Method for promoting plant growth, method for producing plant growth promoter, and nutritional composition for use in plant cultivation

Abstract

To provide a method for promoting plant growth, a method for producing a plant growth promoter, and a nutritional composition for use in plant cultivation.SOLUTION: A method for promoting plant growth comprising a step of preparing a crude extract of cells and / or tissues containing KODA, a step of mixing the crude extract with a soil composition or a nutrient solution to prepare a soil composition or a nutrient solution containing an effective amount of KODA, and a step of cultivating plants using the soil composition or the nutrient solution containing the effective amount of KODA.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] The present disclosure relates to methods for promoting plant growth, methods for producing plant growth promoters, and nutritional compositions for use in cultivating plants. [Background technology]

[0002] KODA (9,10-α-ketol linolenic acid) is a plant-derived physiologically active substance that confers tolerance to various environmental stresses, including drought and disease, and to environmental changes (Non-Patent Documents 1 to 3). KODA is typically present in plants only in trace amounts, and production methods using chemical synthesis or microorganisms are expensive and unsuitable for practical use in the agricultural field. Therefore, a cheap, simple, and highly efficient method for synthesizing KODA has been sought for many years.

[0003] Methods for chemically synthesizing KODA have been reported, but they require numerous reaction steps, resulting in a very low final yield of 0.81% (Non-Patent Document 4). Production using 9-LOX and AOS enzyme proteins extracted from recombinant Escherichia coli or other strains requires the addition of expensive α-linolenic acid as a raw material, resulting in high costs. A method has also been devised for cultivating duckweed (Lemna paucicostata), which is known to accumulate high levels of KODA (Patent Document 1). However, duckweed's tendency to grow planarly results in low productivity per unit area. Furthermore, the laborious process of extracting KODA from cells increases production costs.

[0004] Recently, researchers have succeeded in synthesizing KODA by crushing leaves from Arabidopsis thaliana strains in which the KODA synthesis genes (AOS and 9-LOX) from duckweed have been expressed, and then incubating the crushed leaves for a certain period of time (Patent Document 2, Non-Patent Document 5). However, purifying KODA from crude leaf extracts requires multiple steps due to the high content of contaminants, which increases the final cost of KODA production. [Prior art documents] [Patent documents]

[0005]

Patent Document 1

Patent document 2

Non-licensed literature

[0006] [Non-licensed document 1] Haque et al., “KODA, an α-ketol derivative of linolenic acid provides wide recovery ability of wheat against various abiotic stresses”, Biocatalysis and Agricultural Biotechnology, Vol. 7, pp.67-75, 2016 [Non-licensed document 2] Wang et al., "Oxylipins Other Than Jasmonic Acid Are Xylem-Resident Signals Regulating Systemic Resistance Induced by Trichoderma virens in Maize.", The Plant cell, vol. 32(1), pp.166-185, 2020.. [Non-licensed document 3] Wang et al., "α-Ketol linolenic acid (KODA) application affects endogenous abscisic acid, jasmonic acid and aromatic volatiles in grapes infected by a pathogen (Glomerella cingulata)." Journal of plant physiology vol. 192, pp.90-7, 2016

Non-licensed Document 4

[0007] The present disclosure has been made to solve the above-mentioned problems, and aims to provide a method for promoting plant growth, a method for producing a plant growth promoter, and a nutritional composition for use in plant cultivation. [Means for solving the problem]

[0008] One aspect of the present disclosure is a method for promoting plant growth, comprising the steps of preparing a crude extract of cells and / or tissues containing KODA, mixing the crude extract with a soil composition or nutrient solution to prepare a soil composition or nutrient solution containing an effective amount of KODA, and cultivating a plant using the soil composition or nutrient solution containing the effective amount of KODA. One aspect of the present disclosure is a method for promoting plant growth, comprising the steps of preparing a crude extract of cells and / or tissues transformed with a nucleic acid comprising a nucleotide sequence encoding AOS and / or 9-LOX, mixing the crude extract with a soil composition or nutrient solution, and cultivating a plant using the soil composition or nutrient solution. One aspect of the present disclosure is a method for producing a plant growth-promoting agent, the method comprising preparing a crude extract of cells and / or tissues transformed with a nucleic acid comprising a nucleotide sequence encoding AOS and / or 9-LOX, wherein the growth-promoting agent comprises the crude extract. One aspect of the present disclosure is a nutritional composition for use in cultivating plants, wherein the nutritional composition is a soil composition for use in cultivating plants in soil or a nutrient solution for hydroponic cultivation, the nutritional composition comprising a crude extract of KODA-containing cells and / or tissues, and the nutritional composition comprises an effective amount of KODA. [Effects of the Invention]

[0009] According to the present disclosure, it is possible to provide a method for promoting plant growth, a method for producing a plant growth promoter, and a nutritional composition for use in plant cultivation. [Brief explanation of the drawings]

[0010] [Figure 1] Figure 1 shows a photograph of wild-type Arabidopsis thaliana 41 days after sowing. Nutrient solution (Mock), wild-type Arabidopsis thaliana extract (WT), vector control extract (VC), and KODA-producing extract (KODA-producing strain) were mixed with growth soil to prepare soil compositions, and wild-type Arabidopsis thaliana seeds were sown in each soil composition. Bolt-forming plants were supported with stakes. Continuous light was applied, and the temperature was set at 23°C. DETAILED DESCRIPTION OF THE INVENTION

[0011] Non-limiting embodiments of the present disclosure will be described below. The present disclosure is not limited to the examples in the following embodiments.

[0012] <Methods for promoting plant growth> In one aspect, a method for promoting plant growth is provided. The method for promoting plant growth in this embodiment includes the steps of preparing a crude extract of cells and / or tissues containing KODA, mixing the crude extract with a soil composition or nutrient solution to prepare a soil composition or nutrient solution containing an effective amount of KODA, and cultivating a plant using the soil composition or nutrient solution containing the effective amount of KODA. Using a crude extract containing KODA provides a method for promoting plant growth using KODA that does not require purification of KODA.

[0013] In embodiments, the plant is not particularly limited and may include terrestrial plants, aquatic plants, epiphytes, and rock plants. In embodiments, the plant may include, but is not limited to, plants of the Malvaceae, Rubiaceae, Brassicaceae, Poaceae, Cucurbitaceae, Asteraceae, Moraceae, Pesamiaceae, Araceae, Umbelliferae, Solanaceae, Papayaaceae, Rosaceae, Amaryllidaceae, Fabaceae, Rutaceae, Oleaceae, Amaranthaceae, and Liliaceae families. Brassicaceae, Asteraceae, Apiaceae, Solanaceae, Rosaceae, Amaranthaceae, and Fabaceae are particularly preferred. More specific examples of plants according to the embodiment include kiwifruit (Actinidia deliciosa), onion (Allium cepa), leek (Allium fistulosum), garlic (Allium sativum), celery (Apium graveolens var. dulce), Arabidopsis (Arabidopsis thaliana), peanut (Arachis hypogaea), beet (Beta vulgaris subsp. Vulgaris), cauliflower (Brassica oleracea var. botrytis), cabbage (Brassica oleracea var. capitata), broccoli (Brassica oleracea var. italica), rapeseed (Brassica rapa), bok choy (Brassica rapa var. chinensis), mizuna (Brassica rapa var. nipposinica), and komatsuna (Brassica rapa var. perviridis), turnip (Brassica rapa var.rapa), tea plant (Camellia sinensis), bell pepper (Capsicum annuum), paprika (Capsicum annuum), safflower (Carthamus tinctorius), lime (Citrus aurantifolia), lemon (Citrus limon), orange (Citrus sinensis), grapefruit (Citrus x paradisi), coconut palm (Cocos nucifera), coffee plant (Coffea arabica), taro (Colocasia esculenta), coriander (Coriandrum sativum), mitsuba (Cryptotaenia japonica), cucumber (Cucumis sativus), pumpkin (Cucurbita maxima), persimmon (Diospyros kaki), oil palm (Elaeis spp.), buckwheat (Fagopyrum esculentum), fig (Ficus carica), strawberry (Fragaria × ananassa), garland chrysanthemum (Glebionis coronaria), soybean (Glycine max), sunflower (Helianthus annuus), barley (Hordeum vulgare), sweet potato (Ipomoea batatas), morning glory (Ipomoea nil), lettuce (Lactuca sativa), lentil (Lens culinaris), apple (Malus domestica), peppermint (Mentha × piperita), banana (Musa spp.), watercress (Nasturtium officinale), tobacco (Nicotiana tabacum), basil (Ocimum basilicum), olive (Olea europaea), rice (Oryza sativa), shiso (Perilla frutescens var.crispa), avocado (Persea americana), parsley (Petroselinum crispum), kidney beans (Phaseolus vulgaris), pepper (Piper nigrum), peas (Pisum sativum), pears (Pyrus pyrifolia), rosemary (Rosmarinus officinalis), sugarcane (Saccharum officinarum), tomato (Solanum lycopersicum), eggplant (Solanum melongena), potato (Solanum tuberosum), sorghum (Sorghum bicolor), spinach (Spinacia oleracea), cocoa (Theobroma cacao), thyme (Thymus vulgaris), wheat (Triticum aestivum), blueberries (Vaccinium spp.), grapes (Vitis spp.), adzuki beans (Vigna angularis), corn (Zea Examples of suitable herbs include, but are not limited to, Zingiber mays, Zingiber mioga, Zingiber officinale, and related species. Among these, Arabidopsis thaliana, cabbage, bok choy, mizuna, komatsuna, coriander, peppermint, basil, shiso, lettuce, and spinach are preferred.

[0014] In the present disclosure, "promoting plant growth" may mean increasing one or more selected from the group consisting of plant size, organ size, tissue size, cell size, biomass, growth rate, growth factor, and growth efficiency compared to a plant grown under control growth conditions. Promotion of plant growth may be determined, for example, by comparing plant size, organ size, tissue size, cell size, or biomass with that of a control plant at one or more selected from one week, two weeks, three weeks, one month (30 days), or two months or more after sowing.

[0015] In this disclosure, KODA refers to 9,10-α-ketol linolenic acid (also known as 9-hydroxy-10-oxo-12(Z),15(Z)-octadecadienoic acid or 9,10-ketol-octadecadienoic acid). The structure of KODA is shown below.

[0016] [ka]

[0017] Those skilled in the art will appreciate that KODA may spontaneously exist in an ionic or salt form depending on the equilibrium state in a particular aqueous environment. Therefore, the present disclosure should be understood to encompass both ionic and salt forms of KODA. Salts of KODA may be, for example, sodium, potassium, lithium, ammonium, or other monovalent cation salts.

[0018] The cells and / or tissues in the present disclosure may be cells and / or tissues of organisms, including plants, algae, animals, or microorganisms. These organisms may be wild-type or genetically engineered. A preferred wild-type organism is a plant that produces KODA. An example of such a plant is duckweed. An example of a genetically engineered organism is an organism into which a gene encoding an enzyme involved in KODA synthesis has been introduced. The organism into which the gene is introduced is not limited as long as it is an organism into which gene introduction is possible, but is preferably a plant. Suitable examples of such plants include sugarcane, Arabidopsis, potato, tobacco, tomato, rapeseed, and lettuce. Examples of genes to be introduced include AOS and 9-LOX. The introduced gene may be an endogenous gene or an exogenous gene. The exogenous gene may be artificially introduced into a plant individual, tissue, or cell, for example, by transformation.

[0019] In the embodiments, crude extracts of cells and / or tissues containing KODA may include crude extracts of cells and / or tissues transformed with a nucleic acid comprising a nucleotide sequence encoding AOS and / or 9-LOX. In the embodiments, the cells and / or tissues transformed with a nucleic acid comprising a nucleotide sequence encoding AOS and / or 9-LOX are preferably cells and / or tissues capable of synthesizing alpha-linolenic acid. In the embodiments, crude extracts of cells and / or tissues containing KODA may be prepared from cells and / or tissues containing KODA or from cells and / or tissues containing precursors of KODA.

[0020] The transformed cells and / or tissues may be cells and / or tissues from a plant, such as sugarcane, Arabidopsis, potato, tobacco, tomato, rapeseed, or lettuce. In embodiments, the tissue may be, for example, just a leaf of the plant or the whole plant.

[0021] In the present disclosure, transformation includes introducing an exogenous gene to increase expression of the gene compared to a corresponding non-transformed individual, tissue, or cell. Transformation can be carried out by various techniques known to those skilled in the art, including the Agrobacterium method, the gene gun (particle gun) method, the electroporation method, the PEG method, homologous recombination, and various genome editing techniques including the CRISPR / CAS9 method.

[0022] In one embodiment, the nucleotide sequence encoding 9-LOX (also referred to as a 9-LOX gene) is a nucleotide sequence encoding 9-lipoxygenase (9-LOX). 9-LOX is an enzyme that has the activity of introducing a hydroperoxy group at the 9-position of linolenic acid. Preferably, the 9-LOX encoded by the nucleotide sequence encoding 9-LOX is plant-derived 9-LOX, more preferably duckweed-derived 9-LOX. The 9-LOX encoded by the nucleotide sequence encoding 9-LOX may contain one or more of amino acid substitutions, deletions, and additions, so long as it retains the above-mentioned activity. More specifically, the 9-LOX may be an enzyme having an amino acid sequence identity of 90% or more, 95% or more, or 98% or more to duckweed-derived 9-LOX (amino acid sequence encoded by a cDNA corresponding to GenBank ID: HV534700.1).

[0023] In an embodiment, the nucleotide sequence encoding AOS (also referred to as an AOS gene) is a nucleotide sequence encoding allene oxide synthase (AOS). AOS is an enzyme with allene oxide synthase activity that synthesizes allene oxide from hydroperoxylated fatty acids. Preferably, the AOS encoded by the nucleotide sequence encoding AOS is a plant-derived AOS, more preferably an AOS derived from duckweed. The AOS encoded by the nucleotide sequence encoding AOS may contain one or more of amino acid substitutions, deletions, and additions, as long as it has the above-mentioned activity. More specifically, the AOS may be an enzyme having an amino acid sequence identity of 90% or more, 95% or more, or 98% or more to the AOS derived from duckweed (amino acid sequence encoded by cDNA corresponding to GenBank ID: HV534712.1).

[0024] In the embodiments, the nucleotide sequences encoding 9-LOX and AOS may be operably linked to a functional promoter. The functional promoter is not particularly limited as long as it can induce expression of 9-LOX or AOS in plant individuals, tissues, or cells. Suitable examples include the 35S promoter of cauliflower mosaic virus (CaMV) and its variants. The 9-LOX gene and / or AOS gene linked to a functional promoter may be contained in any vector suitable for transformation. Furthermore, the 9-LOX and / or AOS gene may contain an untranslated region (UTR) in addition to the coding region.

[0025] In one embodiment, a method for promoting plant growth includes preparing a crude extract of cells and / or tissues containing KODA. The crude extract may be prepared by subjecting the cells and / or tissues to one or more treatments selected from disruption and extraction. The preparation of the crude extract may also optionally include one or more treatments selected from solid-liquid separation, incubation, and drying. For example, the crude extract may be a crude extract prepared through all of these treatments, or may be prepared by simply adding a solvent to disrupted plant matter. For example, freeze-dried products prepared by disrupting the above-ground parts of the plant (leaves and stems) may be distributed as a freeze-dried product, which can be mixed with water by the user as needed to obtain the crude extract. This method has the significant advantage of not requiring special solvents or equipment.

[0026] In embodiments, disruption may refer to breaking down plant tissue into pieces, for example, 1 cm or less, 0.5 cm or less, 0.1 cm or less, or 0.01 cm or less in the longest diameter. Disrupting cells and / or tissues in embodiments may be performed using tools and methods known to those skilled in the art, including, for example, a mortar and pestle, freezing, a homogenizer, or a bead mill, and may include powdering the cells and / or tissues. More specifically, disruption in embodiments may include grinding in a mortar under liquid nitrogen to a powder.

[0027] In embodiments, extraction involves extracting KODA from cells and / or tissues using a solvent, such as water or an aqueous solution containing nutrients for plant growth, which may be added to the cells and / or tissues prior to or after disruption.

[0028] In the solid-liquid separation of the embodiment, the solid and liquid components in the suspension can be separated by methods known to those skilled in the art. For example, the solids can be removed from the suspension by filtration using a filter material such as filter paper or filter cloth, and the remaining liquid can be used as a crude extract. Alternatively, filtration can be performed by other methods known to those skilled in the art, such as using various spin columns and centrifuges. Alternatively, as known to those skilled in the art, the suspension can be centrifuged, and the supernatant from which the pellet has been removed can be used as a crude extract.

[0029] Incubation of the embodiment may include incubating the suspension with a solvent for a certain period of time. By allowing KODA synthesis to continue during the incubation step, the KODA content in the sample can be increased. Optionally, α-linolenic acid can also be added to the disrupted material of the embodiment to increase the KODA content.

[0030] Drying in embodiments may be accomplished by freeze-drying, which may be accomplished by drying the frozen sample under vacuum by methods known to those skilled in the art.

[0031] The present disclosure is based on the finding that KODA in a crude extract maintains its activity for a long period of time in nutrient compositions, such as soil, mixed with the crude extract, and provides a method for utilizing a crude extract, such as plant leaves, directly as a growth-promoting agent with the effects of KODA, without the need for a costly KODA purification process. The step of preparing a crude extract of cells and / or tissues containing KODA in the embodiments may include preparing a crude extract of cells and / or tissues transformed with a nucleic acid comprising a nucleotide sequence encoding AOS and / or 9-LOX.

[0032] In the present disclosure, "a soil composition or nutrient solution containing an effective amount of KODA" refers to a soil composition or nutrient solution containing a sufficient amount of KODA to promote growth and / or suppress flower formation compared to a soil composition or nutrient solution that does not contain KODA. For example, two crude extracts of the same tissue, each differing in KODA content, are mixed with soil to prepare soil compositions A and B. If soil composition A does not promote growth and flower formation compared to the soil composition that does not contain KODA, and soil composition B promotes growth and / or flower formation compared to the soil composition that does not contain KODA, then soil composition A can be determined to not contain an effective amount of KODA and soil composition B to contain an effective amount of KODA. These two crude extracts may be prepared from tissues of a plant transformed with a KODA synthetic gene containing AOS and 9-LOX and the corresponding wild-type plant or a plant transformed with an empty vector. Alternatively, the amount of KODA contained in the target soil or nutrient solution may be quantified, and if a corresponding amount of purified KODA is added to soil or nutrient solution that does not contain KODA, and promotion of growth and / or suppression of flower bud formation is observed compared to soil or nutrient solution without added KODA, the target soil or nutrient solution may be determined to be "soil or nutrient solution containing an effective amount of KODA."

[0033] The soil composition or nutrient solution in the present disclosure is not limited as long as it is used for cultivating plants. The soil composition or nutrient solution is collectively referred to as a "nutrient composition" or a "nutrient composition for use in cultivating plants."

[0034] The soil composition of the embodiment may be, for example, natural soil, but is not limited to any soil that can be used for plant cultivation. The soil composition of the embodiment may include a soil composition containing one or more of peat moss, leaf mold, vermiculite, and Akadama soil. As will be apparent to those skilled in the art, the composition of the soil composition used for plant cultivation can be adjusted appropriately depending on the plant species and cultivation conditions.

[0035] The nutrient solution of the present invention may be a nutrient solution known to those skilled in the art, containing salts such as potassium nitrate, calcium nitrate, ammonium nitrate, magnesium nitrate, ammonium sulfate, ammonium chloride, potassium sulfate, magnesium sulfate, potassium dihydrogen phosphate, ammonium dihydrogen phosphate, potassium chloride, calcium chloride, ferrous sulfate, manganese sulfate, zinc sulfate, copper sulfate, and sodium molybdate, as well as boric acid, urea, and chelating agents (e.g., EDTA). The composition and concentration of these nutrient solution components can be adjusted appropriately depending on the species or variety of the plant being cultivated, the cultivation conditions, and the purpose of cultivation. Watering with such a nutrient solution provides the nutrients and moisture necessary for plant growth. To cultivate plants using the nutrient solution of the present invention, a cultivation device known to those skilled in the art, including a cultivation bed, a nutrient solution tank, a supply pump, and water supply and drainage pipes, can be used. The nutrient solution may be supplied to the plants by either a circulating or non-circulating supply method. The nutrient solution can also be supplied by placing plants fixed in a solid medium, such as a urethane medium, in a cultivation bed that is constantly irrigated.

[0036] In embodiments, the step of preparing a soil composition or nutrient solution containing an effective amount of KODA includes blending a crude extract of cells and / or tissues containing KODA with the soil composition or nutrient solution. This step may be carried out, for example, by blending a crude extract containing solids with the soil composition. Alternatively, it may be carried out by blending a crude extract containing no solids with the nutrient solution. The amount of crude extract blended is an amount sufficient to provide a sufficient amount of KODA so that the soil composition or nutrient solution contains an effective amount of KODA. Thus, the higher the concentration of KODA in the crude extract, the smaller the amount of crude extract that can be blended.

[0037] The step of cultivating a plant using a soil composition or nutrient solution containing an effective amount of KODA according to the embodiment can be carried out using any plant cultivation method known to those skilled in the art, with the exception of using a soil composition or nutrient solution containing an effective amount of KODA, and the conditions can be adjusted appropriately depending on the plant species being cultivated. Plant cultivation can be carried out in cultivation facilities known to those skilled in the art, such as fields, greenhouses, and hydroponic cultivation facilities, and can involve irradiation with natural or non-natural light, preferably at an appropriate temperature for each plant species.

[0038] In one embodiment of the present disclosure, there is provided a method for promoting plant growth, the method comprising the steps of preparing a crude extract of cells and / or tissues transformed with a nucleic acid comprising a nucleotide sequence encoding AOS and / or 9-LOX, mixing the crude extract with a soil composition or nutrient solution, and cultivating the plant using the soil composition or nutrient solution. The above descriptions regarding the various elements in this embodiment (plant, gene, transformation, crude extract of cells and / or tissues, soil composition or nutrient solution, plant cultivation, etc.) are applicable.

[0039] In the present disclosure, the steps of preparing a crude cell and / or tissue extract may include disrupting the cells and / or tissue to prepare disrupted cells and / or tissue, suspending the disrupted cells and / or tissue in water to prepare a suspension, and incubating the suspension. In the present disclosure, the steps of disrupting and suspending the disrupted cells and / or tissue may occur simultaneously.

[0040] The above description regarding disruption can be applied to disrupting cells and / or tissues in the step of disrupting cells and / or tissues to prepare disrupted cells and / or tissues according to the embodiment.

[0041] The step of preparing a suspension of the embodiment may include adding various solvents to a sample of crushed or uncrushed plant matter, tissues, or cells to form a suspension and extracting KODA. The above explanation regarding extraction may be applied to such extraction of KODA. The suspension before solid-liquid separation may contain solids and liquid.

[0042] The step of incubating the suspension of the embodiment may include incubating the suspension to allow KODA synthesis to continue. The above description of incubation is applicable to the incubation of the embodiment.

[0043] The step of preparing a crude cell and / or tissue extract of the embodiment may include performing solid-liquid separation of the suspension. The above description of solid-liquid separation is applicable to the solid-liquid separation of the suspension of the embodiment.

[0044] <Method for manufacturing plant growth promoter> In another aspect, there is provided a method for producing a plant growth-promoting agent, the method comprising the step of preparing a crude extract of cells and / or tissues transformed with a nucleic acid comprising a nucleotide sequence encoding AOS and / or 9-LOX, wherein the growth-promoting agent comprises the crude extract. The explanations provided in the section <Method for Promoting Plant Growth> are applicable to the various elements of this production method (e.g., plant, gene, crude cell and / or tissue extract, suspension, incubation, etc.).

[0045] In the embodiment, the method for producing a plant growth-promoting agent comprises a crude extract of cells and / or tissues transformed with a nucleic acid comprising a nucleotide sequence encoding AOS and / or 9-LOX. In the embodiment, a lysate of cells and / or tissues transformed with a nucleic acid comprising a nucleotide sequence encoding AOS and / or 9-LOX may be used as the growth-promoting agent directly, or the lysate may be further suspended in a solvent such as water and extracted to form the growth-promoting agent.

[0046] In the embodiment, the step of preparing a crude extract of cells and / or tissues in the method for producing a plant growth-promoting agent includes the steps of disrupting the cells and / or tissues to prepare disrupted cells and / or tissues, suspending the disrupted cells and / or tissues in water to prepare a suspension, and incubating the suspension. The above descriptions, including the description provided in relation to the section <Method for promoting plant growth>, are applicable to these steps.

[0047] <Nutrition composition for use in plant cultivation> In yet another aspect, there is provided a nutritional composition for use in cultivating a plant, the nutritional composition being a soil composition for use in cultivating a plant in soil culture or a nutrient solution for hydroponics, the nutritional composition comprising a crude extract of cells and / or tissues containing KODA, the nutritional composition comprising an effective amount of KODA, etc. The elements of this production method (plant, gene, nutritional composition, crude extract of cells and / or tissues, effective amount of KODA, etc.) can be applied to the explanations provided in the section entitled "Method for Promoting Plant Growth."

[0048] Nutritional compositions for use in cultivating plants according to embodiments include crude extracts of cells and / or tissues containing KODA. Such nutritional compositions can be prepared by mixing the crude extracts of cells and / or tissues containing KODA with a solid substrate such as a soil composition or a solvent such as a solution. The KODA contained in the crude extracts mixed into the nutritional compositions according to embodiments provides an effective amount of KODA.

[0049] In the nutritional compositions for use in cultivating plants of the embodiments, the crude extract of cells and / or tissues containing KODA includes crude extracts of cells and / or tissues transformed with a nucleic acid comprising a nucleotide sequence encoding AOS and / or 9-LOX.

[0050] In an embodiment of the nutritional composition for use in cultivating a plant, the nutritional composition is a soil composition for use in cultivating a plant in soil culture or a nutrient solution for hydroponic culture, and the nutritional composition comprises a crude extract of cells and / or tissues transformed with a nucleic acid comprising a nucleotide sequence encoding AOS and / or 9-LOX.

[0051] In the nutritional composition for use in cultivating plants of the embodiment, the crude extract of cells and / or tissues comprises an aqueous extract of the cells and / or tissues. The aqueous extract of the embodiment can be prepared using water as a solvent by the method described in the section "Method for promoting plant growth."

[0052] <Method for suppressing flower bud formation in plants> In one aspect of the present disclosure, a method for suppressing flower bud formation in a plant is provided. In this disclosure, suppressing flower bud formation refers to at least temporarily suppressing (e.g., delaying) flower bud formation compared to a control plant to which the method or composition of the present disclosure is not applied. At least temporarily suppressing flower bud formation can maintain, continue, or promote plant growth prior to the flower bud formation stage. The method of the embodiment can increase the biomass of agricultural crops, particularly those for which suppression of flower bud formation is desirable, and is therefore expected to be effective for leafy vegetables. The above descriptions, including those provided in the context of the method for promoting plant growth, can be applied to the various elements of this method (e.g., plant, gene, nutritional composition, crude cell and / or tissue extract, effective amount of KODA, etc.).

[0053] In one embodiment, a method for suppressing flower bud formation in a plant includes preparing a crude extract of cells and / or tissues containing KODA, mixing the crude extract with a soil composition or nutrient solution to prepare a soil composition or nutrient solution containing an effective amount of KODA, and cultivating a plant using the soil composition or nutrient solution containing the effective amount of KODA.

[0054] In an embodiment of the method for suppressing flower bud formation in a plant, the crude extract of cells and / or tissues containing KODA includes a crude extract of cells and / or tissues transformed with a nucleic acid comprising a nucleotide sequence encoding AOS and / or 9-LOX.

[0055] In one embodiment, a method for suppressing flower bud formation in a plant includes preparing a crude extract of cells and / or tissues transformed with a nucleic acid comprising a nucleotide sequence encoding AOS and / or 9-LOX, mixing the crude extract with soil or a nutrient solution, and cultivating a plant using the soil composition or nutrient solution.

[0056] In the embodiment, in the method for inhibiting flower bud formation in a plant, the step of preparing a crude extract of cells and / or tissues includes the steps of disrupting the cells and / or tissues to prepare disrupted cells and / or tissues, suspending the disrupted cells and / or tissues in water to prepare a suspension, and incubating the suspension. The above-mentioned explanations, including the explanations provided in the section <Method for promoting plant growth>, are applicable to these steps.

[0057] <Method of manufacturing plant flower bud formation inhibitor> In another aspect of the present disclosure, there is provided a method for producing a plant flowering inhibitor, the method comprising preparing a crude extract of cells and / or tissues transformed with a nucleic acid comprising a nucleotide sequence encoding AOS and / or 9-LOX, wherein the growth-promoting agent comprises the crude extract. The above descriptions, including those provided in connection with the section <Method for Promoting Plant Growth>, can be applied to various elements of this production method (e.g., plant, gene, crude cell and / or tissue extract, suspension, incubation, etc.).

[0058] In the embodiment, the step of preparing a crude extract of cells and / or tissues in the method for producing a plant flower bud formation inhibitor includes the steps of disrupting the cells and / or tissues to prepare disrupted cells and / or tissues, suspending the disrupted cells and / or tissues in water to prepare a suspension, and incubating the suspension. The above-mentioned explanations, including the explanations provided in the section <Method for promoting plant growth>, are applicable to these steps. [Example]

[0059] Examples of the present disclosure are described below, but the present disclosure is not limited to the examples described below.

[0060] Materials and Methods 1. Transformed Plants Following the method described in Ihara et al.'s paper (Ihara et al., "Developing a platform for production of the oxylipin KODA in plants," Journal of experimental botany, vol. 73(9), pp. 3044-3052, 2022), Arabidopsis transformants producing KODA (KODA-producing strains) were created by introducing mVenus-linked duckweed KODA synthesis genes (AOS and 9-LOX) and a kanamycin resistance-conferring gene. A vector control strain was also created by transformation using a control vector lacking the KODA synthesis gene.

[0061] Specifically, Arabidopsis was transformed using the inflorescence-immersion method. Arabidopsis plants grown until flower buds formed were immersed for 3 minutes in a suspension of Agrobacterium carrying the target gene. The inflorescences were then wrapped in plastic wrap and left overnight under low light. From the following day, the plants were grown under normal conditions, and seeds were harvested. The seeds were then sown on MS medium containing a drug, and the desired transformants were selected using the drug resistance marker gene on the vector.

[0062] 2. Cultivation of Transformed Plants The wild-type strain, vector control strain, and KODA-producing strain were cultivated under normal Arabidopsis growth conditions. Sterilized seeds of the wild-type strain, vector control strain, and KODA-producing strain were sown in sterilized soil (Promix:vermiculite, 1:1 (vol)) and stored at 4°C in the dark for 2–4 days for vernalization. Growth was then performed at 23°C under continuous light.

[0063] 3. Preparation of Crude Extracts from Transformed Plants Approximately 15 g of fresh plant matter was crushed into powder in a mortar under liquid nitrogen, the powder was placed in a 50 mL plastic tube, and suspended in approximately twice the amount of water. The tube was then inverted approximately 10 times to mix, and the suspension was then left to stand in an incubator at 30°C for 2 hours, resulting in a crude extract.

[0064] 4. Growth of Arabidopsis thaliana in soil compositions containing crude extracts Water was added to the crude extract to make a 300 mL volume, which was then mixed with 300 mL of a 2x concentrated nutrient solution for Arabidopsis thaliana growth. 600 mL of this mixture was mixed with 1.5 L of sterilized soil (Promix:vermiculite, 1:1 (vol)) and used as a soil composition for Arabidopsis thaliana growth. Soil compositions were prepared for each of the nutrient solution (mock), wild-type extract (WT), vector control extract (VC), and KODA-producing strain extract (KODA-producing strain). Wild-type Arabidopsis seeds were then sown in each soil, and subsequent growth was compared. After sowing, the soil was watered with only the basic nutrient solution, and no additional fertilizer with or without KODA was applied.

[0065] Results and Discussion Figure 1 shows Arabidopsis plants grown in each soil composition 30 days after sowing. Compared to soil containing a nutrient solution, no differences in growth promotion or flower bud formation were observed in Arabidopsis plants grown in soil containing a wild-type extract or a vector control strain extract. In contrast, wild-type Arabidopsis plants grown in soil containing a KODA-producing strain extract showed delayed bolting and larger plant size than those grown in other soil compositions.

[0066] The results for the model plant Arabidopsis thaliana demonstrate that plants grown in soil containing KODA-containing crude extracts exhibit enhanced growth and suppressed flower bud formation. This method is expected to be effective for leafy vegetables, as it can increase the biomass of crops where suppression of flower bud formation is particularly desirable.

[0067] Although the present disclosure has been described with reference to the above several embodiments, the present disclosure is not limited to the above several embodiments. Various changes can be made to the configuration and details of the present invention within the scope of the present disclosure.

[0068] The present disclosure includes the following embodiments. (Section 1) A method for promoting plant growth, comprising: preparing a crude extract of cells and / or tissues containing KODA; combining the crude extract with a soil composition or nutrient solution to prepare a soil composition or nutrient solution containing an effective amount of KODA; and and cultivating a plant using a soil composition or nutrient solution containing the effective amount of KODA. How to promote plant growth. (Section 2) Item 10. The method of item 1, wherein the crude extract of cells and / or tissues containing KODA comprises a crude extract of cells and / or tissues transformed with a nucleic acid comprising a nucleotide sequence encoding AOS and / or 9-LOX. (Section 3) A method for promoting plant growth, comprising: Preparing crude extracts of cells and / or tissues transformed with a nucleic acid comprising a nucleotide sequence encoding AOS and / or 9-LOX; combining the crude extract with a soil composition or nutrient solution; and and cultivating a plant using the soil composition or nutrient solution. How to promote plant growth. (Section 4) preparing a crude extract of the cells and / or tissues, disrupting the cells and / or tissues to prepare disrupted cells and / or tissues; suspending the disrupted cells and / or tissues in water to prepare a suspension; and incubating the suspension. The method according to item 3. (Section 5) A method for producing a plant growth promoter, comprising: preparing a crude extract of cells and / or tissues transformed with a nucleic acid comprising a nucleotide sequence encoding AOS and / or 9-LOX; The growth promoter comprises the crude extract Manufacturing method. (Section 6) preparing a crude extract of the cells and / or tissues, disrupting the cells and / or tissues to prepare disrupted cells and / or tissues; suspending the disrupted cells and / or tissues in water to prepare a suspension; and incubating the suspension. The method according to item 5. (Section 7) A nutritional composition for use in cultivating plants, comprising: The nutrient composition is a soil composition for use in soil cultivation of plants or a nutrient solution for hydroponics, the nutritional composition comprises a crude extract of cells and / or tissues containing KODA; The nutritional composition comprises an effective amount of KODA. Nutritional composition. (Section 8) Item 8. The nutritional composition of Item 7, wherein the cells and / or tissues containing KODA comprise cells and / or tissues transformed with a nucleic acid comprising a nucleotide sequence encoding AOS and / or 9-LOX. (Section 9) A nutritional composition for use in cultivating plants, comprising: The nutrient composition is a soil composition for use in soil cultivation of plants or a nutrient solution for hydroponics, The nutritional composition comprises a crude extract of cells and / or tissues transformed with a nucleic acid comprising a nucleotide sequence encoding AOS and / or 9-LOX. Nutritional composition. (Section 10) Item 10. The nutritional composition according to Item 9, wherein the crude extract of the cells and / or tissues is a water extract of the cells and / or tissues. (Section 11) A method for inhibiting flower bud formation in a plant, comprising: preparing a crude extract of cells and / or tissues containing KODA; combining the crude extract with a soil composition or nutrient solution to prepare a soil composition or nutrient solution containing an effective amount of KODA; and and cultivating a plant using a soil composition or nutrient solution containing the effective amount of KODA. A method for inhibiting flower bud formation in plants. (Section 12) Item 12. The method of Item 11, wherein the crude extract of cells and / or tissues containing KODA comprises a crude extract of cells and / or tissues transformed with a nucleic acid comprising a nucleotide sequence encoding AOS and / or 9-LOX. (Section 13) A method for inhibiting flower bud formation in a plant, comprising: Preparing crude extracts of cells and / or tissues transformed with a nucleic acid comprising a nucleotide sequence encoding AOS and / or 9-LOX; mixing the crude extract with soil or nutrient solution; and and cultivating a plant using the soil composition or nutrient solution. A method for inhibiting flower bud formation in plants. (Section 14) preparing a crude extract of the cells and / or tissues, disrupting the cells and / or tissues to prepare disrupted cells and / or tissues; suspending the disrupted cells and / or tissues in water to prepare a suspension; and incubating the suspension. Item 14. The method according to item 13. (Section 15) A method for producing a plant flower bud formation inhibitor, comprising: preparing a crude extract of cells and / or tissues transformed with a nucleic acid comprising a nucleotide sequence encoding AOS and / or 9-LOX; The growth promoter comprises the crude extract Manufacturing method. (Section 16) preparing a crude extract of the cells and / or tissues, disrupting the cells and / or tissues to prepare disrupted cells and / or tissues; suspending the disrupted cells and / or tissues in water to prepare a suspension; and incubating the suspension. Item 16. The method according to item 15.

Claims

1. A method for promoting plant growth, comprising: preparing a crude extract of cells and / or tissues containing KODA; combining the crude extract with a soil composition or nutrient solution to prepare a soil composition or nutrient solution containing an effective amount of KODA; and and cultivating a plant using a soil composition or nutrient solution containing the effective amount of KODA. How to promote plant growth.

2. The method of claim 1, wherein the crude extract of cells and / or tissues containing KODA comprises a crude extract of cells and / or tissues transformed with a nucleic acid comprising a nucleotide sequence encoding AOS and / or 9-LOX.

3. A method for promoting plant growth, comprising: Preparing crude extracts of cells and / or tissues transformed with a nucleic acid comprising a nucleotide sequence encoding AOS and / or 9-LOX; combining the crude extract with a soil composition or nutrient solution; and and cultivating a plant using the soil composition or nutrient solution. How to promote plant growth.

4. preparing a crude extract of the cells and / or tissues, disrupting the cells and / or tissues to prepare disrupted cells and / or tissues; suspending the disrupted cells and / or tissues in water to prepare a suspension; and incubating the suspension. The method of claim 3.

5. A method for producing a plant growth promoter, comprising: preparing crude extracts of cells and / or tissues transformed with a nucleic acid comprising a nucleotide sequence encoding AOS and / or 9-LOX; The growth promoter comprises the crude extract Manufacturing method.

6. preparing a crude extract of the cells and / or tissues, disrupting the cells and / or tissues to prepare disrupted cells and / or tissues; suspending the disrupted cells and / or tissues in water to prepare a suspension; and incubating the suspension. The method of claim 5.

7. A nutritional composition for use in cultivating plants, comprising: The nutrient composition is a soil composition for use in soil cultivation of plants or a nutrient solution for hydroponics, the nutritional composition comprises a crude extract of cells and / or tissues containing KODA; The nutritional composition comprises an effective amount of KODA. Nutritional composition.

8. The nutritional composition of claim 7, wherein the crude extract of cells and / or tissues containing KODA comprises a crude extract of cells and / or tissues transformed with a nucleic acid comprising a nucleotide sequence encoding AOS and / or 9-LOX.

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