (15nt) <dnf-471>(Agilent Technologies) and analysis software: ProSize (Agilent Technologies) were used to confirm that there were no problems with the quality of the RNA samples, before 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 according to the following steps. 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 ligation, size selection, and amplification are performed

[0053] (B) Analysis by next-generation sequencer Sequence analysis Equipment: DNBSEQ T7 (MGI) Sequence (outline) Sequencing was performed using DNBSEQ T7 according to the following steps. 1. Addition of Sequencing Reagents 2. Single-base extension reaction 3. Removal of unreacted base 4. Fluorescent signal acquisition 5. Removal of protecting groups and fluorescence 6 Repeat the cycle 2 cycles...3 cycles... until you reach 300 cycles.

[0054] (C) Data analysis Primary data analysis Analysis computer OS: Ubuntu Desktop 22.04.4 LTS Analysis software: FastQC, hisat2, samtools, HTSeq, DESeq2 The analysis was performed using the software mentioned above. 1. Check the quality of your leads FastQC was used to check the quality of the bases contained in the resulting fastq files. 2 Mapping and Sorting The data were mapped to the Oryza sativa reference genome, Oryza sativa Japonica Group (assembly IRGSP-1.0), using hisat2. The files were then sorted and converted to binary data using samtools. 3 Lead Count The number of mapped reads was counted using htseq-count in HTSeq. 4 Statistical analysis The DESeq2 package in the statistical package R was used to compare gene expression levels between the control and cocoa extract-added groups. Tertiary data analysis Genes reported to be involved in various environmental stress tolerances in previous studies were searched from the Rice Annotation Project (https: / / rapdb.dna.affrc.go.jp / ). The searched genes were then searched from the gene expression list created in this experiment. Selection of target genes and plotting gene expression levels Various environmental stress tolerance genes present in known databases were collected. Various stress tolerance genes were then extracted from the list of genes with elevated expression levels, and their expression levels were displayed as heat maps and bar graphs. In the heat maps shown in Figures 3, 5, 7, 9, 11, and 13, CTS indicates the control group, and CCh-bS indicates the cocoa extract-added group.

[0055] Gene expression analysis showed that the expression levels of several heat stress tolerance genes tended to be higher in the shoots of plants treated with cocoa extract than in the control group (Figure 3). In particular, the expression level of the heat stress tolerance gene Os03g0854500 was increased in the shoots (Figure 4). The following findings have been reported regarding these molecules related to heat stress tolerance, and they are thought to contribute to heat stress tolerance. It has been suggested that molecules homologous to Os03g0854500 are induced by high temperature stress and play an important role in plant survival under high temperature conditions (Reference 1). This gene and similar genes have also been reported to be widely present in plants other than Arabidopsis and rice, such as sunflower, lettuce, and common wheat (Reference 2).

[0056] Gene expression analysis showed that the expression levels of several drought stress tolerance genes tended to be higher in the shoots of the cocoa extract-treated area than in the control area (Figure 5). In particular, the expression level of the drought stress tolerance gene Os02g0115700 was increased in the shoots (Figure 6). These drought stress tolerance-related molecules are thought to contribute to drought stress tolerance, as the following findings have been reported previously. It has been reported that the expression of a molecule homologous to Os02g0115700 is induced by drought stress, and the activity of the protein translated from this gene increases (Reference 3). This gene and similar genes have also been reported to be widely present in sunflower, cassava, bread wheat, maize, and other plants in addition to Arabidopsis and rice (Reference 4).

[0057] Gene expression analysis showed that the expression levels of several cold stress tolerance genes tended to be higher in the shoots of the cocoa extract-added area than in the control area (Figure 7). In particular, the expression level of the cold stress tolerance gene Os03g0285800 increased in the shoots (Figure 8). The following findings have been reported regarding these molecules related to cold stress tolerance, and therefore they are thought to contribute to cold stress tolerance. It has been suggested that a MAP kinase cascade contributes to cold tolerance (Reference 5). This gene and similar genes have been reported to be widely present in soybean, wild cotton, and common wheat in addition to Arabidopsis and rice (Reference 6).

[0058] Gene expression analysis showed that the expression levels of several salt stress tolerance genes tended to be higher in the shoots of the cocoa extract-added area than in the control area (Figure 9). In particular, the expression level of the salt stress tolerance gene Os02g0543000 was increased in the shoots (Figure 10). The following findings have been reported regarding these molecules related to salt stress tolerance, and therefore they are thought to contribute to salt stress tolerance. Overexpression of a homologous gene to Os02g0543000 has been reported to increase salt stress tolerance (Reference 7). This gene and similar genes have been reported to be widely present in soybeans, sunflowers, lettuce, and other plants in addition to Arabidopsis and rice (Reference 8).

[0059] Gene expression analysis showed that the expression levels of several osmotic stress tolerance genes tended to be higher in the shoots of the cocoa extract-added area than in the control area (Figure 11). In particular, the expression level of the stress tolerance gene Os02g0666200 was increased in the shoots (Figure 12). The following findings have been reported regarding molecules related to osmotic stress tolerance, and therefore this gene is thought to contribute to osmotic stress tolerance. The homologous molecule of Os02g0666200 promotes water uptake (Reference 9). This gene and similar genes have been reported to be widely present in Arabidopsis thaliana, rice, and other plants, including rapeseed, wheat, and maize (Reference 10).

[0060] Gene expression analysis showed that the expression levels of several high-light stress tolerance genes tended to be higher in the shoots of the cocoa extract-treated area than in the control area (Figure 13). In particular, the expression level of the high-light stress tolerance gene Os05g0592400 was increased in the shoots (Figure 14). The following findings have been reported regarding these molecules related to high-light stress tolerance, and therefore they are thought to contribute to high-light stress tolerance. A homologous gene to Os05g0592400 has been reported to be involved in the removal of DNA damaged by ultraviolet light (Reference 11). This gene and similar genes have been reported to be widely present in plants other than Arabidopsis and rice, including pepper, tobacco, and common wheat (Reference 12).

[0061] From the above, it is believed that cocoa extract has the effect of improving tolerance to one or more stresses selected from the group consisting of high temperature stress, drought stress, low temperature stress, salt stress, 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, 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 biostimulant containing only one or more active ingredients selected from the group consisting of cocoa extract and cocoa powder.

2. The biostimulant according to claim 1, which has one or more plant growth-promoting effects selected from the group consisting of elongation of stems, leaves or roots, increased leaf number, promotion of flowering or fruiting, increased number of flowers or fruits, increased plant weight or crop yield, greening, and promotion of tillering.

3. The biostimulant according to claim 1 or 2, which has the effect of improving environmental stress tolerance against one or more stresses selected from the group consisting of temperature stress, nutritional stress, chemical stress, light stress, drought stress, pH stress, salt stress, hypoxic stress, herbicide stress, physical stress, and disease stress.

4. The biostimulant according to claim 1 or 2, which activates genes in the stress response system.

5. A method for improving environmental stress tolerance, comprising applying a biostimulant containing, as an active ingredient, one or more species selected from the group consisting of cocoa extract and cocoa powder to a plant or to soil or culture solution in which the plant grows.

6. The method for improving environmental stress tolerance according to claim 5, wherein the amount of the biostimulant used is 0.001 to 50% by mass or 0.001 to 50% by volume relative to the culture medium or dilution solvent.

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