Plant growth stimulants and methods for growing plants using plant growth stimulants

Abstract

This invention provides a plant growth aid that allows plant cells to efficiently absorb bioactive substances for plant use. [Solution] A plant growth aid comprising gelatin particles containing gelatin (A1) and a bioactive substance for plants, wherein the volume average particle diameter of the gelatin particles is 50 to 1,100 nm.

Description

[Technical Field]

[0001] This invention relates to a plant growth aid. Furthermore, this invention relates to a method for growing plants. [Background technology]

[0002] In agricultural applications, technologies are known that promote crop growth by delivering plant-derived physiologically active substances such as plant hormones, functional nucleic acids, and transformation nucleic acids to plant cells in order to stably produce crops (plants) (see, for example, Patent Document 1). However, the plant growth efficiency was not sufficient and needed improvement. [Prior art documents] [Patent Documents]

[0003] [Patent Document 1] Special Publication No. 2001-501959 [Overview of the Initiative] [Problems that the invention aims to solve]

[0004] The object of the present invention is to provide a plant growth aid that can efficiently allow plant cells to take up bioactive substances for plant use. [Means for solving the problem]

[0005] The present inventors have arrived at the present invention as a result of their research to achieve the above objective. Specifically, the present invention relates to a plant growth stimulant comprising gelatin (A1) and gelatin particles containing a bioactive substance for plants, wherein the volume average particle diameter of the gelatin particles is 50 to 1,100 nm; and a method for growing plants using the above plant growth stimulant. [Effects of the Invention]

[0006] The plant growth aid of the present invention can efficiently allow plant cells to take up bioactive substances for plants. [Modes for carrying out the invention]

[0007] The present invention relates to a plant growth stimulant comprising gelatin (A1) and gelatin particles containing a bioactive substance for plants, wherein the volume average particle diameter of the gelatin particles is 50 to 1,100 nm, and a method for growing plants using the plant growth stimulant.

[0008] The characteristics of the plant growth aid of the present invention will be described in detail below.

[0009] <Gelatin (A1)> The gelatin (A1) contained in the gelatin particles that make up the plant growth aid of the present invention may be commercially available or manufactured by known methods. Examples of methods for producing gelatin (A1) include decomposing collagen derived from pigskin or bovine bone with an acid or alkaline solution. Furthermore, gelatin (A1) may be gelatin modified with a compound having 2 moles or more amino groups per mole of compound, and it is preferable to use gelatin in which the carboxyl group of the gelatin and the compound having 2 moles or more amino groups per mole of compound are amide-bonded.

[0010] Gelatin modified with a compound having 2 moles or more amino groups per mole of the compound can be obtained, for example, by the following method.

[0011] To an aqueous solution prepared by swelling gelatin with an isoelectric point of 4-10 with water at room temperature for 10-60 minutes, and stirring at 35-45°C for 90-150 minutes, a compound having 2 or more amino groups per mole of compound is added, and the pH is adjusted to 4.5-5.2 using 1M HCl. Subsequently, a condensing agent (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride, N,N-dicyclohexylcarbodiimide, N,N'-diisopropylcarbodiimide, or ions with two or more valents (such as chlorides of calcium, zinc, copper, etc. In this case, gelatin and a compound having two or more amino groups per mole of compound are ionically bonded) is added, and the mixture is stirred at 35-45°C for 10-24 hours. After that, by dialysis in water and freeze-drying for 3-5 days, gelatin modified with a compound having two or more amino groups per mole of compound can be obtained.

[0012] Compounds containing 2 or more amino groups per mole include compounds containing 2 moles of amino groups per mole (compounds with 2 to 12 carbon atoms, specifically ethylenediamine and putrescine), compounds containing 3 moles of amino groups per mole (compounds with 2 to 12 carbon atoms, specifically spermidine), and compounds containing 4 or more amino groups per mole (compounds with 2 to 12 carbon atoms, specifically spermine).

[0013] In the reaction described above, the number of moles of the compound having 2 or more amino groups per mole of the compound is preferably 1 to 75 times the number of moles of carboxyl groups present in the gelatin before the reaction. Furthermore, in the above reaction, the number of moles of the condensing agent is preferably 2 to 5 times the number of moles of carboxyl groups present in the gelatin before the reaction.

[0014] Furthermore, gelatin (A1) may be gelatin modified with a compound having 2 moles or more carboxyl groups per mole of the compound, and it is preferable to use gelatin in which the amino group of the gelatin and the compound having 2 moles or more carboxyl groups per mole of the compound are amide-bonded.

[0015] Gelatin modified with a compound having 2 moles or more carboxyl groups per mole of the compound can be obtained, for example, by the following method.

[0016] Gelatin with an isoelectric point of 4 to 10 is swollen at room temperature for 10 to 60 minutes using dimethyl sulfoxide, and stirred at 35 to 45 °C for 1 to 2 days. A solution in which a compound having 2 or more moles of carboxy groups per mole of compound 1 is dissolved in dimethyl sulfoxide is added to the aqueous solution thus prepared, and the pH is adjusted to 4.5 to 5.2 using 1M HCl. Thereafter, it is stirred at 35 to 45 °C for 0.5 to 3 hours. Thereafter, dialysis against water for 3 to 5 days and freeze-drying are performed to obtain gelatin modified with a compound having 2 or more moles of carboxy groups per mole of compound 1.

[0017] Examples of the compound having 2 or more moles of carboxy groups per mole of compound 1 include a compound having 2 moles of carboxy groups per mole of compound 1 (a compound having 2 to 12 carbon atoms. Specifically, succinic acid, glutaric acid, etc.), a compound having 3 moles of carboxy groups per mole of compound 1 (a compound having 2 to 12 carbon atoms. Specifically, propane tricarboxylic acid, etc.), a compound having 4 or more moles of carboxy groups per mole of compound 1 (a compound having 2 to 12 carbon atoms. Specifically, butane tetracarboxylic acid, etc.), and the like.

[0018] In the above-described reaction, the number of moles of the compound having 2 or more moles of carboxy groups per mole of compound 1 is preferably an amount that is 1 to 75 times based on the number of moles of amino groups possessed by the gelatin before the reaction.

[0019] Gelatin (A1) (particularly, gelatin modified with a compound having 2 or more moles of amino groups per mole of compound 1) preferably has amino groups from the viewpoints of interaction with cell membranes and growth promotion. The ratio of amino groups is preferably 30 to 140 mol / mol, more preferably 60 to 90 mol / mol, based on the number of moles of gelatin. In this specification, the "number of moles of gelatin" means a value obtained by dividing the weight of gelatin by the weight-average molecular weight obtained by the method for measuring the weight-average molecular weight of gelatin described below.

[0020] In the present invention, the weight-average molecular weight of gelatin (A1) is preferably 80,000 to 120,000. When the weight-average molecular weight is 120,000 or less, the efficiency of delivery into plants tends to improve. Furthermore, when the weight-average molecular weight is 80,000 or higher, there is a tendency for the gelatin particles to more easily retain bioactive substances for plants. Furthermore, from the viewpoint of decomposition rate, the weight-average molecular weight of gelatin (A1) is more preferably between 90,000 and 110,000.

[0021] The weight-average molecular weight of gelatin (A1) can be measured using gel permeation chromatography (GPC) under conditions such as the following. <Measurement conditions> Device: "HLC-8120GPC" [Manufactured by Tosoh Corporation] Column: "Guardcolumn H XL -H (1 unit) [Manufactured by Tosoh Corporation], "Shodex OHpak SB-806M HQ (8.0mm I.D. x 300mm)" (2 units) [Manufactured by Shodex Corporation] Sample solution: 0.1% by weight gelatin aqueous solution Solution injection volume: 100μL Flow rate: 1mL / min Measurement temperature: 40℃ Detection device: Refractive index detector Reference substance: pullulan

[0022] <Cross-linked gelatin (A2)> In the plant growth aid of the present invention, the gelatin particles may include cross-linked gelatin (A2) in which gelatin (A1) particles are cross-linked with each other. Crosslinked gelatin (A2) may have a crosslinked structure formed by crosslinking via amino groups and / or carboxyl groups of gelatin (A1).

[0023] One example of a method for obtaining cross-linked gelatin (A2) by cross-linking gelatin (A1) is the following method (a method for obtaining it as particles containing cross-linked gelatin (A2) as described below).

[0024] To obtain cross-linked gelatin (A2), first, a poor solvent (such as alcohol, amine, or acetone) is added dropwise to an aqueous solution containing gelatin (A1). Preferably, gelatin (A1) is gelatin modified with a compound having 2 moles or more amino groups per mole of the compound. Subsequently, an aqueous solution containing a crosslinking agent (a compound having 2 moles or more of groups that bind to the groups of gelatin per mole of the compound; specifically, glutaraldehyde, etc.) is added, and the mixture is stirred at 35-45°C for 6-12 hours to obtain crosslinked gelatin. It is preferable to add the crosslinking agent in an amount of 0.00664-0.16 mmol per mole of gelatin. Subsequently, to optionally block unreacted aldehyde groups, a solution of a neutral compound (such as glycine) with a chemical formula weight of 50 to 200 amino groups may be added, and the mixture may be stirred at 18 to 25°C for 0.5 to 2 hours. Preferably, the amount of neutral compound added is 0.125 to 3.01 mmol per mole of gelatin. Furthermore, if necessary, aggregates may be removed using a 40 μm cell strainer or the like, and the desired volume-average particle size may be adjusted by repeating centrifugation. Methods of centrifugation include centrifuging at 15,000 to 180,000 rpm for 5 to 15 minutes and then removing the supernatant.

[0025] The weight-average molecular weight of the cross-linked gelatin (A2) in this invention is 1.5 × 10⁻⁶. 8 ~2.0×10 12 It is preferable that this be the case. The weight-average molecular weight of cross-linked gelatin (A2) is 2.0 × 10⁻⁶. 12 The following conditions tend to improve delivery efficiency into plants. Furthermore, the weight-average molecular weight of cross-linked gelatin (A2) is 1.5 × 10⁻⁶. 8 The above conditions tend to make it easier to retain bioactive substances for plants within gelatin particles. The weight-average molecular weight of cross-linked gelatin (A2) can be measured using the same method as described above for measuring the weight-average molecular weight of gelatin (A1).

[0026] <Physiologically active substances for plants> The plant bioactive substance contained in the gelatin particles constituting the plant growth aid of the present invention preferably includes at least one selected from the group consisting of plant growth hormones, functional nucleic acids, and transformation nucleic acids.

[0027] <Plant hormones> Preferred plant hormones include PSK, CLV3, TDIF, RGF, IDA, CLE25, CLE40, FON2, LjCLE-RS1, PSY1, CEP1, NtHypSys I, EPF1, LURE, stomagen, RALF, SCR / SP11, systemin, AtPep1, POLARIS, ROT4, etc. It is even more preferable that the plant hormone has the amino acid sequence shown in SEQ ID NO: 1, the amino acid sequence shown in SEQ ID NO: 2, or an amino acid sequence with 80% or more homology to these amino acid sequences. When using, for example, a peptide having the amino acid sequence shown in SEQ ID NO: 1 or a peptide (CLE25) having the amino acid sequence shown in SEQ ID NO: 2 as a plant hormone, it is preferable that the gelatin (A1) is a gelatin in which the carboxyl group of the aforementioned gelatin is amide-bonded to a compound having 2 moles or more amino groups per mole of the compound.

[0028] The molecular weight of plant growth hormones determined by SDS-PAGE (SDS-polyacrylamide gel electrophoresis) is preferably between 1,000 and 30,000, from the viewpoint of delivery efficiency into plants.

[0029] <Functional nucleic acid> Examples of functional nucleic acids include functional DNA and functional RNA. Functional RNA may also be an RNA interference (RNAi) molecule. RNA interference (RNAi) molecules include RNA silencing molecules targeting target RNA in plant cells, small interfering RNAs (siRNAs), short hairpin RNAs (shRNAs), microRNAs (miRNAs), Piwi-interacting RNAs (piRNAs), and trans-acting siRNAs (tasiRNAs).

[0030] From the viewpoint of delivery efficiency into plants, the number of base pairs in functional nucleic acids is preferably 10 to 100 bp.

[0031] <Nucleic acids for transformation> DNA and RNA can be used as nucleic acids for transformation. The nucleic acid used for transformation may be an exogenous protein gene, an agricultural gene, a marker gene, etc. Furthermore, the nucleic acid used for transformation may be a nucleic acid for expressing a specific protein, a nucleic acid for male sterility, a nucleic acid for conferring herbicide resistance, a nucleic acid for conferring insect damage resistance, a nucleic acid for conferring resistance to bacterial diseases, a nucleic acid for conferring resistance to fungal diseases, a nucleic acid for conferring resistance to viral diseases, etc.

[0032] From the viewpoint of delivery efficiency into the plant, the number of base pairs in the nucleic acid for transformation is preferably 10 to 100 bp.

[0033] When using functional nucleic acids and transforming nucleic acids, gelatin (A1) is preferably gelatin in which a carboxyl group of gelatin is amide-bonded to a compound having 2 moles or more amino groups per mole of the compound.

[0034] <Gelatin particles> The gelatin particles constituting the plant growth aid of the present invention are particles with a volume-average particle diameter of 50 to 1,100 nm. If the volume-average particle size of the gelatin particles is less than 50 nm, the recovery efficiency of the gelatin particles and the encapsulation efficiency of bioactive substances for plants will deteriorate. When the volume-average particle size of gelatin particles exceeds 1,100 nm, the efficiency of delivery into the plant body deteriorates. Furthermore, from the viewpoint of delivery efficiency into plants, the volume-average particle diameter of the gelatin particles is preferably 50 to 1,000 nm, and more preferably 50 to 600 nm. The volume-average particle size of gelatin particles can be measured by dynamic light scattering (DLS). Methods for adjusting the volume-average particle size of gelatin particles include, as mentioned above, removing aggregates using a cell strainer or the like, and adjusting to the desired volume-average particle size by repeatedly centrifuging.

[0035] From the viewpoint of promoting plant growth, the proportion of bioactive substances for plants contained in gelatin particles is preferably 0.6 to 36 pmol / μg, and more preferably 5.5 to 17 pmol / μg, based on the weight of the gelatin particles.

[0036] The zeta potential of gelatin particles is preferably +0.1 to +6.0 mV, from the viewpoint of interaction with the cell membrane and promotion of plant growth. The zeta potential of gelatin particles can be adjusted (please instruct us on how to adjust the zeta potential) by, for example, modifying the gelatin particles with a compound to which a charged functional group is attached (such as a compound having 2 moles or more amino groups per mole of the above compound, or a compound having 2 moles or more carboxyl groups per mole of the compound). The zeta potential can be measured using a Zetasizer Ultra Red Label (manufactured by Malvern Panlytical) with a solution containing gelatin particles (for example, the plant growth stimulant of the present invention) diluted with phosphate buffer (PB solution) to a particle concentration of 10 μg / mL.

[0037] Gelatin particles can be obtained, for example, by the following method. Gelatin particles according to the present invention are obtained by adding a dispersion of gelatin (A1) and / or cross-linked gelatin (A2) to an aqueous solution of a bioactive substance for plants and allowing it to impregnate for 5 to 30 minutes. If necessary, centrifugation may then be performed to remove the supernatant and replace it with phosphate-buffered saline (PBS). Centrifugation is preferably carried out at, for example, 15,000 to 18,000 rpm for 20 to 60 minutes. Furthermore, the zeta potential of the gelatin (A1) and / or cross-linked gelatin (A2) used in the above-described process is preferably +0.1 to +10 mV, and more preferably +0.1 to +6.0 mV. The zeta potential can be measured using the same method as the method for measuring the zeta potential of gelatin particles described above.

[0038] <Other Ingredients> The plant growth aid of the present invention comprises gelatin (A1) and gelatin particles containing a bioactive substance for plants, wherein the volume average particle diameter of the gelatin particles is 50 to 1,100 nm. The plant growth aid of the present invention may contain, in addition to gelatin particles, a spreading agent (nonionic surfactant, anionic surfactant, cationic surfactant, silicone-based spreading agent, etc.) and water. The spreading agent may be a compound with a molecular weight of 2,000 or more (preferably 2,000 to 80,000).

[0039] Next, a method for growing plants using the plant growth aid of the present invention will be described. In the plant cultivation method of the present invention, the plant growth stimulant is preferably used by spraying or applying it to the plant, more preferably by spraying or applying it to the leaves, and particularly preferably by spraying it to the leaves of the plant. The plant growth stimulant of the present invention is suitably used by foliar spraying or application, and is suitably used by foliar spraying. When applying the plant growth stimulant of the present invention to a plant, it is preferable to use the plant growth stimulant in the form of an aqueous dispersion. Furthermore, when applying to leaves, the plant growth stimulant of the present invention may be applied to a portion of the leaves of the plant, or to all of the leaves. When the plant growth stimulant of the present invention is an aqueous dispersion, from the viewpoint of sprayability and plant growth, the weight percentage of gelatin particles is preferably 0.001 to 15% by weight, more preferably 0.001 to 10% by weight, based on the weight of the aqueous dispersion [plant growth stimulant]. Furthermore, when applied to leaves by spraying, the amount of the plant growth stimulant of the present invention used per leaf area (preferably sprayed) is preferably 0.001 to 200 g / m² as the amount of gelatin particles used (sprayed). 2 Comfortable 0.001~100g / m 2 That is the case. Foliar spraying or application may be performed on either the upper or lower surface of the leaf, or on both sides.

[0040] The plant growth stimulant of the present invention can efficiently allow plants to take up bioactive substances for plants, has appropriate elution properties of bioactive substances for plants from gelatin particles, exhibits excellent sustained release of bioactive substances for plants, and has excellent sustained efficacy. Therefore, it is effective in promoting plant growth and is particularly useful for agricultural applications. Furthermore, although it is a hypothesis, it is thought that the plant growth stimulant is effective in promoting growth because it is taken up by plants through pores and exerts the above-mentioned effects. The plant growth method of the present invention can be used as a method for promoting plant growth.

[0041] Furthermore, the following mechanism is hypothesized for the delivery of gelatin particles to plants. It is assumed that a complex of gelatin fragments from the breakdown of gelatin particles and plant-derived bioactive substances, or the particles themselves, are delivered to the surface or inside plant cells, and that the plant-derived bioactive substances contained in the gelatin particles exert their effects.

[0042] Plants that have leaves are preferred as the plants to which the plant growth stimulant of the present invention is used. Examples of plants include rice, wheat (barley, wheat, rye, oats, etc.), fruit vegetables, leafy vegetables, root vegetables, flowers, and roses. Examples of fruit vegetables include tomatoes, bell peppers, peas, cucumbers, watermelons, edamame, melons, strawberries, okra, eggplants, green beans, pumpkins, broad beans, and corn. Examples of leafy vegetables include butterbur, green onions, Japanese ginger, garlic, lettuce, shallots, broccoli, cabbage, perilla, Chinese cabbage, bok choy, Japanese parsley, Japanese angelica tree, spinach, pickled mustard greens, cauliflower, lettuce, Brussels sprouts, asparagus, Japanese parsley, onions, parsley, chives, garland chrysanthemum, and celery. Root vegetables include radishes, turnips, burdock, carrots, potatoes, taro, sweet potatoes, yams, ginger, and lotus root. Roses include Arabidopsis thaliana. In particular, cruciferous crops such as rice, tomatoes, watermelons, cucumbers, strawberries, Chinese cabbage, and Arabidopsis thaliana are preferred.

[0043] The plant growth stimulant of the present invention is particularly preferable to be used during the seedling stage. When using the plant growth stimulant during the seedling stage, the seedling sheet material described below may be used as needed. A seedling sheet having the seedling sheet material and the seedling sheet base material can also be used. The seedling sheet can be used by placing it on the surface of the soil or in the soil. The seedling sheet material can be used by placing it on the surface of the soil or in the soil, or by mixing it in. A method of growing plants using a plant growth stimulant, the seedling sheet material, and / or the seedling sheet during the seedling stage is one of the preferred embodiments of the present invention. The plant growth method of the present invention is suitably used as a plant seedling method. The plants are preferably the plants described above.

[0044] Seedling cultivation includes sowing seeds, germination, and greening, and may also include hardening after greening. In the present invention, it is preferable to apply (preferably spray) the plant growth stimulant to the leaves of the seedlings during the greening period. When hardening is performed, the plant growth stimulant may be applied to the leaves during the greening and / or hardening period, and it is preferable to apply it to the leaves during the greening period. The following are examples of preferred embodiments when using the plant growth method of the present invention during the seedling cultivation period. Seeds are sown in the soil on which the seedling sheet material and / or seedling sheet is installed, germination is allowed, and the seedlings are greened. During the greening period, a plant growth stimulant is applied to the leaves of the seedlings. The application of the plant growth stimulant to the leaves (preferably by foliar spraying) may be done on either the upper or lower side of the leaf, or on both sides of the leaf. The seedling sheet material and / or seedling sheet can be installed in seedling trays or the like, and it is preferable to place soil (soil) on top of the seedling sheet material and / or seedling sheet and sow the seeds. Seeds of the above-mentioned plants are preferred. Before sowing, preliminary measures may be taken for the seeds (such as seed selection, soaking, and germination). The conditions for the soil, seed sowing, germination, greening, and hardening should be selected or adjusted according to the type of plant. After hardening, it is preferable to transplant the seedlings to the main paddy field or field.

[0045] The following explanation uses rice seeds as the seed for seedling cultivation as an example. Normally, seedlings planted by a rice transplanter are grown in a bed called a seedling bed, but the following seedling cultivation process is preferable.

[0046] 1) Preliminary measures for seeds 1-1) Seed selection: After disinfection, remove floating grains by salt water selection, then wash with water and dry. 1-2) Soaking the seeds: Soak the seeds for 5 days to ensure even water absorption. During this time, the water is changed daily, and oxygen is supplied by draining the water. 1-3) Germination: After supplying oxygen, soak the seeds in 32°C warm water for 10 hours until they reach a pigeon-chest state.

[0047] 2) Preparation of the soil Select soil with a granular structure that has good aeration and drainage, and adjust the pH to 5. Mix in base fertilizer.

[0048] 3) Adding soil Place the soil in the seedling bed, compact it, and make it level.

[0049] 4) Seeding Spread the seeds evenly in the seedbed and water them to allow the seeds to settle. Fix any unevenness in the grain.

[0050] 5) Covering with soil Add soil to a thickness of 5 mm and level it to make a flat surface.

[0051] 6) Equalizing seedling emergence using a seedling incubator Leave it undisturbed at 32°C for two days, and cultivate it until the sheath leaves are about 1.2 cm long.

[0052] 7) Preliminary greening After supplying oxygen and watering, expose the seedlings to sunlight in the nursery bed and manage them at 25°C for one day and 20°C for one day.

[0053] 8) Greening In a greenhouse, maintain a daytime temperature of 30°C and a nighttime temperature of 12°C for 8 days, watering thoroughly several times a day until the plants develop 2.5 leaves.

[0054] 9) Hardening Under controlled conditions of 20°C during the day and 10°C at night, the seedlings are gradually grown to a 3.5-leaf stage, adapting to natural conditions over 10 days.

[0055] The seedling sheet is preferably placed inside the seedling bed before "3) Soil filling". The location and method of placement are optional, but it is preferable to place it at the bottom.

[0056] Plant growth stimulants are more preferably applied during the "8) Greening" period, but may also be applied during the "9) Hardening" period.

[0057] <Materials for seedling cultivation sheets> The seedling sheet material preferably contains a water-absorbing polymer material (D), and may also contain fertilizer (E) as needed. Furthermore, it is preferable that the seedling sheet material contains a filler (F).

[0058] The water-absorbing polymer material (D) can be a water-absorbing resin or the like, and is not particularly limited, but it is preferably a hydrophilic crosslinked polymer containing a carboxyl group, and polyacrylic acid (salt) is more preferred. Furthermore, it is not limited to a form in which the entire amount (100% by weight) is polymer. Polyacrylic acid (salt) refers to a polymer whose main component is acrylic acid (salt) as a repeating unit. Specifically, it refers to a polymer that contains, as monomers excluding the crosslinking agent, preferably 50 to 100 mol%, more preferably 70 to 100 mol%, even more preferably 90 to 100 mol%, and particularly preferably substantially 100 mol%, of acrylic acid (salt). The salt used as the polymer is preferably an alkali metal salt, an alkaline earth metal salt, or an ammonium salt, with monovalent salts and alkali metal salts being preferred among these, and potassium salts and sodium salts being particularly preferred. Furthermore, the shape of the polyacrylic acid (salt) is not particularly limited, but it is preferably in particulate or powder form.

[0059] The water absorption ratio of ion-exchanged water at 25°C to the superabsorbent polymer material (D) is generally 80 to 1000 times, preferably 90 to 670 times, more preferably 120 to 530 times, and even more preferably 130 to 480 times. If the water absorption ratio is less than 80 times, the water retention capacity of the water-retaining agent will be low, requiring the use of large quantities, which may increase costs or necessitate frequent water replenishment. A higher water absorption ratio is preferable because it allows for the use of smaller quantities, but superabsorbent polymer materials with a water absorption ratio exceeding 1000 times have the problem of low water permeability, which can negatively affect vegetation. The water absorption ratio is measured using the following method.

[0060] [Method for measuring the water absorption ratio of ion-exchanged water] A sample L (g) of the superabsorbent polymer material (D) is placed in a nylon mesh bag (250 mesh), and the bag is immersed in an excess of deionized water. After 60 minutes of immersion, the bag is lifted into the air and left to stand for 15 minutes to drain the water. The weight M (g) is then measured, and the water absorption ratio is calculated using the following formula. Perform the same procedure as above using only the mesh bag, and subtract the weight N(g) of this bag as a blank. Water absorption ratio of deionized water = (MN) / L

[0061] From the viewpoint of vegetation, the pH value of the absorbent material when 100 parts by weight of ion-exchanged water at 25°C is absorbed by 1 part by weight of the superabsorbent polymer material (D) is preferably 4.5 to 7.5, and more preferably 5.0 to 7.0. The pH value is measured using the following method.

[0062] [Method for measuring pH value] Add 1 part by weight of superabsorbent polymer material (D) to 100 parts by weight of ion-exchanged water at 25°C, and leave in a constant temperature bath at 25°C for 8 hours to swell the superabsorbent polymer material and create an absorbent body. Confirm that the temperature of the absorbent body is 25°C with a thermometer, insert a pH meter into the absorbent body, and after confirming that the pH value has stabilized, read the value. If the water absorption ratio of the superabsorbent polymer material is small, the absorbent body of the superabsorbent polymer material and the ion-exchanged water will separate into two phases. In this case, stir to homogenize, then insert a pH meter and measure the value. If the two phases separate again immediately after stirring and homogenization, insert a pH meter while stirring and measure the value.

[0063] Examples of fertilizers (E) include ordinary fertilizers such as nitrogenous fertilizers, phosphate fertilizers, potassium fertilizers, organic fertilizers, compound fertilizers, calcareous fertilizers, silicate fertilizers, magnesium fertilizers, manganese fertilizers, boron fertilizers, and trace element compound fertilizers, as well as other special fertilizers (such as slow-release fertilizers). These fertilizer components are in liquid or solid form such as powder, and can be added to the water-absorbing polymer material (D) or contained in the water used to soak the water-absorbing polymer material (D) to create the seedling sheet material or to be present in the seedling sheet.

[0064] The addition amount of the fertilizer (E) can be arbitrarily determined in consideration of the type of crop to be cultivated, the type of fertilizer to be used, etc. However, per unit area (m 2 ) of the seedling-raising sheet, for example, it is 1 to 500 g, preferably 3 to 300 g. In the case of the material for the seedling-raising sheet, it is preferable to blend the fertilizer (E) into the material for the seedling-raising sheet so that the usage amount of the fertilizer (E) per unit area (m 2 ) is within the above range.

[0065] The filler (F) is preferably a powdery, particulate, fibrous or cottony filler. The filler (F) is preferably one having appropriate air permeability so as not to inhibit the germination and growth of seeds, one that does not adversely affect the soil when attached to the ground, and / or one having a property of being easily decomposed on the soil surface or inside the soil, and examples thereof include those described below. For example, inorganic porous materials such as perlite, vermiculite, and rock fiber, wood chips, rice husks, buckwheat husks, rice bran, cotton, straw, peat, wool, sawdust, pulp, and paper scraps.

[0066] The addition amount of the filler (F) is preferably 1 to 500 g, more preferably 3 to 300 g, per unit area (m 2 ) of the seedling-raising sheet in order to ensure air permeability and thickness. In the case of the material for the seedling-raising sheet, it is preferable to blend the filler (F) into the material for the seedling-raising sheet so that the usage amount of the filler (F) per unit area (m 2 ) is within the above range.

[0067] <Seedling-raising sheet The seedling-raising sheet is a seedling-raising sheet having the above-mentioned material for the seedling-raising sheet and a seedling-raising sheet base material. Examples of the seedling-raising sheet base material include a sheet (G).

[0068] Sheet (G) may be, for example, a permeable sheet, a water-disintegrating sheet, or a water-soluble sheet, and may be a combination of two or more of these types layered together. Preferably, sheet (G) has a thickness of 0.01 to 9 mm after being formed into a seedling sheet, and more preferably 0.02 to 3 mm. The weight of sheet (G) is such that, in order to ensure the shape retention and thickness of the seedling sheet, it is such that, for example, the weight of the sheet is equal to the unit area (m²) of the seedling sheet. 2 The amount per serving is preferably 5 to 300 g, more preferably 10 to 100 g.

[0069] Examples of permeable sheets include woven or nonwoven fabrics (cloths) of cellulose fibers, paper, woven or knitted fabrics or films of water-soluble polyvinyl alcohol fibers, and cardboard. Among these, it is preferable that the degree of permeability is such that the water absorption rate is 5 minutes or less according to Method A of water absorption rate described in JIS L 1096. Furthermore, it is preferable that the permeable sheet has adequate breathability so as not to inhibit seed germination and growth. It is also preferable that the permeable sheet has properties that make it easily decomposed on or within the soil surface when it is attached to the ground. From this perspective, permeable sheets are preferably made of cellulose-based paper or nonwoven fabric.

[0070] Examples of water-disintegrating sheets include paper in which pulp fibers are bonded together with a water-soluble or hydrophilic adhesive, a water-swellable polymer, etc., so that the pulp fibers disintegrate upon contact with water (such as "Dissolvo MDP" manufactured by Mishima Paper Co., Ltd.), and paper in which a heat sealant is used in combination with this to improve moldability (heat adhesion) (such as "Dissolvo MDP-P" manufactured by Mishima Paper Co., Ltd.). These papers are characterized by their rapid disintegration speed upon water absorption.

[0071] Examples of water-soluble sheets include water-soluble polyvinyl alcohol (PVA) films, starch films, carrageenan films, and water-soluble nonwoven fabrics made from PVA fibers (such as "Ecomold" and "Ecosolve" manufactured by Nippon Vilene Co., Ltd.). When compared at the same thickness, these sheets have a lower rate of dissolution (decomposition) in water than the water-disintegrating paper mentioned above, but they have the advantage of greater sheet strength in a dry state.

[0072] As a combination of two or more types of sheets, such as a water-permeable sheet, a water-disintegrating sheet, or a water-soluble sheet, a laminated sheet made by bonding water-disintegrating paper and a water-soluble film can be used. Examples of laminate sheets made by bonding water-disintegrating paper and a water-soluble film include at least one type of bonded and laminated water-disintegrating paper and a water-soluble film (such as "Dissolvo A" manufactured by Mishima Paper Co., Ltd., which is made by bonding a polyval film to "Dissolvo MDP"). These laminate sheets are characterized by their rapid dissolution (disintegration) in water and high film strength. This is because the thickness of the water-soluble film to be bonded can be reduced by the strength of the paper, thus improving both the dissolution (disintegration) speed and film strength overall. Among these water-soluble or water-disintegrating sheets, water-disintegrating paper and water-soluble nonwoven fabrics are preferred. Furthermore, the time required for these water-soluble or water-disintegrating sheets to disintegrate or dissolve in water is, for example, within 5 minutes, preferably within 2 minutes, and more preferably within 1 minute.

[0073] The seedling sheet is preferably a sheet containing a water-absorbent polymer material (D), and more preferably a seedling sheet comprising a water-absorbent polymer material (D) and a sheet (G), wherein the water-absorbent polymer material (D) is present on or inside at least one sheet (G). In this seedling sheet, fertilizer (E) and filler (F) can be present in at least one sheet (G).

[0074] The seedling sheet comprises a superabsorbent polymer material (D), at least one sheet (G), and fertilizer (E) and / or filler (F), and the sheet may be such that the superabsorbent polymer material (D) and fertilizer (E) and / or filler (F) are present on or inside at least one sheet (G). When using two or more sheets (G) as seedling trays, it is sufficient that the superabsorbent polymer material (D) is present on the surface or inside at least one sheet (G) overall.

[0075] Examples of seedling sheets using two or more sheets (G) include, for example, a five-layer structure in which a layer of sheet (G), a layer of filler (F) and water-absorbing polymer material (D), a layer of sheet (G), a layer of fertilizer (E), and a layer of sheet (G) are stacked in that order, and a four-layer structure in which two layers of sheet (G), a layer of water-absorbing polymer material (D), a layer of mixed fertilizer (E) and filler (F), and a layer of sheet (G) are stacked in that order.

[0076] Methods for manufacturing seedling sheets include known methods such as immersing a sheet (G) in a mixture of a water-absorbing polymer material (D), fertilizer (E), and filler (F), or applying the mixture to the surface of the sheet (G).

[0077] When using two sheets (G), in addition to the method of stacking two sheets prepared in the same way as described above, there are two other methods, for example: (a) A method of uniformly spreading a mixture of a water-absorbent polymer material (D), fertilizer (E), and filler (F) onto one sheet (G), then placing the other sheet (G) on top, and further applying pressure molding such as embossing. (b) A method of coating one sheet (G) with a mixture of a water-absorbing polymer material (D), fertilizer (E), and filler (F) added to a suitable binder (H) as described below, then overlapping the other sheet (G) and processing and molding it, followed by drying.

[0078] Examples of binders (H) used to fix the superabsorbent polymer material (D), fertilizer (E), and filler (F) onto the seedling sheet include natural polymers, synthetic resins, and natural or synthetic rubber. Examples of natural polymers include starch, carboxymethylcellulose, methylcellulose, ethylcellulose, hydroxymethylcellulose, hydroxyethylcellulose, sodium alginate, guar gum, xanthan gum, bean gum, carrageenan, and gluten. Examples of synthetic resins include acrylic resins other than superabsorbent polymer materials (D), polyurethane resins, unsaturated polyester resins, polyamide resins, and ethylene copolymer resins. Examples of natural or synthetic rubbers include natural rubber, acrylic rubber, butyl rubber, polyisobutylene rubber, styrene-butadiene rubber, ethylene-propylene rubber, and chloroprene rubber. These can be used individually or in combination of two or more. Preferred among these are water-soluble natural polymers such as starch, carboxymethylcellulose, and sodium alginate.

[0079] This specification contains the following information:

[0080] (1) of this disclosure is a plant growth aid comprising gelatin (A1) and gelatin particles containing a bioactive substance for plants, wherein the volume average particle size of the gelatin particles is 50 to 1,100 nm.

[0081] Disclosure (2) is a plant growth aid according to Disclosure (1), wherein the gelatin (A1) has an amide bond formed by bonding a carboxyl group to a compound having 2 moles or more amino groups per mole of the compound, and the zeta potential of the gelatin particles is +0.1 to +6.0 mV.

[0082] Disclosure (3) is a plant growth aid according to Disclosure (1) or (2), wherein the gelatin particles include cross-linked gelatin (A2) formed by cross-linking the gelatin (A1) particles together.

[0083] Disclosure (4) is a plant growth aid according to any one of Disclosures (1) to (3), wherein the above-mentioned bioactive substance for plants comprises at least one selected from the group consisting of plant growth hormones, functional nucleic acids, and transformation nucleic acids.

[0084] This disclosure (5) is a plant growth aid according to this disclosure (4), wherein the plant growth hormone is a peptide and the molecular weight of the plant growth hormone determined by SDS-PAGE (SDS polyacrylamide gel electrophoresis) is 1,000 to 30,000.

[0085] This disclosure (6) is a plant growth aid according to this disclosure (5), wherein the plant growth hormone is a peptide having the amino acid sequence shown in SEQ ID NO: 1, the amino acid sequence shown in SEQ ID NO: 2, or an amino acid sequence having 80% or more homology to these amino acid sequences.

[0086] This disclosure (7) is a plant growth aid described in this disclosure (4), wherein the functional nucleic acid is an RNA interference (RNAi) molecule.

[0087] The present disclosure (8) is a plant growth aid according to the present disclosure (7), wherein the RNA interference (RNAi) molecule is at least one selected from the group consisting of RNA silencing molecules, small interfering RNA (siRNA), short hairpin RNA (shRNA), microRNA (miRNA), Piwi-interacting RNA (piRNA), and trans-acting siRNA (tasiRNA) that target RNA in plant cells.

[0088] This disclosure (9) is a plant growth aid according to this disclosure (4), wherein the transforming nucleic acid is at least one selected from the group consisting of foreign protein genes, agricultural genes, and marker genes.

[0089] The present disclosure (10) is a plant growth aid according to the present disclosure (4), wherein the transforming nucleic acid is at least one selected from the group consisting of nucleic acids for expressing a predetermined protein, nucleic acids for male sterility, nucleic acids for conferring herbicide resistance, nucleic acids for conferring insect pest resistance, nucleic acids for conferring resistance to bacterial diseases, nucleic acids for conferring resistance to fungal diseases, and nucleic acids for conferring resistance to viral diseases.

[0090] Disclosure (11) is a method for growing plants using a plant growth stimulant described in any of Disclosures (1) to (10). [Examples]

[0091] The present invention will be further described below with reference to examples and comparative examples, but the present invention is not limited thereto.

[0092] <Production Examples 1A-32A and Comparative Production Examples 1A-2A: Preparation of Amine-Modified Gelatin or Carboxylic Acid-Modified Gelatin> Two g of the raw gelatin listed in Tables 1-1 to 1-5 was dissolved in 50 ml of milli-Q water. Then, the amines or carboxylic acids of the types listed in Tables 1-1 to 1-5 were added in the amounts listed in Tables 1-1 to 1-5, and the pH of each sample was adjusted to 5 using hydrochloric acid. When an amine was added as a raw material, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (molecular weight 191.7) was then added in a molar amount 50 times the amount of carboxyl groups present in the gelatin. This mixed solution was stirred at 37°C for 12 hours. After the reaction was complete, amine-modified gelatin or carboxylic acid-modified gelatin according to Production Examples 1A to 32A and Comparative Production Examples 1A to 2A were obtained by dialysis in water and freeze-drying for 3 days.

[0093] The gelatin used as a raw material, as listed in Tables 1-1 to 1-5, was the following: PI9: Gelatin with an isoelectric point of 9.0, weight-average molecular weight: 100,000, carboxyl group content per mole of gelatin: 99 mol / mol, amino group content per mole of gelatin: 33 mol / mol, manufactured by Nitta Gelatin Co., Ltd. PI5: Gelatin with an isoelectric point of 5.0, weight-average molecular weight: 99000, carboxyl group content per mole of gelatin: 120 mol / mol, amino group content per mole of gelatin: 32 mol / mol, manufactured by Nitta Gelatin Co., Ltd. Fish gelatin: Manufactured by Nitta Gelatin Co., Ltd., weight-average molecular weight: 300,000, carboxyl group content per mole of gelatin: 100 mol / mol, amino group content per mole of gelatin: 33 mol / mol Food-grade gelatin: Manufactured by MP Biomedicals, Inc., weight-average molecular weight: 100,000, carboxyl group content per mole of gelatin: 119.9 mol / mol, amino group content per mole of gelatin: 30.6 mol / mol

[0094] Furthermore, the introduction rates of amino groups and carboxyl groups in the amine-modified gelatin or carboxylic acid-modified gelatin for production examples 1A to 32A and comparative production examples 1A to 2A were measured as follows.

[0095] In a 2 ml tube, 200 μL of PBS (Fujifilm Wako Pure Chemical Industries), 200 μL of aqueous solution containing each amine-modified gelatin or carboxylic acid-modified gelatin at a concentration of 1 mg / mL, 200 μL of sodium trinitrobenzenesulfonate (Fujifilm Wako Pure Chemical Industries), and 200 μL of NaHCO3 (Nacalai Tesque) were added in order and the mixture was allowed to react (room temperature, protected from light, 60 minutes).

[0096] Next, 100 μL of 1M HCl (Fujifilm Wako Pure Chemical Industries) and 200 μL of 10 wt% sodium dodecyl sulfate aqueous solution (Fujifilm Wako Pure Chemical Industries) were added sequentially to the above mixed solution to stop the reaction. Finally, the solution was transferred to a 96-well plate in 200 μL / well increments, and the absorbance at a wavelength of 415 nm was measured using a plate reader (Thermo Fisher Scientific). For quantitative analysis, a β-alanine aqueous solution was used as a calibration curve to calculate the molar concentration of amino groups relative to absorbance. The introduction rate was then calculated using 33 mol of amino groups and 99 mol of carboxyl groups per mole of gelatin. The results are shown in Tables 1-1 to 1-5.

[0097] [Table 1-1]

[0098] [Table 1-2]

[0099] [Table 1-3]

[0100] [Table 1-4]

[0101] [Table 1-5]

[0102] <Manufacturing Examples 1B-32B and Comparative Manufacturing Examples 1B-2B: Preparation of Gelatin Particles> Amine-modified gelatin or carboxylic acid-modified gelatin from Production Examples 1A to 32A and Comparative Production Examples 1A to 2A was dissolved in water to a concentration of 50 mg / mL to prepare an aqueous solution of amine-modified gelatin or carboxylic acid-modified gelatin. Aqueous solutions of amine-modified gelatin or carboxylic acid-modified gelatin were heated to 40°C and stirred for 2 hours. Next, 1.25 mL of the amine-modified gelatin or carboxylic acid-modified gelatin aqueous solution was dispensed into a 10 mL vial, and 5 mL of acetone was added dropwise using a 10 mL syringe and a 24 G needle in a 40°C bath. Then, 20 μL of 25 wt% glutaraldehyde aqueous solution was added to the resulting particles, and the gelatin particles were chemically crosslinked by stirring at 40°C for 6 hours.

[0103] Subsequently, to block unreacted aldehyde groups, 2 mL of a 100 mM glycine solution was added to the particles, and the mixture was stirred at room temperature for at least 1 hour. After the reaction was complete, aggregates were removed using a 40 μm cell strainer, the mixture was centrifuged at 16000 G, the supernatant was removed, and the mixture was redispersed in 1 mL of Milli-Q water. This procedure was repeated twice, and finally, the supernatant obtained by centrifuging at 1000 G was collected as a dispersion of amine-modified gelatin or carboxylic acid-modified gelatin particles.

[0104] Following the above steps, gelatin particle dispersions relating to Production Examples 1B to 32B and Comparative Production Examples 1B to 2B were prepared.

[0105] The volume-average particle size was measured using a Zetasizer Ultra Red Label (Malvern Panlytical) prepared by diluting the obtained amine-modified gelatin or carboxylic acid-modified gelatin particle dispersion to 1 μg / mL with PBS solution. Furthermore, the zeta potential of the obtained amine-modified gelatin or carboxylic acid-modified gelatin particle dispersion was measured using a Zetasizer Ultra Red Label (Malvern Panlytical) with a solution diluted in PB solution to a particle concentration of 10 μg / mL. The results are shown in Tables 1-1 to 1-5.

[0106] <Manufacturing Examples 33B-34B: Preparation of Gelatin Particles> A gelatin particle dispersion was obtained in the same manner as in Production Example 1B, except that "1.25 mL of 50 mg / mL aqueous solution of amine or carboxylic acid-modified gelatin that had been preheated to 40°C and stirred for 2 hours" was replaced with "1.25 mL of 50 mg / mL aqueous solution of PI9 that had been preheated to 40°C and stirred for 2 hours" or "1.25 mL of aqueous solution of PI5 that had been preheated to 40°C and stirred for 2 hours". Subsequently, the volume-average particle size and zeta potential were measured in the same manner as in Production Example 1B. The results are shown in Table 1-4.

[0107] <Manufacturing example 1P> The peptide (Stomagen) with the amino acid sequence shown in SEQ ID NO: 1 was synthesized using the F-moc method with a peptide synthesizer (PreludeX, Gyros Protein Technologies, Inc.). After deprotection with a deprotection solution containing TFA, the peptide was precipitated with diethyl ether to obtain the crude peptide. Subsequently, it was purified by HPLC (M600 pump, Waters Japan Co., Ltd.) using an ODS column (Cosmosil 5C18-AR-II, 20 × 250 mm, Nacalai Tesque Inc.). Ultrapure water containing 0.1% TFA and acetonitrile containing 0.1% TFA were used as solvents. Gradient elution was performed linearly at 1% / min, and elution was performed at 10 mL / min. The molecular weight of the peptide with the amino acid sequence shown in Sequence ID No. 1, determined by SDS-PAGE (SDS-polyacrylamide gel electrophoresis), was 5.1 kDa.

[0108] <Manufacturing example 2P> The peptide (CLE25) with the amino acid sequence shown in Sequence ID No. 2 was prepared using a peptide synthesizer in the same manner as in Production Example 1P. The molecular weight of the peptide with the amino acid sequence shown in Sequence ID No. 2, determined by SDS-PAGE (SDS-polyacrylamide gel electrophoresis), was 1.3 kDa.

[0109] <Examples 1C-34C and Comparative Examples 1C-2C: Preparation of plant growth aids containing peptide-encapsulated gelatin particles> 10 μL of a 25 μM peptide aqueous solution [using Stomagen as the peptide] was added to a 1.5 mL Eppendorf tube, and 20 μL of the gelatin particle dispersions according to Production Examples 1B-34B and Comparative Production Examples 1B-2B, which were adjusted to 1 mg / mL by adding water, were added and impregnated at room temperature for 15 minutes. Then, the mixture was centrifuged at 4°C and 16000 G for 4 minutes, the supernatant was collected, and replaced with 20 μL of PBS(-) to obtain the plant growth stimulants according to Examples 1C-34C and Comparative Examples 1C-2C.

[0110] The zeta potential was evaluated using the plant growth stimulants prepared in Examples 1C to 34C and Comparative Examples 1C to 2C by the following method. The zeta potential was measured using a Zetasizer Ultra Red Label (Malvern Panlytical) with a solution of the above growth-promoting agent diluted to 10 μg / mL with PB solution (results are shown in Tables 1-1 to 1-5).

[0111] The amount of peptide encapsulation was evaluated using the plant growth stimulants prepared in Examples 1C to 34C and Comparative Examples 1C to 2C by the following method. 20 μL of the plant growth stimulant obtained above was left to stand in a test tube at 37°C. Six hours after standing at 37°C, 20 μL of PBS(+) containing 2 mg / mL of collagenase D from Clostridium histolyticum (Roche) as a gelatin particle-degrading enzyme was added. After 72 hours, test tubes containing the plant growth stimulant were centrifuged at 4°C and 16,000 rpm. The absorbance of the recovered supernatant at 280 nm was measured using NanoDrop to calculate the peptide content (peptide weight per gram of particle). The results are shown in Tables 1-1 to 1-5. Furthermore, a higher value indicates better internal capacity performance.

[0112] <Examples 1D-34D and Comparative Examples 1D-2D: Preparation of plant growth aids containing peptide-encapsulated gelatin particles> In Examples 1C to 34C and Comparative Examples 1C to 2C, plant growth stimulants for Examples 1D to 34D and Comparative Examples 1D to 2D were obtained in the same manner as in Examples 1C to 34C and Comparative Examples 1C to 2C, except that the peptide used in the "25 μM peptide aqueous solution" was changed from Stomagen to CLE25. Subsequently, the zeta potential and content were measured in the same manner as in Examples 1C to 34C and Comparative Examples 1C to 2C. The results are shown in Tables 1-1 to 1-5.

[0113] Using the plant growth stimulants according to Examples 4C, 4D, 9C, 9D, 11C, 13C, 32C, and 32D, the amount of gelatin degradation and the amount of peptide sustained release were measured by the following method. 20 μL of each plant growth stimulant was allowed to stand in a test tube at 37°C. After 6 hours, 20 μL of PBS(+) containing 2 mg / mL of collagenase D from Clostridium histolyticum (Roche) as a gelatin particle-degrading enzyme was added, and the following sampling procedures were subsequently performed at the time intervals shown in Table 2. From the absorbance at 280 nm measured during each sampling operation, the amount of peptide encapsulation and the amount of gelatin degradation were calculated. Based on the amounts of peptide and gelatin used in the experiment, the sustained release rate and degradation rate were calculated. The results are shown in Table 2. The sustained release rate and decomposition rate were determined by visually inspecting the amount of gelatin removed, with the amount of sustained release and decomposition at that point being set to 100%.

[0114] <Sampling operation> Test tubes containing a plant growth stimulant were centrifuged at 4°C and 16,000 rpm. The supernatant was sampled in its entirety, and its absorbance at 280 nm was measured using NanoDrop. After sampling the supernatant, 20 μL of PBS(-) was added to the test tube, and the particles were redispersed in the liquid by osmosis, and the static test was continued.

[0115] [Table 2]

[0116] <Examples 1E-30E, 33E-34E, and Comparative Example 2E: Preparation of plant growth aids containing RNA-encapsulated gelatin particles> As the RNA used, we selected a miRNAmimic (a double-stranded RNA consisting of RNA with the sequence shown in SEQ ID NO: 3 and RNA with the sequence shown in SEQ ID NO: 4) targeting the mRNA of the pho2 gene, which is known to regulate the function of phosphate transporters, and used it after FITC modification. The size of the double-stranded RNA was 19 bp.

[0117] First, double-stranded RNAs, specifically RNA having the sequence shown in Sequence ID No. 3 and RNA having the sequence shown in Sequence ID No. 4, were dissolved in water to a concentration of 100 nM to prepare an RNA aqueous solution. Next, 20 μL of each gelatin particle dispersion, adjusted to 1 mg / mL, was added to a 1.5 mL Eppendorf tube. The prepared RNA aqueous solution was then added to achieve the N / P ratios listed in Tables 1-1 to 1-5. The mixture was then made up to 100 μL with milliQ water and allowed to stand at room temperature for 15 minutes. After that, the mixture was centrifuged at 4°C and 16000 G for 4 minutes, the supernatant was collected, and replaced with 20 μL of PBS(-) to obtain the plant growth stimulants according to Examples 1E to 30E, Examples 33E to 34E, and Comparative Example 2E. The amount of unencapsulated nucleic acid was quantified by measuring the absorbance at 260 nm of the recovered supernatant, and the encapsulation rate was calculated.

[0118] The zeta potential was evaluated using the plant growth stimulants described in Examples 1E-30E, Examples 33E-34E, and Comparative Example 2E, by the following method. Each plant growth stimulant was diluted to 10 μg / mL with PB solution, and the zeta potential was measured using a Zetasizer Ultra Red Label (Malvern Panlytical). The results are shown in Tables 1-1 to 1-5.

[0119] Using the plant growth stimulants described in Examples 4E, 9E, 11E, and 13E, the amount of gelatin degradation and the amount of sustained release of nucleic acids were evaluated by the following method. Each plant growth stimulant (20 μL) was placed in a test tube and allowed to stand at 37°C. After standing at 37°C for 6 hours or more, 20 μL of PBS(+) containing 2 mg / mL of collagenase D from Clostridium histolyticum (Roche) as a gelatin particle-degrading enzyme was added. Subsequently, the following sampling procedures were performed at the time intervals shown in Table 3. The amount of nucleic acid released was measured from the absorbance at 260 nm measured during each sampling operation, and the amount of gelatin decomposition was measured from the absorbance at 280 nm. The sustained release rate and decomposition rate were calculated by defining the point at which no particle precipitate was observed during centrifugation as 100% decomposition rate. The results are shown in Table 3.

[0120] <Sampling operation> Test tubes containing plant growth stimulants were centrifuged at 4°C and 16,000 rpm. The supernatant was sampled in its entirety, and the absorbance at 260 nm and 280 nm was measured using NanoDrop. After sampling the supernatant, 20 μL of PBS(-) was added to the test tube, and the particles were redispersed in the liquid by osmosis, and the static test was continued.

[0121] [Table 3]

[0122] The cultivation method for Arabidopsis thaliana used to evaluate plant growth stimulants (containing plant hormones) is shown below.

[0123] <Preparation of plant growth stimulants for the cultivation of Arabidopsis thaliana> The plant growth stimulant according to Example 2C was diluted with water, and the concentration of Stomagen in the diluted solution was adjusted to the value shown in Table 4-1. In addition, a gelatin particle dispersion according to Production Example 2B was prepared as a control. Similarly, the plant growth stimulants for Examples 3C, 4C, 7C to 9C, 33C, and 34C were prepared so that the concentration of Stomagen in the diluted solution was as shown in Tables 4-1 and 4-2. Furthermore, as controls, gelatin particle dispersions corresponding to Production Example 3B, Production Example 4B, Production Examples 7B to 9B, Production Example 33B, and Production Example 34B were prepared, respectively. Furthermore, for comparison, a Stomagen aqueous solution without gelatin particles was prepared as a plant growth aid, with the concentrations shown in Table 4-2.

[0124] [Table 4-1]

[0125] [Table 4-2]

[0126] <Making a pot> Plant growing pots were prepared by pouring sucrose-containing MS agar medium (MS medium with 0.8% (w / v) sucrose and 2% (w / v) agar added) into cylindrical and rectangular prism-shaped pots with open tops and bottoms, and allowing them to solidify. These pots were placed in an incubator under long-day conditions of 25°C, 18 hours of light, and 6 hours of darkness.

[0127] <Hypochlorous acid treatment of seeds> Arabidopsis thaliana seeds (Col-0) were purchased from Inplanta Innovations Inc. The seeds were sterilized by immersing them in 1% hypochlorite and stirring for 1 minute, after which the hypochlorite was removed by centrifugation. After hypochlorite treatment, the seeds were washed three times with sterile water, sown on top of cylindrical pots, and stored in the dark at 4°C for 24 hours.

[0128] <Hydroponic cultivation of plants> Multiple cylindrical pots were prepared and grown for 14 days in an incubator under long-day conditions of 25°C, 16 hours of light, and 8 hours of darkness. After 14 days of growth, the grown Arabidopsis thaliana seedlings were transplanted into the aforementioned rectangular prism pots and grown for 7 days in an incubator under long-day conditions of 25°C, 16 hours of light, and 8 hours of darkness.

[0129] <Addition of plant growth stimulants to Arabidopsis thaliana> After transplanting Arabidopsis thaliana seedlings into rectangular prism pots and allowing 7 days to pass, 3 μL of the plant growth stimulants and Stomagen aqueous solution listed in Tables 4-1 and 4-2 were added to the leaves of 5 leaves of each seedling using a pipette. Stem length and the number of withered leaves were evaluated 37 days after the addition of the plant growth stimulants in an incubator under long-day conditions of 25°C, 16 hours of light and 8 hours of darkness. The results are shown in Tables 4-1 and 4-2. The longer the stem, the more the plant's growth was promoted. The more dead leaves there are, the more it indicates that the plant's growth is impaired.

[0130] Furthermore, in Tables 4-1 and 4-2, "◎" in "Evaluation of stem length" and "Evaluation of number of withered leaves" means that the result is significantly better than when using a stomagen-free aqueous solution with a stomagen concentration of 0.0 μM, "〇" means that the result is the same as or better than when using a stomagen-free aqueous solution with a stomagen concentration of 0.0 μM, and "×" means that the result is worse than when using a stomagen-free aqueous solution with a stomagen concentration of 0.0 μM.

[0131] <Cucumber cultivation> The cultivation method for cucumbers used to evaluate plant growth stimulants (containing plant hormones) is shown below.

[0132] <Preparation of plant growth stimulants to be used in cucumber cultivation> The plant growth stimulant according to Example 2C was diluted with water, and the concentration of Stomagen in the diluted solution was adjusted to the value shown in Table 5. In addition, a gelatin particle dispersion according to Production Example 2B was prepared as a control. Similarly, the plant growth stimulants for Examples 4C, 9C, 32C, and Comparative Example 1C were prepared so that the concentration of Stomagen in the diluted solution was as shown in Table 5. In addition, gelatin particle dispersions corresponding to Production Example 4B, Production Example 9B, Production Example 32B, and Comparative Production Example 1B were prepared as controls for each example. Furthermore, for comparison, we prepared Stomagen aqueous solutions without gelatin particles at the concentrations shown in Table 5 as plant growth aids.

[0133] [Table 5]

[0134] The cucumber seeds used were of the Eterno variety. Sowing was carried out using cell trays (Tokai Kasei TO plug trays, 128 cells) and sterilized Yanmar vegetable soil. One cucumber seed was sown in each cell tray, and the seedlings were grown in an incubator under long-day conditions of 25°C, 16 hours of light and 8 hours of darkness.

[0135] <Spraying of plant growth stimulants on cucumbers> After sowing the cucumber seeds and growing them in an incubator until the true leaves appeared, the plant growth stimulants and Stomagen aqueous solution listed in Table 5 were sprayed onto the leaves at a rate of 3 μL per leaf (one application) using a hand sprayer. Subsequently, the plants were grown in an incubator under long-day conditions of 25°C, 16 hours of light and 8 hours of darkness.

[0136] <Observation of stomatal density in cucumbers> Replicas of five leaves were collected from each seedling by transferring the underside of the plant leaves onto silicone resin. The transfers were performed at 0, 7, and 14 days after peptide application, and the leaves were collected from the same location each time. Subsequently, nail polish topcoat was applied to the obtained silicone resin, and the stomatal density of the transferred replica surface was measured using an optical microscope, and the percentage increase or decrease from the stomatal density on day 0 was calculated. The results are shown in Table 5. In Table 5, the stomatal density is shown as the relative density, with the stomatal density on day 0 set to 1.

[0137] The cultivation method for Arabidopsis thaliana used for evaluating plant growth stimulants (containing nucleic acids) is shown below.

[0138] <Preparation of plant growth stimulants for the cultivation of Arabidopsis thaliana> Water was added to the plant growth stimulants according to Examples 4E and 9E to prepare the solutions so that the N / P ratio was as shown in Tables 6-1, 6-2, and 7.

[0139] <Comparative Example 3E: Preparation of a plant growth aid containing RNA-encapsulated particles for comparison> In Example 1E, the procedure was carried out in the same manner as in Example 1E, except that 20 μL of Lipofectamine 2000 (a dispersion of cationic lipid particles manufactured by Invitrogen) was used instead of 20 μL of the gelatin particle dispersion according to Production Example 1B, to obtain the plant growth stimulant according to Comparative Example 3E. The concentration of RNA contained in the plant growth stimulant was adjusted to be the same as in Example 1E.

[0140] Furthermore, RNA aqueous solutions and water at the concentrations shown in Tables 6-1, 6-2, and 7 were prepared as plant growth aids for comparison.

[0141] [Table 6-1]

[0142] [Table 6-2]

[0143] [Table 7]

[0144] <Dispersion of nucleic acid-encapsulated gelatin particles onto Arabidopsis thaliana> The process from "pot preparation" to "hydroponic cultivation of plants" described above was carried out in the same manner. The plants were then transplanted into rectangular prism-shaped pots, and after 7 days, 3 μL of each plant growth stimulant listed in Tables 6-1, 6-2, and 7 was added to the leaf surface using a pipette. Subsequently, the plants were grown in an incubator under long-day conditions of 25°C, 16 hours of light and 8 hours of darkness.

[0145] <qRT-PCR of Arabidopsis thaliana> Using tweezers or a cork polarizer, 25–50 mg of leaves were collected from each seedling after the cultivation time indicated in Tables 6-1 and 6-2. Next, total RNA was isolated from Arabidopsis thaliana using ISOSPIN Plant RNA (Nippon Gene Co., Ltd.). Then, cDNA was synthesized from the isolated total RNA using Prime Script (Takara, Prime Script RT reagent kit). Quantitative real-time PCR (qRT PCR) reactions were set up using TaqMan® Universal Master Mix II, NMT1, NMT2, and 18 Taqman® probes purchased from Life Technologies (Carlsbad, CA), with three replicates for each reaction. qRT PCR was performed using Step one Real-time PCR (Thermo Fisher Scientific), and the Ct value of the target gene, the pho2 gene (a double-stranded nucleic acid containing the sequence shown in SEQ ID NO: 5 and the sequence shown in SEQ ID NO: 6), was measured. Furthermore, the Ct value of the housekeeping gene CBP gene (a double-stranded nucleic acid consisting of the sequence shown in SEQ ID NO: 7 and the sequence shown in SEQ ID NO: 8) was also measured by performing quantitative real-time PCR using the cDNA in question. Then, using the Ct value of the pho2 gene when water is sprayed and the plants are grown for 24 hours as the baseline, the values ​​for each plant growth stimulant used are compared. -ddCt The values ​​were calculated. The results are shown in Tables 6-1 and 6-2. Note 2 -ddCt A lower value indicates a higher gene expression suppression effect.

[0146] The following kits were used: KOD SYBR qPCR Mix (Toyobo): 6.6μL 10 μM Forward Primer: 1 μL (SEQ ID NOs. 5 and 7) 10 μM Reverse Primer: 1 μL (SEQ ID NOs. 6 and 8) 50 x ROX reference dye: 0.4 μL (manufactured by Bio-Rad)

[0147] <Measurement of stomatal density in Arabidopsis thaliana> Replicas of the underside of Arabidopsis thaliana leaves were obtained by transferring the underside of the leaf onto a polyurethane membrane. Subsequently, nail polish topcoat was applied to the resulting polyurethane membrane, and the replica surface was transferred to it. The stomatal density was observed under an optical microscope, and the stomatal density was measured on day 0 and day 14 after application of each plant growth stimulant. The results are shown in Table 7. [Industrial applicability]

[0148] The plant growth stimulant of the present invention is excellent in promoting plant growth and is extremely useful for horticultural and agricultural applications.

Claims

1. It contains gelatin (A1) and gelatin particles containing a bioactive substance for plants, A plant growth aid comprising gelatin particles having a volume-average particle diameter of 50 to 1,100 nm.

2. The plant growth aid according to claim 1, wherein the gelatin (A1) has an amide bond formed by bonding a carboxyl group to a compound having 2 moles or more amino groups per mole of the compound, and the zeta potential of the gelatin particles is +0.1 to +6.0 mV.

3. The plant growth aid according to claim 1 or 2, wherein the gelatin particles include cross-linked gelatin (A2) formed by cross-linking the gelatin (A1) particles together.

4. The plant growth aid according to claim 1 or 2, wherein the bioactive substance for plants comprises at least one selected from the group consisting of plant growth hormones, functional nucleic acids, and transformative nucleic acids.

5. The plant growth aid according to claim 4, wherein the plant growth hormone is a peptide, and the molecular weight of the plant growth hormone determined by SDS-PAGE (SDS polyacrylamide gel electrophoresis) is 1,000 to 30,000.

6. The plant growth aid according to claim 5, wherein the plant growth hormone is a peptide having the amino acid sequence shown in SEQ ID NO: 1, the amino acid sequence shown in SEQ ID NO: 2, or an amino acid sequence having 80% or more homology to these amino acid sequences.

7. The plant growth aid according to claim 4, wherein the functional nucleic acid is an RNA interference (RNAi) molecule.

8. The plant growth aid according to claim 7, wherein the RNA interference (RNAi) molecule is at least one selected from the group consisting of RNA silencing molecules, small interfering RNA (siRNA), short hairpin RNA (shRNA), microRNA (miRNA), Piwi-interacting RNA (piRNA), and trans-acting siRNA (tasiRNA) that target RNA in plant cells.

9. The plant growth aid according to claim 4, wherein the nucleic acid for transformation is at least one selected from the group consisting of foreign protein genes, agricultural genes, and marker genes.

10. The plant growth aid according to claim 4, wherein the transformation nucleic acid is at least one selected from the group consisting of nucleic acids for expressing a predetermined protein, nucleic acids for male sterility, nucleic acids for conferring herbicide resistance, nucleic acids for conferring insect pest resistance, nucleic acids for conferring resistance to bacterial diseases, nucleic acids for conferring resistance to fungal diseases, and nucleic acids for conferring resistance to viral diseases.

11. A method for growing plants using the plant growth stimulant described in claim 1 or 2.

Images