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WO2025005176A1 - Plant callus inducing agent, method for inducing callus, method for producing callus, and method for producing plant body - Google Patents

Plant callus inducing agent, method for inducing callus, method for producing callus, and method for producing plant body Download PDF

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Publication number
WO2025005176A1
WO2025005176A1 PCT/JP2024/023315 JP2024023315W WO2025005176A1 WO 2025005176 A1 WO2025005176 A1 WO 2025005176A1 JP 2024023315 W JP2024023315 W JP 2024023315W WO 2025005176 A1 WO2025005176 A1 WO 2025005176A1
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Prior art keywords
callus
plant
producing
inducer
inducing
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French (fr)
Japanese (ja)
Inventor
敦司 岡澤
素啓 園田
宏太郎 藤野
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University Public Corporation Osaka
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University Public Corporation Osaka
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    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
    • A01H3/00—Processes for modifying phenotypes, e.g. symbiosis with bacteria
    • A01H3/04—Processes for modifying phenotypes, e.g. symbiosis with bacteria by treatment with chemicals
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
    • A01H4/00—Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
    • A01H6/00—Angiosperms, i.e. flowering plants, characterised by their botanic taxonomy
    • A01H6/20—Brassicaceae, e.g. canola, broccoli or rucola
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
    • A01H6/00—Angiosperms, i.e. flowering plants, characterised by their botanic taxonomy
    • A01H6/46—Gramineae or Poaceae, e.g. ryegrass, rice, wheat or maize
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
    • A01H6/00—Angiosperms, i.e. flowering plants, characterised by their botanic taxonomy
    • A01H6/54—Leguminosae or Fabaceae, e.g. soybean, alfalfa or peanut
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N47/00—Biocides, pest repellants or attractants, or plant growth regulators containing organic compounds containing a carbon atom not being member of a ring and having no bond to a carbon or hydrogen atom, e.g. derivatives of carbonic acid
    • A01N47/08—Biocides, pest repellants or attractants, or plant growth regulators containing organic compounds containing a carbon atom not being member of a ring and having no bond to a carbon or hydrogen atom, e.g. derivatives of carbonic acid the carbon atom having one or more single bonds to nitrogen atoms
    • A01N47/28—Ureas or thioureas containing the groups >N—CO—N< or >N—CS—N<
    • A01N47/34—Ureas or thioureas containing the groups >N—CO—N< or >N—CS—N< containing the groups, e.g. biuret; Thio analogues thereof; Urea-aldehyde condensation products
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01P—BIOCIDAL, PEST REPELLANT, PEST ATTRACTANT OR PLANT GROWTH REGULATORY ACTIVITY OF CHEMICAL COMPOUNDS OR PREPARATIONS
    • A01P21/00—Plant growth regulators

Definitions

  • the present invention relates to a callus inducer for plants, a method for inducing callus using the callus inducer, a method for producing callus, and a method for producing a plant body.
  • plants can form a mass of dedifferentiated plant cells (callus) from differentiated plant tissues, and this is used in a wide range of fields, such as the production of useful substances and the creation of transformants.
  • callus dedifferentiated plant cells
  • auxins such as indole-3-acetic acid (hereinafter sometimes referred to as IAA) and 2,4-dichlorophenoxyacetic acid (hereinafter sometimes referred to as 2,4-D), which act on germination, flowering, phototropism, etc.
  • cytokinins such as kinetin and zeatin, which act on lateral bud formation, aging, nutritional response, etc.
  • the composition of these plant hormones for callus induction needs to be examined for each plant species.
  • Patent Document 1 discloses a two-step culture method comprising a callus preparation step of preparing callus from a plant body and a plant organ formation step of forming plant organs from the callus, in which culture conditions such as light intensity and glucose concentration are controlled to efficiently produce a plant body.
  • Patent Document 2 discloses a callus inducer containing a 1,4-disubstituted piperazine derivative or a 1-substituted piperazine derivative having a basic structure different from that of conventional callus inducers, specifically fipexide, as an active ingredient, and describes that the callus inducer is applicable to a wide range of plants.
  • Patent Document 3 discloses a method for preparing differentiated embryogenic callus (DEC) tissue of sorghum using a callus induction medium supplemented with one or more auxins, one or more cytokinins, and one or more agents that reduce oxidative browning, such as lipoic acid.
  • the present inventors attempted to search for a novel callus inducer that is applicable to a wide range of plants and is easier to use.
  • Non-Patent Document 1 aryloxyacetylthiourea derivatives to develop agents to inhibit the root elongation of parasitic weeds. They discovered that compounds with specific structures among these derivatives have the activity of inducing callus in plants, which led to the completion of the present invention.
  • a plant callus inducer comprising a compound represented by the formula: ⁇ 2>
  • ⁇ 4> A method for inducing callus, comprising the step of contacting a plant body, a plant cell, a plant tissue fragment or a plant seed with the callus inducer described in ⁇ 1> to induce callus.
  • ⁇ 5> The method for inducing callus according to ⁇ 4>, wherein the halogen atom of the compound represented by formula (I) or a salt thereof is chlorine or bromine.
  • ⁇ 6> The method for inducing callus according to ⁇ 4> or ⁇ 5>, wherein the plant is any one of a Gramineae plant, a Brassicaceae plant, a Leguminosae plant, a Solanaceae plant, and a Phytolacca family plant.
  • a method for producing callus comprising the step of contacting a plant body, a plant cell, a plant tissue fragment or a plant seed with the callus inducer described in ⁇ 1> to induce and proliferate callus.
  • ⁇ 10> Use of a compound represented by formula (I) or a salt thereof for inducing callus in a plant.
  • ⁇ 11> The use according to ⁇ 10>, wherein the halogen atom of the compound represented by formula (I) or a salt thereof is chlorine or bromine.
  • ⁇ 12> The use according to ⁇ 10> or ⁇ 11>, wherein the plant is any one of a Gramineae plant, a Cruciferae plant, a Leguminosae plant, a Solanaceae plant, and a Phytolacca family plant.
  • a method for producing a plant body comprising the steps of: contacting a plant body, a plant cell, a plant tissue fragment or a plant seed with the callus inducer described in ⁇ 1> to induce callus; and transplanting the callus obtained by callus induction in the above step into a shoot induction medium to induce shoots.
  • ⁇ 15> The method for producing a plant according to ⁇ 13> or ⁇ 14>, wherein the plant is any one of a Gramineae plant, a Cruciferae plant, a Leguminosae plant, a Solanaceae plant, and a Phytolacca family plant.
  • the callus inducer of the present invention can be applied to a wide range of plants, including plants for which callus induction was previously difficult. It is particularly significant that the agent can be applied to sorghum, which has high resistance to drought and soil toxicity.
  • Photographs showing the results of GUS staining of Arabidopsis thaliana callus induced in a medium containing compound (II) and CIM (Example 2).
  • 1 is a photograph of sorghum callus induced in a medium containing compound (II) (Example 3).
  • a) Photograph of red clover seedlings grown in a medium containing compound (II), Kinetion, IPA, and Zeatin, and CIM (control) Example 4
  • b) Partially enlarged photograph of IPA and compound (II) in a. 1 shows photographs of Arabidopsis thaliana callus induced on a medium containing compound (II) and a medium containing FPX (Example 5).
  • FIG. 1 shows photographs of callus of Sorghum bicolor induced in a medium containing Compound (II) and a medium containing FPX (Example 6).
  • FIG. 1 is a schematic diagram showing the steps of explant preparation, callus induction and shoot induction of tissue explants of Arabidopsis thaliana (Example 7).
  • 1 shows photographs of Arabidopsis root explants in which callus was induced on a medium containing Compound (II) and CIM (Example 7).
  • 1 shows photographs of Arabidopsis root explants that were induced into shoots after callus induction on a medium containing Compound (II) and CIM (Example 7).
  • 1 is a top view of a plate on which explants of Nicotiana benthamiana and its tissue fragments were prepared (Example 8). 1 shows photographs of shoot, hypocotyl, root and root tip explants of Nicotiana benthamiana for which callus was induced on a medium containing compound (II) and CIM (Example 8). 1 is a photograph of Phytolacca americana in which callus was induced in a medium containing compound (II) (Example 9).
  • the callus inducer of the present invention is characterized by having callus induction activity in plants and containing as an active ingredient a compound represented by the following formula (I) or a salt thereof (hereinafter also referred to as compound (I) or a salt thereof).
  • R represents a halogen atom.
  • halogen atom in the compound represented by (I) examples include fluorine, chlorine, bromine and iodine. Among these, chlorine or bromine is preferred, and chlorine is even more preferred.
  • the formula (II) shows the case where the halogen atom R is chlorine (Cl), and the formula (III) shows the case where the halogen atom R is bromine (Br).
  • the compound represented by the formula (I) can be synthesized by the method described in Tetrahedron135(2023)133333'Synthesis of aryloxyacetylthiourea derivatives for the development of radicle elongation inhibitor of parasitic weeds' (Non-Patent Document 1) by the present inventors.
  • the compound represented by the formula (II) (hereinafter also referred to as compound (II)) is 3-[2-(4-Chlorophenoxy)acetyl]-1-(3,4-dichlorophenyl)thiourea, which corresponds to compound "3ab" in the same document.
  • the compound represented by the formula (III) is 3-[2-(4-Bromophenoxy)acetyl]-1-(3,4-dichlorophenyl)thiourea, which corresponds to compound "3ac" in the same document.
  • the salt of the compound so long as it is a salt that has the callus-inducing activity of the present invention.
  • the callus inducer of the present invention may contain compound (I) or a salt thereof as an active ingredient, and may contain other ingredients other than compound (I) or a salt thereof to the extent that the callus induction activity is not inhibited.
  • other ingredients include known pharmaceutical additives such as excipients, emulsifiers, and wetting agents.
  • the form of the callus inducer of the present invention is not particularly limited, and may be any form that allows callus induction, such as an emulsion, liquid, oil, aqueous solution, wettable powder, flowable, powder, microgranule, granule, aerosol, paste, or jelly.
  • the plant to which the callus inducer of the present invention is applied is not particularly limited.
  • it may be either angiosperms or gymnosperms, and in the case of angiosperms, it may be either monocotyledonous or dicotyledonous.
  • gymnosperms include Cycadaceae (cycads, etc.), Ginkgoaceae (ginkgo, etc.), Pinaceae (pine, Japanese black pine, fir, spruce, etc.), Cupressaceae (cedar, etc.), and Taxaceae (yew, etc.), but are not limited to these.
  • examples of monocotyledonous plants include, but are not limited to, Poaceae (rice, wheat, barley, oat, rye, millet, foxtail millet, barnyard millet, corn, finger millet, sorghum, bamboo, reed, Miscanthus, amaranth, miscanthus, switchgrass, sorghum, brachypodium, etc.), Araceae (taro, duckweed, etc.), Palmaceae (palm, date palm, etc.), Musaceae (banana, etc.).
  • Poaceae rice, wheat, barley, oat, rye, millet, foxtail millet, barnyard millet, corn, finger millet, sorghum, bamboo, reed, Miscanthus, amaranth, miscanthus, switchgrass, sorghum, brachypodium, etc.
  • Araceae taro, duckweed, etc.
  • Palmaceae
  • dicotyledonous plants include, but are not limited to, Brassicaceae (Arabidopsis thaliana, rapeseed, cabbage, broccoli, cauliflower, etc.), Solanaceae (eggplant, tomato, potato, tobacco, etc.), Pyretaceae (American pokeweed, etc.), Cucurbitaceae (melon, pumpkin, etc.), Fabaceae (soybean, red clover, etc.), Malvaceae (cotton, etc.), Asteraceae (chrysanthemum, carrot, etc.), Salicaceae (poplar, etc.), Vitaceae (grape, etc.), Rosaceae (rose, apple, etc.), Orchidaceae (orchid, etc.), Liliaceae (lily, tulip, onion, etc.), Salicaceae (poplar, etc.), Myrtaceae (eucalyptus, etc.), Euphorbiaceae (cassava, etc.), and Oro
  • the "method for inducing callus” of the present invention refers to a method comprising the step of contacting a plant body, a plant cell, a plant tissue fragment, a plant seed, or the like with the callus inducer of the present invention to induce callus.
  • the "method for producing callus” of the present invention refers to a method comprising the steps of contacting a plant body, a plant cell, a piece of plant tissue, or a plant seed with the callus inducer of the present invention to induce callus, and then further growing the induced callus. These methods may include a step of sterilizing the plant seed to be induced in advance, or a step of germinating the plant seed after sterilization.
  • the "plant body, plant cell, plant tissue piece, or plant seed” used in the "callus induction method” or “callus production method” of the present invention may be any that is capable of callus induction. It may be the entire plant, and in the case of plant tissue, it may be, for example, the shoot tip, stem, leaf, shoot, embryo cell, root petal, fruit, epidermis, etc. It is preferable that these plant bodies are plants that have been grown by aseptic sowing, or plants that have been sterilized with 70% ethanol, sodium hypochlorite aqueous solution, etc., and made sterile.
  • the callus induction medium is not particularly limited as long as it contains the compound represented by formula (I) or a salt thereof and is capable of inducing callus.
  • the compound represented by formula (I) may be added to the callus induction medium in one type or in a combination of two or more types.
  • the compound represented by formula (I) or its salt may be used in a medium at a concentration that allows the callus induction effect of the present invention to be exhibited depending on the target plant and treatment conditions.
  • the concentration is not particularly limited, but may be added in the range of, for example, 0.01 to 100 ⁇ M.
  • the concentration is preferably 0.1 to 10 ⁇ M, and more preferably 0.2 to 5 ⁇ M.
  • the total amount of these compounds may be adjusted to be within the above concentration range.
  • the callus induction medium may contain other components in addition to these compounds or their salts. Such components include sugars, gelling agents, inorganic salts, and other components commonly used in callus induction.
  • auxin and cytokinin may also be used in combination with the callus inducer of the present invention, as long as they do not interfere with the action of the callus inducer.
  • the compound represented by formula (I) or its salt can induce callus alone, without being used in combination with components that have been required for conventional callus induction, such as auxin, kinetin, and cytokinin.
  • the culturing for inducing callus is preferably carried out under sterile conditions.
  • the temperature during the culturing is preferably 20 to 25°C, and the light conditions are preferably between constant light irradiation and constant darkness.
  • the culturing can be carried out until callus is induced, but callus induction is usually observed 2 to 4 weeks after the culturing.
  • the subculture method includes a method of replacing the medium with a callus induction medium containing the callus inducer of the present invention every other month.
  • the callus induced by the method of the present invention can be used for producing genetically modified crops, etc.
  • a genetically modified plant can be produced by infecting the callus induced by the present invention with Agrobacterium having a plasmid containing a target gene, isolating the callus in which the plasmid has been inserted into the chromosome, and redifferentiating the callus.
  • Test method Callus induction was carried out on Arabidopsis thaliana by the following steps, followed by shoot induction. (1) Germination Treatment Arabidopsis seeds were sterilized by treating with 70% ethanol for 2 minutes and then with 1% sodium hypochlorite for 10 minutes. The seeds were sown on 1/2 MS medium and grown at 25°C for 4 to 5 days under a 16-h light/8-h dark cycle.
  • GUS staining solution (per 1 mL)> 0.2 M phosphate buffer (pH 7.0) 500 ⁇ L 20 ⁇ L 0.5M EDTA 0.05M Fe(II) 100 ⁇ L 0.05M Fe(III) 100 ⁇ L 1% Triton 100 ⁇ L 50 ⁇ g/ml X-gluc 20 ⁇ L DW 160 ⁇ L
  • Example 3 Evaluation of callus inducing activity of the callus inducer of the present invention on Sorghum bicolor was carried out according to the following steps. 1. Germination treatment and callus induction Sorghum bicolor seeds were sterilized by treating with 70% ethanol for 2 minutes, and then with 1% sodium hypochlorite for 60 minutes. The seeds were sown in a medium containing compound (II) (1/2 MS medium, 1% sucrose, 2.6 ⁇ M compound (II)). For comparison, the seeds were also sown in CIM. After sowing, the seeds were cultured in the dark at 25° C. for 4 weeks to induce callus.
  • II a medium containing compound
  • Example 4 Evaluation of callus inducing activity of the callus inducer of the present invention on red clover (Trifolium pratense) was carried out by the following steps. 1. Callus induction Red clover seeds were sterilized by treating with 70% ethanol for 2 minutes, and then with 1% sodium hypochlorite for 60 minutes. The seeds were sown in a medium containing compound (II) (1/2 MS medium, 1% sucrose, 1 ppm (2.6 ⁇ M)-compound (II)).
  • the seeds were also sown in a medium, CIM, containing 1 ppm kinetin, zeatin, 1 ppm or 10 ppm IPA instead of compound (II). After sowing, the seeds were cultured for 4 weeks to induce callus.
  • Example 5 Comparison of callus induction activity in Arabidopsis thaliana between compound (II) and fipexide (1-[(p-chlorophenoxy)acetyl]-4-piperonylpiperazine, 1-(4-chlorophenoxyacetyl)-4-(1,3-benzodioxole-5-ylmethyl)piperazine, hereinafter referred to as FPX) Callus induction in Arabidopsis thaliana was carried out by the following steps. 1.
  • Callus induction Arabidopsis seeds were sown in a medium containing compound (II) (1/2 MS medium, 1% sucrose, 0.1 or 1.0 ⁇ M compound (II)) or a medium containing FPX (1/2 MS medium, 1% sucrose, 1.0 ⁇ M FPX). These were cultured at 25° C. for 4 weeks under a 16-h light/8-h dark period to induce callus.
  • a medium containing compound (II) 1/2 MS medium, 1% sucrose, 0.1 or 1.0 ⁇ M compound (II)
  • a medium containing FPX 1/2 MS medium, 1% sucrose, 1.0 ⁇ M FPX
  • Example 6 Comparison of callus induction activity of compound (II) and FPX in Sorghum bicolor
  • Callus induction in Sorghum bicolor was carried out according to the following steps. 1.
  • Callus induction Sorghum bicolor seeds were sterilized by treating with 70% ethanol for 2 minutes, and then with 1% sodium hypochlorite for 60 minutes. The seeds were sown in a medium containing compound (II) (1/2 MS medium, 1% sucrose, 3.0 ⁇ M compound (II)) or in a medium containing FPX (1/2 MS medium, 1% sucrose, 3.0 ⁇ M FPX). After sowing, the seeds were cultured in the dark at 25° C. for 4 weeks to induce callus.
  • compound (II) 1/2 MS medium, 1% sucrose, 3.0 ⁇ M compound (II)
  • a medium containing FPX 1/2 MS medium, 1% sucrose, 3.0 ⁇ M FPX
  • Example 7 Evaluation of the callus induction activity of the callus inducer of the present invention on Arabidopsis thaliana root explants and its effect on shoot induction
  • Callus induction was performed on Arabidopsis thaliana root explants and shoot induction was then performed according to the following steps.
  • Test method (1) Germination treatment Arabidopsis seeds were sterilized by treating with 70% ethanol for 2 minutes and then with 1% sodium hypochlorite for 10 minutes. The seeds were sown on 1/2 MS medium and grown at 25°C for 7 days under a 16-h light/8-h dark cycle.
  • the state of callus obtained by inducing callus from Arabidopsis roots and then inducing shoots is shown in Figure 11.
  • the shoot induction rate of callus induced by CIM was 22% (4 out of 18).
  • the shoot induction rate of callus induced by the medium containing compound (II) was 56% (14 out of 25). From this morphological observation result and the morphological observation result of Example 2, it was found that the callus inducer of the present invention increases the shoot induction rate of callus obtained by callus induction, compared with the conventional callus induction medium CIM. This indicates that plant bodies can be reconstructed with high frequency by using the callus inducer of the present invention.
  • Example 8 Evaluation of callus inducing activity of the callus inducer of the present invention on Nicotiana benthamiana explants
  • Test method (1) Germination treatment Nicotiana benthamiana seeds were sterilized by treating with 70% ethanol for 2 minutes and then with 1% sodium hypochlorite for 10 minutes. The seeds were sown on 1/2 MS medium and grown at 25°C for 7 days under a 16-h light/8-h dark period. (2) Preparation of explants and callus induction The N.
  • benthamiana grown for 7 days was cut into tissue pieces of shoot, hypocotyl, root, and root tip, and then transplanted into a medium containing compound (II) (1/2 MS medium, 1% sucrose, 1.0 ⁇ M compound (II)) to prepare explants as shown in FIG. 12.
  • a medium containing compound (II) 1/2 MS medium, 1% sucrose, 1.0 ⁇ M compound (II)
  • explants transplanted into CIM were also prepared. These were cultured at 25° C. for 4 weeks under a 16-h light/8-h dark period to induce callus.
  • Example 9 Evaluation of callus inducing activity of the callus inducer of the present invention on explants of Phytolacca americana
  • Callus induction of explants of Phytolacca americana was carried out by the following steps. 1. Germination treatment and callus induction The seed coat of pokeweed seeds was removed, and the seeds were treated with 70% ethanol for 2 minutes, and then sterilized by treating with 1% sodium hypochlorite for 45 minutes. The seeds were sown on MS medium and grown for 30 days at 25°C under a 16-h light/8-h dark period. The pokeweed grown for 30 days was cut into leaves and stems of about 0.5 to 1.0 cm.
  • the leaves and stems of about 0.5 to 1.0 cm were then transplanted into a medium containing compound (II) (MS medium, 3% sucrose, 2.6 ⁇ M compound (II)) to prepare explants.
  • the explants were cultured in the light at 25°C for 4 weeks to induce callus.
  • the callus inducer of the present invention can be applied to a wide range of plants, making it possible to induce callus in plants where it was previously impossible to induce callus. It is particularly significant that callus can be induced in sorghum, which has high resistance to drought and soil toxicity. Callus obtained using the callus inducer of the present invention can be used for a variety of purposes, including food, pharmaceuticals, cosmetics, lumber, construction materials, feed, ornamental purposes, biofuel, and testing and research.

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Abstract

The present invention addresses the problem of providing: a novel callus inducing agent; a method for inducing a callus using the callus inducing agent; a method for producing a callus; and a method for producing a plant body. Provided is a plant callus inducing agent comprising a compound represented by formula (I) (wherein R represents a halogen atom) or a salt thereof. Also provided are: a method for inducing a callus, the method including a step for bringing a plant body, a plant cell, a plant tissue piece or a plant seed into contact with the callus inducing agent to induce the formation of a callus; a method for producing a callus, the method further including a step for growing the induced callus; and a method for producing a plant body, the method including a step for inducing a callus and a step for inducing a shoot.

Description

植物のカルス誘導剤、カルスの誘導方法、カルスの製造方法及び植物体の製造方法Plant callus inducer, callus induction method, callus production method, and plant body production method

　本発明は、植物のカルス誘導剤、当該カルス誘導剤を用いたカルスの誘導方法、カルスの製造方法及び植物体の製造方法に関する。 The present invention relates to a callus inducer for plants, a method for inducing callus using the callus inducer, a method for producing callus, and a method for producing a plant body.

　植物は分化後の植物組織から脱分化した植物細胞の塊（カルス）を形成できることが知られており、有用物質の生産や、形質転換体の作出等、広い分野で利用されている。
　一般的にカルスの誘導には、発芽、開花、光屈性等に働くインドール－３－酢酸（以下、ＩＡＡと示す場合がある）、２，４－ジクロロフェノキシ酢酸（以下、２，４－Ｄと示す場合がある）等のオーキシンと、側芽形成、老化、栄養反応などに働くカイネチン、ゼアチン等のサイトカイニンを植物に対して同時に処理する必要がある。しかし、カルス誘導の為のこれらの植物ホルモンの組成は植物種ごとに検討する必要があった。 It is known that plants can form a mass of dedifferentiated plant cells (callus) from differentiated plant tissues, and this is used in a wide range of fields, such as the production of useful substances and the creation of transformants.
Generally, to induce callus, it is necessary to simultaneously treat a plant with auxins such as indole-3-acetic acid (hereinafter sometimes referred to as IAA) and 2,4-dichlorophenoxyacetic acid (hereinafter sometimes referred to as 2,4-D), which act on germination, flowering, phototropism, etc., and cytokinins such as kinetin and zeatin, which act on lateral bud formation, aging, nutritional response, etc. However, the composition of these plant hormones for callus induction needs to be examined for each plant species.

　例えば、特許文献１には、植物体からカルスを作製するカルス作製工程と、前記カルスから植物器官を形成する植物器官形成工程の二段階培養方法において、光強度やグルコース濃度等の培養条件を制御することで、植物体を効率よく生産できる方法が開示されている。
　また、特許文献２には、従来のカルス誘導剤とは異なる基本構造を有する１，４－二置換ピペラジン誘導体又は１－置換ピペラジン誘導体、具体的にはフィペキシドを有効成分とするカルス誘導剤が開示されており、適用できる植物の範囲が広いことが記載されている。
　さらに特許文献３には、１種以上のオーキシン、１種以上のサイトカイニン、及び、リポ酸等の酸化褐変を低減する１種以上の薬剤が補充されたカルス誘導培地を用いてソルガムの分化胚形成カルス（ＤＥＣ）組織を調製する方法が開示されている。
　本発明者らは、適用する植物の範囲が広く、より簡便な新規カルス誘導剤の探索を試みた。 For example, Patent Document 1 discloses a two-step culture method comprising a callus preparation step of preparing callus from a plant body and a plant organ formation step of forming plant organs from the callus, in which culture conditions such as light intensity and glucose concentration are controlled to efficiently produce a plant body.
Furthermore, Patent Document 2 discloses a callus inducer containing a 1,4-disubstituted piperazine derivative or a 1-substituted piperazine derivative having a basic structure different from that of conventional callus inducers, specifically fipexide, as an active ingredient, and describes that the callus inducer is applicable to a wide range of plants.
Furthermore, Patent Document 3 discloses a method for preparing differentiated embryogenic callus (DEC) tissue of sorghum using a callus induction medium supplemented with one or more auxins, one or more cytokinins, and one or more agents that reduce oxidative browning, such as lipoic acid.
The present inventors attempted to search for a novel callus inducer that is applicable to a wide range of plants and is easier to use.

特開2019-24375号公報JP 2019-24375 A 国際公開第2015/186591号パンフレットInternational Publication No. 2015/186591 Brochure 特表2019-520820号公報Patent Publication No. 2019-520820

Tetrahedron135(2023)133333Tetrahedron135(2023)133333

　本発明は、新規のカルス誘導剤、当該カルス誘導剤を用いたカルスの誘導方法及びカルスの製造方法を提供することを課題とする。 The objective of the present invention is to provide a novel callus inducer, a method for inducing callus using said callus inducer, and a method for producing callus.

　本発明者らはこれまでに寄生雑草の幼根伸長抑制剤開発のためアリールオキシアセチルチオ尿素誘導体の合成を行ってきた（非特許文献１）。当該誘導体のうち特定構造を有する化合物に植物のカルス誘導活性を見出し、本発明を完成するに至った。 The inventors have previously synthesized aryloxyacetylthiourea derivatives to develop agents to inhibit the root elongation of parasitic weeds (Non-Patent Document 1). They discovered that compounds with specific structures among these derivatives have the activity of inducing callus in plants, which led to the completion of the present invention.

　即ち、本発明は以下の構成を有する。
＜１＞次式（Ｉ）：

Figure JPOXMLDOC01-appb-C000002
（式中、Ｒはハロゲン原子を示す。）
で示される化合物又はその塩を含有する植物のカルス誘導剤。
＜２＞式（Ｉ）で示される化合物又はその塩のハロゲン原子が塩素又は臭素である＜１＞に記載のカルス誘導剤。
＜３＞植物がイネ科植物、アブラナ科植物、マメ科植物、ナス科植物又はヤマゴボウ科植物のいずれかである＜１＞又は＜２＞に記載のカルス誘導剤。
＜４＞植物体、植物細胞、植物組織片又は植物種子を、＜１＞に記載のカルス誘導剤と接触させ、カルス誘導する工程を含む、カルスの誘導方法。
＜５＞式（Ｉ）で示される化合物又はその塩のハロゲン原子が塩素又は臭素である＜４＞に記載のカルスの誘導方法。
＜６＞植物がイネ科植物、アブラナ科植物、マメ科植物、ナス科植物又はヤマゴボウ科植物のいずれかである＜４＞又は＜５＞に記載のカルスの誘導方法。
＜７＞植物体、植物細胞、植物組織片又は植物種子を、＜１＞に記載のカルス誘導剤と接触させ、カルス誘導し、増殖する工程を含む、カルスの製造方法。
＜８＞式（Ｉ）で示される化合物又はその塩のハロゲン原子が塩素又は臭素である＜７＞に記載のカルスの製造方法。
＜９＞植物がイネ科植物、アブラナ科植物、マメ科植物、ナス科植物又はヤマゴボウ科植物のいずれかである＜７＞又は＜８＞に記載のカルスの製造方法。
＜１０＞
式（Ｉ）で示される化合物又はその塩の植物のカルス誘導のための使用。 
＜１１＞
式（Ｉ）で示される化合物又はその塩のハロゲン原子が塩素又は臭素である＜１０＞に記載の使用。
＜１２＞
植物がイネ科植物、アブラナ科植物、マメ科植物、ナス科植物又はヤマゴボウ科植物のいずれかである＜１０＞又は＜１１＞に記載の使用。
＜１３＞
植物体、植物細胞、植物組織片又は植物種子を、＜１＞に記載のカルス誘導剤と接触させ、カルス誘導する工程と、前記工程においてカルス誘導して得られたカルスをシュート誘導培地に移植し、シュート誘導する工程と、を含む、植物体の製造方法。
＜１４＞
式（Ｉ）で示される化合物又はその塩のハロゲン原子が塩素又は臭素である＜１３＞に記載の植物体の製造方法。
＜１５＞
植物がイネ科植物、アブラナ科植物、マメ科植物、ナス科植物又はヤマゴボウ科植物のいずれかである＜１３＞又は＜１４＞に記載の植物体の製造方法。 That is, the present invention has the following configuration.
<1> The following formula (I):
Figure JPOXMLDOC01-appb-C000002
 (In the formula, R represents a halogen atom.)
A plant callus inducer comprising a compound represented by the formula:
<2> The callus inducer according to <1>, wherein the halogen atom of the compound represented by formula (I) or a salt thereof is chlorine or bromine.
<3> The callus inducer according to <1> or <2>, wherein the plant is any one of a Gramineae plant, a Brassicaceae plant, a Leguminosae plant, a Solanaceae plant, and a Phytolacca family plant.
<4> A method for inducing callus, comprising the step of contacting a plant body, a plant cell, a plant tissue fragment or a plant seed with the callus inducer described in <1> to induce callus.
<5> The method for inducing callus according to <4>, wherein the halogen atom of the compound represented by formula (I) or a salt thereof is chlorine or bromine.
<6> The method for inducing callus according to <4> or <5>, wherein the plant is any one of a Gramineae plant, a Brassicaceae plant, a Leguminosae plant, a Solanaceae plant, and a Phytolacca family plant.
<7> A method for producing callus, comprising the step of contacting a plant body, a plant cell, a plant tissue fragment or a plant seed with the callus inducer described in <1> to induce and proliferate callus.
<8> The method for producing callus described in <7>, wherein the halogen atom of the compound represented by formula (I) or its salt is chlorine or bromine.
<9> The method for producing a callus according to <7> or <8>, wherein the plant is any one of a Gramineae plant, a Brassicaceae plant, a Leguminosae plant, a Solanaceae plant, and a Phytolaccaea plant.
＜10＞
Use of a compound represented by formula (I) or a salt thereof for inducing callus in a plant.
<11>
The use according to <10>, wherein the halogen atom of the compound represented by formula (I) or a salt thereof is chlorine or bromine.
＜12＞
The use according to <10> or <11>, wherein the plant is any one of a Gramineae plant, a Cruciferae plant, a Leguminosae plant, a Solanaceae plant, and a Phytolacca family plant.
＜13＞
A method for producing a plant body, comprising the steps of: contacting a plant body, a plant cell, a plant tissue fragment or a plant seed with the callus inducer described in <1> to induce callus; and transplanting the callus obtained by callus induction in the above step into a shoot induction medium to induce shoots.
＜14＞
The method for producing a plant body according to <13>, wherein the halogen atom of the compound represented by formula (I) or a salt thereof is chlorine or bromine.
＜15＞
The method for producing a plant according to <13> or <14>, wherein the plant is any one of a Gramineae plant, a Cruciferae plant, a Leguminosae plant, a Solanaceae plant, and a Phytolacca family plant.

　本発明のカルス誘導剤は適用できる植物の範囲が広く、従来、カルスの誘導が困難だった植物も適用対象となり得る。特にそのうちでも、干ばつと土壌毒性に対する耐性が高いソルガムを適用対象とすることができる意義は大きい。 The callus inducer of the present invention can be applied to a wide range of plants, including plants for which callus induction was previously difficult. It is particularly significant that the agent can be applied to sorghum, which has high resistance to drought and soil toxicity.

ａ．化合物（II）を含む培地及びＣＩＭでカルス誘導されたシロイヌナズナのカルスの写真である（実施例２）。ｂ．化合物（II）を含む培地、化合物（III）を含む培地及びＣＩＭでカルス誘導されたシロイヌナズナのカルスの写真である。a) Photographs of Arabidopsis callus induced in a medium containing compound (II) and CIM (Example 2). b) Photographs of Arabidopsis callus induced in a medium containing compound (II), a medium containing compound (III), and CIM. 化合物（II）を含む培地及びＣＩＭでカルス誘導された後シュート誘導されたシロイヌナズナの写真である（実施例２）。1 shows photographs of Arabidopsis thaliana in which callus was induced on a medium containing compound (II) and CIM, and then shoots were induced (Example 2). ａ．化合物（II）を含む培地及びＣＩＭでカルス誘導された後シュート誘導されたシロイヌナズナを上から見た写真である（実施例２）。ｂ．シュート誘導率及びシュートの模式図である。Photographs of Arabidopsis thaliana in which shoots were induced after callus induction in a medium containing compound (II) and CIM (Example 2), and a schematic diagram of shoot induction rate and shoots. 化合物（II）を含む培地及びＣＩＭでカルス誘導されたシロイヌナズナのカルスＧＵＳ染色結果を示す写真である（実施例２）Photographs showing the results of GUS staining of Arabidopsis thaliana callus induced in a medium containing compound (II) and CIM (Example 2). 化合物（II）を含む培地でカルス誘導されたソルガムのカルスの写真である（実施例３）。1 is a photograph of sorghum callus induced in a medium containing compound (II) (Example 3). ａ．化合物（II）、Ｋｉｎｅｔｉｏｎ、ＩＰＡ、Ｚｅａｔｉｎを含む培地、ＣＩＭ（ｃｏｎｔｒｏｌ）で育成されたムラサキツメクサの実生の写真である（実施例４）。ｂ．ａのうちＩＰＡと化合物（II）の一部拡大写真である。a) Photograph of red clover seedlings grown in a medium containing compound (II), Kinetion, IPA, and Zeatin, and CIM (control) (Example 4). b) Partially enlarged photograph of IPA and compound (II) in a. 化合物（II）を含む培地及びＦＰＸを含む培地でカルス誘導されたシロイヌナズナのカルスの写真である（実施例５）。1 shows photographs of Arabidopsis thaliana callus induced on a medium containing compound (II) and a medium containing FPX (Example 5). 化合物（II）を含む培地及びＦＰＸを含む培地でカルス誘導されたソルガム・ビコロのカルスの写真である（実施例６）。1 shows photographs of callus of Sorghum bicolor induced in a medium containing Compound (II) and a medium containing FPX (Example 6). シロイヌナズナの組織片の外植体の調製、カルス誘導及びシュート誘導の流れを示した模式図である（実施例７）。FIG. 1 is a schematic diagram showing the steps of explant preparation, callus induction and shoot induction of tissue explants of Arabidopsis thaliana (Example 7). 化合物（II）を含む培地及びＣＩＭでカルス誘導されたシロイヌナズナの根の外植体の写真である（実施例７）。1 shows photographs of Arabidopsis root explants in which callus was induced on a medium containing Compound (II) and CIM (Example 7). 化合物（II）を含む培地及びＣＩＭでカルス誘導された後、シュート誘導されたシロイヌナズナの根の外植体の写真である（実施例７）。1 shows photographs of Arabidopsis root explants that were induced into shoots after callus induction on a medium containing Compound (II) and CIM (Example 7). ベンサミアナタバコとその組織片の外植体を調製したプレートを上から見た写真である（実施例８）。1 is a top view of a plate on which explants of Nicotiana benthamiana and its tissue fragments were prepared (Example 8). 化合物（II）を含む培地及びＣＩＭでカルス誘導されたベンサミアナタバコのシュート、胚軸、根及び根端の外植体の写真である（実施例８）。1 shows photographs of shoot, hypocotyl, root and root tip explants of Nicotiana benthamiana for which callus was induced on a medium containing compound (II) and CIM (Example 8). 化合物（II）を含む培地でカルス誘導されたヨウシュヤマゴボウの写真である（実施例９）。1 is a photograph of Phytolacca americana in which callus was induced in a medium containing compound (II) (Example 9).

　本発明のカルス誘導剤は、植物に対してカルス誘導活性を有し、次式（Ｉ）で示される化合物又はその塩（以下、化合物（Ｉ）又はその塩ともいう。）を有効成分とすることを特徴とする。 The callus inducer of the present invention is characterized by having callus induction activity in plants and containing as an active ingredient a compound represented by the following formula (I) or a salt thereof (hereinafter also referred to as compound (I) or a salt thereof).

Figure JPOXMLDOC01-appb-C000003
（式中、Ｒはハロゲン原子を示す。）
Figure JPOXMLDOC01-appb-C000003
 (In the formula, R represents a halogen atom.)

（Ｉ）で示される化合物中のハロゲン原子としては、例えばフッ素、塩素、臭素、ヨウ素が挙げられる。このうちでも塩素又は臭素が好ましく、塩素がよりいっそう好ましい。
　ハロゲン原子Ｒが塩素（Ｃｌ）の場合を式（II）、臭素（Ｂｒ）の場合を式（III）に示す。 Examples of the halogen atom in the compound represented by (I) include fluorine, chlorine, bromine and iodine. Among these, chlorine or bromine is preferred, and chlorine is even more preferred.
The formula (II) shows the case where the halogen atom R is chlorine (Cl), and the formula (III) shows the case where the halogen atom R is bromine (Br).

Figure JPOXMLDOC01-appb-C000004
Figure JPOXMLDOC01-appb-C000004

Figure JPOXMLDOC01-appb-C000005
Figure JPOXMLDOC01-appb-C000005

　前記式（Ｉ）で示される化合物は、本発明者らによるTetrahedron135(2023)133333‘Synthesis of aryloxyacetylthiourea derivatives for the development of radicle elongation inhibitor of parasitic weeds’（非特許文献１）に記載された方法により合成することができる。前記式（II）で示される化合物（以下、化合物（II）ともいう。）は、3-[2-(4-Chlorophenoxy)acetyl]-1-(3,4-dichlorophenyl)thioureaであり、同文献の化合物「３ａｂ」に相当する。また、前記式（III）で示される化合物（以下、化合物（III）ともいう。）は、3-[2-(4-Bromophenoxy)acetyl]-1-(3,4-dichlorophenyl)thioureaであり、同文献の化合物「３ａｃ」に相当する。前記化合物の塩としては、本発明のカルス誘導活性を有する塩であれば特に限定されない。 The compound represented by the formula (I) can be synthesized by the method described in Tetrahedron135(2023)133333'Synthesis of aryloxyacetylthiourea derivatives for the development of radicle elongation inhibitor of parasitic weeds' (Non-Patent Document 1) by the present inventors. The compound represented by the formula (II) (hereinafter also referred to as compound (II)) is 3-[2-(4-Chlorophenoxy)acetyl]-1-(3,4-dichlorophenyl)thiourea, which corresponds to compound "3ab" in the same document. The compound represented by the formula (III) (hereinafter also referred to as compound (III)) is 3-[2-(4-Bromophenoxy)acetyl]-1-(3,4-dichlorophenyl)thiourea, which corresponds to compound "3ac" in the same document. There are no particular limitations on the salt of the compound, so long as it is a salt that has the callus-inducing activity of the present invention.

　本発明のカルス誘導剤は、化合物（Ｉ）又はその塩を有効成分として含むものであればよく、カルスの誘導活性を阻害しない範囲で化合物（Ｉ）又はその塩以外のその他の成分を含むものであっても良い。その他の成分として、例えば、賦形剤、乳化剤、湿潤剤等の公知の製剤用添加剤等が挙げられる。
　また、本発明のカルス誘導剤の形態は特に限定されず、カルス誘導可能な形態であればいかなる形態であってもよい。例えば、乳剤、液剤、油剤、水溶液、水和剤、フロアブル、粉剤、微粒剤、粒剤、顆粒剤、エアゾール、ペースト剤、又はゼリー剤等の形態等が挙げられる。 The callus inducer of the present invention may contain compound (I) or a salt thereof as an active ingredient, and may contain other ingredients other than compound (I) or a salt thereof to the extent that the callus induction activity is not inhibited. Examples of other ingredients include known pharmaceutical additives such as excipients, emulsifiers, and wetting agents.
The form of the callus inducer of the present invention is not particularly limited, and may be any form that allows callus induction, such as an emulsion, liquid, oil, aqueous solution, wettable powder, flowable, powder, microgranule, granule, aerosol, paste, or jelly.

　本発明のカルス誘導剤の適用対象となる植物は、特に限定されない。例えば、被子植物及び裸子植物のいずれであってもよく、被子植物の場合、単子葉植物及び双子葉植物のいずれであってもよい。
　裸子植物の場合、例えば、ソテツ科（ソテツ等）、イチョウ科（イチョウ等）、マツ科（アカマツ、クロマツ、モミ、トウヒ等）、スギ科（スギ等）、イチイ科（イチイ等）が挙げられるが、これらに限定されるものではない。 The plant to which the callus inducer of the present invention is applied is not particularly limited. For example, it may be either angiosperms or gymnosperms, and in the case of angiosperms, it may be either monocotyledonous or dicotyledonous.
Examples of gymnosperms include Cycadaceae (cycads, etc.), Ginkgoaceae (ginkgo, etc.), Pinaceae (pine, Japanese black pine, fir, spruce, etc.), Cupressaceae (cedar, etc.), and Taxaceae (yew, etc.), but are not limited to these.

　被子植物の場合、単子葉植物としては、例えば、イネ科（イネ、コムギ、オオムギ、カラスムギ、ライムギ、キビ、アワ、ヒエ、トウモロコシ、シコクビエ、モロコシ、タケ、ヨシ、ススキ、アマランサス、ミスカンサス、スイッチグラス、ソルガム、ブラキポディウム等）、サトイモ科（サトイモ、ウキクサ等）、ヤシ科（ヤシ、ナツメヤシ等）、バショウ科（バナナ等）が挙げられるが、これらに限定されるものではない。
　また、双子葉植物としては、例えば、アブラナ科（シロイヌナズナ、ナタネ、キャベツ、ブロッコリー、カリフラワー等）、ナス科（ナス、トマト、ジャガイモ、タバコ等）、ヤマゴボウ科（ヨウシュヤマゴボウ等）、ウリ科（メロン、カボチャ等）、マメ科（ダイズ、ムラサキツメクサ等）、アオイ科（ワタ等）、キク科（キク、ニンジン等）、ヤナギ科（ポプラ等）、ブドウ科（ブドウ等）、バラ科（バラ、リンゴ等）、ラン科（ラン等）、ユリ科（ユリ、チューリップ、タマネギ等）、ヤナギ科（ポプラ等）、フトモモ科（ユーカリ等）、トウダイグサ科（キャッサバ等）、ハマウツボ科（ストライガ）が挙げられるが、これらに限定されるものではない。 In the case of angiosperms, examples of monocotyledonous plants include, but are not limited to, Poaceae (rice, wheat, barley, oat, rye, millet, foxtail millet, barnyard millet, corn, finger millet, sorghum, bamboo, reed, Miscanthus, amaranth, miscanthus, switchgrass, sorghum, brachypodium, etc.), Araceae (taro, duckweed, etc.), Palmaceae (palm, date palm, etc.), Musaceae (banana, etc.).
Examples of dicotyledonous plants include, but are not limited to, Brassicaceae (Arabidopsis thaliana, rapeseed, cabbage, broccoli, cauliflower, etc.), Solanaceae (eggplant, tomato, potato, tobacco, etc.), Pyretaceae (American pokeweed, etc.), Cucurbitaceae (melon, pumpkin, etc.), Fabaceae (soybean, red clover, etc.), Malvaceae (cotton, etc.), Asteraceae (chrysanthemum, carrot, etc.), Salicaceae (poplar, etc.), Vitaceae (grape, etc.), Rosaceae (rose, apple, etc.), Orchidaceae (orchid, etc.), Liliaceae (lily, tulip, onion, etc.), Salicaceae (poplar, etc.), Myrtaceae (eucalyptus, etc.), Euphorbiaceae (cassava, etc.), and Orobanchaceae (Striga).

　本発明のカルス誘導剤の適用対象となる植物は、好ましくは、イネ科植物、アブラナ科植物、マメ科植物、ナス科植物又はヤマゴボウ科植物のいずれかである。
　イネ科植物として適用対象となるソルガムには、ソルガム・ビコロ（Sorghum bicolor）に加えて、例えば、ソルガム・ハレペンセ（Sorghum halepense）、ソルガム・アルマム（Sorghum almum）、スーダングラス（Sorghum sudanense）、ソルガム・プロピンキューム（Sorghum propinquum）等の任意の種も含まれる。 The plant to which the callus inducer of the present invention is applied is preferably any one of a Gramineae plant, a Cruciferae plant, a Leguminosae plant, a Solanaceae plant, and a Phytolaccaea plant.
Sorghum to be applied as a grass plant includes, in addition to Sorghum bicolor, any species such as Sorghum halepense, Sorghum almum, Sorghum sudanense, and Sorghum propinquum.

　本発明の「カルスの誘導方法」は、植物体、植物細胞、植物組織片又は植物種子等を、本発明のカルス誘導剤と接触させ、カルス誘導する工程を含む方法のことをいう。
　また、本発明の「カルスの製造方法」は、植物体、植物細胞、植物組織片又は植物種子を、本発明のカルス誘導剤と接触させ、カルス誘導し、誘導されたカルスをさらに増殖する工程を含む方法のことをいう。これらの方法には、誘導対象となる植物種子をあらかじめ滅菌する工程や、滅菌後発芽させる工程等を含むことができる。 The "method for inducing callus" of the present invention refers to a method comprising the step of contacting a plant body, a plant cell, a plant tissue fragment, a plant seed, or the like with the callus inducer of the present invention to induce callus.
The "method for producing callus" of the present invention refers to a method comprising the steps of contacting a plant body, a plant cell, a piece of plant tissue, or a plant seed with the callus inducer of the present invention to induce callus, and then further growing the induced callus. These methods may include a step of sterilizing the plant seed to be induced in advance, or a step of germinating the plant seed after sterilization.

　本発明の「カルスの誘導方法」又は「カルスの製造方法」において、植物体等と本発明のカルス誘導剤とを接触させる方法は特に制限はなく、植物の種類、対象器官、カルス誘導剤の剤形等に応じて、浸漬、塗布、散布、培地への添加等を適宜選択することができる。特に、本発明のカルス誘導剤を含有する培地で植物体等を培養することにより行うことが好ましい。 In the "callus induction method" or "callus production method" of the present invention, there is no particular limitation on the method of contacting the plant body, etc. with the callus inducer of the present invention, and methods such as immersion, coating, spraying, addition to a medium, etc. can be appropriately selected depending on the type of plant, the target organ, the dosage form of the callus inducer, etc. In particular, it is preferable to carry out this by culturing the plant body, etc. in a medium containing the callus inducer of the present invention.

　本発明の「カルスの誘導方法」又は「カルスの製造方法」に使用する「植物体、植物細胞、植物組織片又は植物種子」はカルス誘導が可能なものであればどのようなものでもよい。植物の個体全体であってもよく、植物組織の場合は、例えば、茎頂、茎、葉、苗条、胚細胞、根弁、果実、表皮等であってもよい。これらの植物体等は無菌播種により育成した植物体等であるか、７０％エタノールや次亜塩素酸ナトリウム水溶液等で滅菌し、無菌状態にした植物体等であることが好ましい。 The "plant body, plant cell, plant tissue piece, or plant seed" used in the "callus induction method" or "callus production method" of the present invention may be any that is capable of callus induction. It may be the entire plant, and in the case of plant tissue, it may be, for example, the shoot tip, stem, leaf, shoot, embryo cell, root petal, fruit, epidermis, etc. It is preferable that these plant bodies are plants that have been grown by aseptic sowing, or plants that have been sterilized with 70% ethanol, sodium hypochlorite aqueous solution, etc., and made sterile.

　本発明の「カルスの誘導方法」又は「カルスの製造方法」において、カルス誘導培地は、前記式（Ｉ）で示される化合物又はその塩を含み、カルスを誘導できるものであれば特に限定されない。式（Ｉ）で示される化合物は、カルス誘導培地に１種類添加しても良く、２種類以上を組み合わせて添加しても良い。 In the "callus induction method" or "callus production method" of the present invention, the callus induction medium is not particularly limited as long as it contains the compound represented by formula (I) or a salt thereof and is capable of inducing callus. The compound represented by formula (I) may be added to the callus induction medium in one type or in a combination of two or more types.

　前記式（Ｉ）で示される化合物又はその塩の培地中の濃度は、対象とする植物や処理条件に応じて本発明のカルス誘導効果が発揮される濃度で使用すればよい。当該濃度は、特に限定されないが、例えば、０．０１～１００μＭの範囲で添加することができる。好ましくは、０．１～１０μＭであり、さらに好ましくは０．２～５μＭである。２種類以上の化合物を組み合わせる場合（例えば化合物(II)と化合物（III））は、これらの合計量が上記濃度範囲となるように調整すればよい。
　また、カルス誘導培地には、これらの化合物又はその塩以外の、その他の成分を含むことができる。このような成分として、糖類、ゲル化剤、無機塩類などカルス誘導に一般的に用いられるものが挙げられる。また、本発明のカルス誘導剤の作用を妨げない範囲で、オーキシン、サイトカイニン等の他の植物ホルモンを併用しても良い。前記式（Ｉ）で示される化合物又はその塩は、オーキシン、カイネチン、サイトカイニン等の従来カルス誘導で必要とされていた成分と併用しなくても、単独でカルスを誘導することができる。 The compound represented by formula (I) or its salt may be used in a medium at a concentration that allows the callus induction effect of the present invention to be exhibited depending on the target plant and treatment conditions. The concentration is not particularly limited, but may be added in the range of, for example, 0.01 to 100 μM. The concentration is preferably 0.1 to 10 μM, and more preferably 0.2 to 5 μM. When two or more compounds are combined (e.g., compound (II) and compound (III)), the total amount of these compounds may be adjusted to be within the above concentration range.
The callus induction medium may contain other components in addition to these compounds or their salts. Such components include sugars, gelling agents, inorganic salts, and other components commonly used in callus induction. Other plant hormones such as auxin and cytokinin may also be used in combination with the callus inducer of the present invention, as long as they do not interfere with the action of the callus inducer. The compound represented by formula (I) or its salt can induce callus alone, without being used in combination with components that have been required for conventional callus induction, such as auxin, kinetin, and cytokinin.

　植物体等を本発明のカルス誘導剤を含有する培地で培養する場合、カルスを誘導するための培養は無菌条件下で行うことが好ましい。また、培養時の温度は２０～２５℃とすることが好ましく、光条件は恒常的光照射から恒常的暗所条件の間とすることが好ましい。培養はカルスが誘導されるまで行うことができるが、通常、培養から２～４週間でカルスの誘導が認められる。
　本発明の「カルスの製造方法」ではさらに、誘導されたカルスを増殖するために継代培養等を行うことができる。継代培養としては、例えば、一月おきに、培地を本発明のカルス誘導剤を含むカルス誘導培地に交換する等の方法が挙げられる。 When culturing a plant body or the like in a medium containing the callus inducer of the present invention, the culturing for inducing callus is preferably carried out under sterile conditions. The temperature during the culturing is preferably 20 to 25°C, and the light conditions are preferably between constant light irradiation and constant darkness. The culturing can be carried out until callus is induced, but callus induction is usually observed 2 to 4 weeks after the culturing.
In the "method for producing callus" of the present invention, furthermore, subculture or the like can be carried out to grow the induced callus. For example, the subculture method includes a method of replacing the medium with a callus induction medium containing the callus inducer of the present invention every other month.

　本発明の方法によって製造されたカルスより、シュートを誘導し、植物器官を形成することもできる。形成される植物器官は特に制限されず、例えば、葉、根、茎等が挙げられる。カルスから形成した植物器官は、必要に応じて所定の条件下でさらに培養して植物体を形成してもよい。
　シュートの誘導や、植物器官を形成するための培養条件は特に制限されず、従来と同様の培養条件であってもよく、対象となる植物に応じて改良された培養条件であってもよい。
　前記形成された植物器官や植物体はいずれの用途にも使用することができる。例えば、食品用、医薬品用、化粧品用、木材用、建設資材用、飼料用、鑑賞用、バイオ燃料用、試験研究用等に使用することができる。 Shoots can also be induced from the callus produced by the method of the present invention to form plant organs. The plant organs formed are not particularly limited, and examples thereof include leaves, roots, stems, etc. The plant organs formed from the callus may be further cultured under specified conditions as necessary to form a plant body.
The culture conditions for inducing shoots and forming plant organs are not particularly limited, and may be the same as conventional culture conditions or may be culture conditions that have been improved depending on the target plant.
The plant organs and plants thus formed can be used for any purpose, such as food, medicine, cosmetics, lumber, construction materials, feed, ornamental purposes, biofuel, and research purposes.

　本発明の方法によって誘導されたカルスは、遺伝子組み換え作物の作製等に利用することができる。例えば、目的遺伝子を含むプラスミドを有するアグロバクテリウムを本発明により誘導されたカルスに感染させた後、プラスミドが染色体に挿入されたカルスを分離し、これを再分化させることで、遺伝子組み換え植物体を作製することができる。
　以下、本発明について下記の実施例にもとづき具体的に説明するが、当該実施例に限定されるものではない。 The callus induced by the method of the present invention can be used for producing genetically modified crops, etc. For example, a genetically modified plant can be produced by infecting the callus induced by the present invention with Agrobacterium having a plasmid containing a target gene, isolating the callus in which the plasmid has been inserted into the chromosome, and redifferentiating the callus.
The present invention will now be described in detail with reference to the following examples, but is not limited to these examples.

［実施例１］カルス誘導剤の調整
　化合物（II）及び化合物(III)をカルス誘導活性を有する物質として以下の実施例の試験に用いた。化合物（II）及び化合物(III)は、本発明者らによるTetrahedron135(2023)133333‘Synthesis of aryloxyacetylthiourea derivatives for the development of radicle elongation inhibitor of parasitic weeds’に記載された方法により合成した。化合物（II）は、3-[2-(4-Chlorophenoxy)acetyl]-1-(3,4-dichlorophenyl)thioureaであり、同文献の化合物「３ａｂ」に相当する。また、化合物（III）は、3-[2-(4-Bromophenoxy)acetyl]-1-(3,4-dichlorophenyl)thioureaであり、同文献の化合物「３ａｃ」に相当する。 [Example 1] Preparation of callus inducer Compound (II) and compound (III) were used in the following examples as substances having callus induction activity. Compound (II) and compound (III) were synthesized by the present inventors according to the method described in Tetrahedron135(2023)133333'Synthesis of aryloxyacetylthiourea derivatives for the development of radicle elongation inhibitor of parasitic weeds'. Compound (II) is 3-[2-(4-Chlorophenoxy)acetyl]-1-(3,4-dichlorophenyl)thiourea, which corresponds to compound "3ab" in the same document. Compound (III) is 3-[2-(4-Bromophenoxy)acetyl]-1-(3,4-dichlorophenyl)thiourea, which corresponds to compound "3ac" in the same document.

Figure JPOXMLDOC01-appb-C000006
Figure JPOXMLDOC01-appb-C000006

Figure JPOXMLDOC01-appb-C000007
Figure JPOXMLDOC01-appb-C000007

［実施例２］本発明のカルス誘導剤のシロイヌナズナ（Arabidopsis thaliana）に対するカルス誘導活性の評価
１．試験方法
　次の工程により、シロイヌナズナ（Arabidopsis thaliana）のカルス誘導を行い、さらにシュート誘導を行った。
（１）発芽処理
　シロイヌナズナの種子を７０％エタノールで２分間処理した後、１％次亜塩素酸ナトリウムで１０分間処理することによって滅菌した。この種子を１／２ＭＳ培地に播種して１６ｈ明期／８ｈ暗期、２５℃で４～５日間生育させた。
（２）カルス誘導
　前記発芽処理をされたシロイヌナズナの根端から１ｃｍ程度を化合物（II）を含む培地（１／２ＭＳ培地、１％スクロース、０．２６μＭ又は２．６μＭ_化合物（II））又は化合物（III）を含む培地（１／２ＭＳ培地、１％スクロース、０.２３μＭ_化合物（III））に移植した。
　また、比較として従来のカルス誘導培地（以下ＣＩＭという、１／２ＭＳ培地、１％スクロース、０．４６μＭ　カイネチン、２．２６μＭ_２，４－Ｄ）にも移植した。
　これらを１６ｈ明期／８ｈ暗期、２５℃で４週間培養し、カルスの誘導を行った。
（３）シュート誘導
　前記カルス誘導をされたカルスをシュート誘導培地（１／２ＭＳ培地、１．１４μＭ_ＩＡＡ、１２．３μＭ_ＩＰＡ(isopentenyl adenine）に移植し、１６ｈ明期／８ｈ暗期、２５℃でシュート誘導を行った。
（４）カルスＧＵＳ染色
　前記カルス誘導されたシロイヌナズナのカルスについてＧＵＳ染色を行った。ＧＵＳ染色は次の手順により行った。植物体を９０％アセトンに１５分間４℃で浸漬（固定）し、リン酸ｂｕｆｆｅｒで１分間ずつ３回洗浄した。その後暗室下でＧＵＳ染色液処理し、減圧下（約８００ｈＰａ）で３０分間静置した。その後暗室下３７℃で一晩静置し、７０％エタノールで洗浄した。倒立顕微鏡により側根形成誘導部位の観察を行った。ＧＵＳ染色液の組成を以下に示す。
＜ＧＵＳ染色液（１mLあたり）＞
0.2 Mリン酸buffer(pH 7.0)　 500μL
0.5 M EDTA　　　　　　　　　20μL
0.05 M Fe(II)　　　　　　　 100μL
0.05 M Fe(III)　　　　　　　100μL
1%トリトン　　　　　　　　　100μL
50 μg/ml X-gluc　　　　　　20μL
DW　　　　　　　　　　　　　160μL Example 2 Evaluation of the callus inducing activity of the callus inducer of the present invention on Arabidopsis thaliana 1. Test method Callus induction was carried out on Arabidopsis thaliana by the following steps, followed by shoot induction.
(1) Germination Treatment Arabidopsis seeds were sterilized by treating with 70% ethanol for 2 minutes and then with 1% sodium hypochlorite for 10 minutes. The seeds were sown on 1/2 MS medium and grown at 25°C for 4 to 5 days under a 16-h light/8-h dark cycle.
(2) Callus induction About 1 cm from the root tip of the germinated Arabidopsis was transplanted into a medium containing compound (II) (1/2 MS medium, 1% sucrose, 0.26 μM or 2.6 μM compound (II)) or a medium containing compound (III) (1/2 MS medium, 1% sucrose, 0.23 μM compound (III)).
For comparison, the callus was also transferred to a conventional callus induction medium (hereinafter referred to as CIM, 1/2 MS medium, 1% sucrose, 0.46 μM kinetin, 2.26 μM 2,4-D).
These were cultured under a 16-hour light/8-hour dark cycle at 25° C. for 4 weeks to induce callus.
(3) Shoot induction The callus induced as described above was transferred to a shoot induction medium (1/2 MS medium, 1.14 μM IAA, 12.3 μM IPA (isopentenyl adenine)) and shoot induction was carried out at 25° C. under a 16-hour light/8-hour dark period.
(4) Callus GUS staining The callus of Arabidopsis thaliana induced by the callus induction was stained with GUS. GUS staining was performed according to the following procedure. The plant body was immersed (fixed) in 90% acetone at 4°C for 15 minutes, and washed three times with phosphate buffer for 1 minute each. It was then treated with GUS staining solution in a dark room and left to stand for 30 minutes under reduced pressure (about 800 hPa). It was then left to stand overnight at 37°C in a dark room, and washed with 70% ethanol. The lateral root formation induction site was observed using an inverted microscope. The composition of the GUS staining solution is shown below.
<GUS staining solution (per 1 mL)>
0.2 M phosphate buffer (pH 7.0) 500 μL
20μL 0.5M EDTA
0.05M Fe(II) 100μL
0.05M Fe(III) 100μL
1% Triton 100μL
50 μg/ml X-gluc 20 μL
DW 160μL

２．形態観察結果
　カルス誘導における培養開始１４日目及び２０日目のシロイヌナズナのカルスの状態を図１に示した。本図によれば本発明の化合物(II)及び（III）は、カルス誘導活性を有することを確認できた。特に培養開始２０日目には、ＣＩＭに比較して明らかに大きなカルスが形成されていることが確認できる。
　また、シュート誘導されたシロイヌナズナの状態を図２及び図３に示した。その結果、ＣＩＭによって誘導されたカルスのシュート誘導率は２５％（４個中１個）であったのに対して、化合物（II）によって誘導されたカルスのシュート誘導率は１００％（８個中８個）であった（図３ｂ）。また、本発明のカルス誘導剤で誘導されたカルスは、培地に接触している部分（つまり、カルスの下側）からシュートが誘導された。それに対し、ＣＩＭで誘導されたカルスは、カルスの上部からシュートが再生されるという違いが観察された（図３ａ、ｂ）。 2. Morphological Observation Results The state of Arabidopsis callus on the 14th and 20th days after the start of culture in callus induction is shown in Figure 1. From this figure, it was confirmed that the compounds (II) and (III) of the present invention have callus inducing activity. In particular, it was confirmed that a callus was formed that was clearly larger than that of CIM on the 20th day after the start of culture.
The state of shoot-induced Arabidopsis thaliana is shown in Figures 2 and 3. As a result, the shoot induction rate of the callus induced by CIM was 25% (1 out of 4), whereas the shoot induction rate of the callus induced by compound (II) was 100% (8 out of 8) (Figure 3b). In addition, in the callus induced by the callus inducer of the present invention, shoots were induced from the part in contact with the medium (i.e., the lower side of the callus). In contrast, in the callus induced by CIM, shoots were regenerated from the upper part of the callus, a difference being observed (Figures 3a and 3b).

　カルス誘導における培養開始２日目、７日目及び１０日目のシロイヌナズナのカルスのＧＵＳ染色結果を図４に示す。本図によれば０．２６μＭの化合物(II)を含む培地でカルス誘導されたシロイヌナズナは、培養開始７日目から主根及び側根の頂端における特異的なＧＵＳ染色が観察されたのに対し、ＣＩＭでカルス誘導されたシロイヌナズナは、培養開始７日目から側根形成誘導部位が広い範囲にわたって染色されている様子が観察された。同様の染色は２．６μＭの化合物(II)を含む培地でカルス誘導されたシロイヌナズナの培養開始１０日目にも観察された。 The results of GUS staining of Arabidopsis callus on the 2nd, 7th, and 10th days after the start of culture during callus induction are shown in Figure 4. According to this figure, specific GUS staining was observed in the apex of the primary root and lateral roots from the 7th day after the start of culture in Arabidopsis callus induced on a medium containing 0.26 μM compound (II), whereas in Arabidopsis callus induced in CIM, staining was observed over a wide area of the lateral root formation induction site from the 7th day after the start of culture. Similar staining was also observed on the 10th day after the start of culture in Arabidopsis callus induced on a medium containing 2.6 μM compound (II).

［実施例３］本発明のカルス誘導剤のソルガム・ビコロ（Sorghum bicolor）に対するカルス誘導活性の評価
　次の工程により、ソルガム・ビコロ（Sorghum bicolor）のカルス誘導を行った。
１．発芽処理及びカルス誘導
　ソルガム・ビコロの種子を７０％エタノールで２分間処理した後、１％次亜塩素酸ナトリウムで６０分間処理することによって滅菌した。この種子を化合物（II）を含む培地（１／２ＭＳ培地、１％スクロース、２．６μＭ_化合物（II））に播種した。また、比較としてＣＩＭにも播種した。播種後暗所、２５℃で４週間培養し、カルス誘導を行った。 Example 3 Evaluation of callus inducing activity of the callus inducer of the present invention on Sorghum bicolor Callus induction in Sorghum bicolor was carried out according to the following steps.
1. Germination treatment and callus induction Sorghum bicolor seeds were sterilized by treating with 70% ethanol for 2 minutes, and then with 1% sodium hypochlorite for 60 minutes. The seeds were sown in a medium containing compound (II) (1/2 MS medium, 1% sucrose, 2.6 μM compound (II)). For comparison, the seeds were also sown in CIM. After sowing, the seeds were cultured in the dark at 25° C. for 4 weeks to induce callus.

２．形態観察結果
　カルス誘導における培養開始４日目、１０日目及び２０日目のソルガム・ビコロのカルスの状態を図５に示した。本発明のカルス誘導剤は、ソルガムについてもカルス誘導活性があることを確認した。 2. Morphological Observation Results The state of callus of Sorghum bicolor on days 4, 10, and 20 after the start of culture in callus induction is shown in Fig. 5. It was confirmed that the callus inducer of the present invention also has callus inducing activity for sorghum.

［実施例４］本発明のカルス誘導剤のムラサキツメクサ（Trifolium pratense）に対するカルス誘導活性の評価
　次の工程により、ムラサキツメクサ（Trifolium pratense）のカルス誘導を行った。
１．カルス誘導
　ムラサキツメクサの種子を７０％エタノールで、２分間処理した後、１％次亜塩素酸ナトリウムで６０分間処理することによって滅菌した。この種子を化合物（II）を含む培地（１／２ＭＳ培地、１％スクロース、１ｐｐｍ（２．６μＭ）_化合物（II））に播種した。また、比較として化合物（II）の代りに１ｐｐｍのカイネチン、ゼアチン、１ｐｐｍ又は１０ｐｐｍのＩＰＡをそれぞれ含む培地、ＣＩＭにも播種した。播種後４週間培養し、カルス誘導を行った。 Example 4 Evaluation of callus inducing activity of the callus inducer of the present invention on red clover (Trifolium pratense) Callus induction on red clover (Trifolium pratense) was carried out by the following steps.
1. Callus induction Red clover seeds were sterilized by treating with 70% ethanol for 2 minutes, and then with 1% sodium hypochlorite for 60 minutes. The seeds were sown in a medium containing compound (II) (1/2 MS medium, 1% sucrose, 1 ppm (2.6 μM)-compound (II)). For comparison, the seeds were also sown in a medium, CIM, containing 1 ppm kinetin, zeatin, 1 ppm or 10 ppm IPA instead of compound (II). After sowing, the seeds were cultured for 4 weeks to induce callus.

２．形態観察結果
　カルス誘導における培養開始７日目の各培地により培養されたムラサキツメクサの状態を図６ａに示した。図６ａに示したムラサキツメクサのうち、ＩＰＡを含む培地で培養されたムラサキツメクサと化合物（II）を含む培地で培養されたムラサキツメクサの一部拡大図を図６ｂに示した。本発明のカルス誘導剤はムラサキツメクサについてもカルス誘導活性があることを確認した。一方、カイネチン、ゼアニン、ＩＰＡ等のサイトカイニンを添加した培地では、根の伸長が抑制されるもののカルス誘導活性は確認できなかった。 2. Morphological Observation Results The state of red clover cultured in each medium on the 7th day after the start of culture in callus induction is shown in Figure 6a. Of the red clover shown in Figure 6a, a partial enlargement of the red clover cultured in a medium containing IPA and the red clover cultured in a medium containing compound (II) is shown in Figure 6b. It was confirmed that the callus inducer of the present invention also has callus induction activity in red clover. On the other hand, in the medium containing cytokinins such as kinetin, zeanin, and IPA, root elongation was suppressed, but callus induction activity was not confirmed.

［実施例５］化合物（II）とフィペキシド（fipexide; 1-[(p-chlorophenoxy)acetyl]-4-piperonylpiperazine、1-(4-chlorophenoxyacetyl)-4-(1,3-benzodioxole-5-ylmethyl)piperazine、以下ＦＰＸという。）のシロイヌナズナ（Arabidopsis thaliana）に対するカルス誘導活性の比較
　次の工程により、シロイヌナズナ（Arabidopsis thaliana）のカルス誘導を行った。
１．カルス誘導
　シロイヌナズナの種子を化合物（II）を含む培地（１／２ＭＳ培地、１％スクロース、０．１又は１．０μＭ_化合物（II））又はＦＰＸを含む培地（１／２ＭＳ培地、１％スクロース、１．０μＭ_ＦＰＸ）に播種した。これらを１６ｈ明期／８ｈ暗期、２５℃で４週間培養し、カルス誘導を行った。 Example 5 Comparison of callus induction activity in Arabidopsis thaliana between compound (II) and fipexide (1-[(p-chlorophenoxy)acetyl]-4-piperonylpiperazine, 1-(4-chlorophenoxyacetyl)-4-(1,3-benzodioxole-5-ylmethyl)piperazine, hereinafter referred to as FPX) Callus induction in Arabidopsis thaliana was carried out by the following steps.
1. Callus induction Arabidopsis seeds were sown in a medium containing compound (II) (1/2 MS medium, 1% sucrose, 0.1 or 1.0 μM compound (II)) or a medium containing FPX (1/2 MS medium, 1% sucrose, 1.0 μM FPX). These were cultured at 25° C. for 4 weeks under a 16-h light/8-h dark period to induce callus.

２．形態観察結果
　カルス誘導における培養開始７日目及び２８日目のシロイヌナズナの状態を図７に示した。１．０μＭの化合物（II）を含む培地で培養した場合に培養開始２８日目にカルスが形成されていることが確認できる（円で囲んだ部分）。一方で、同濃度（１．０μＭ）のＦＰＸを含む培地で培養した場合には培養開始２８日目においてもカルスは形成されなかった。 2. Morphological Observation Results The state of Arabidopsis thaliana on the 7th and 28th days after the start of culture in callus induction is shown in Figure 7. When cultured in a medium containing 1.0 μM compound (II), callus was confirmed to have formed on the 28th day after the start of culture (circled area). On the other hand, when cultured in a medium containing the same concentration (1.0 μM) of FPX, no callus was formed even on the 28th day after the start of culture.

［実施例６］化合物（II）とＦＰＸのソルガム・ビコロ（Sorghum bicolor）に対するカルス誘導活性の比較
　次の工程により、ソルガム・ビコロ（Sorghum bicolor）のカルス誘導を行った。
１．カルス誘導
　ソルガム・ビコロの種子を７０％エタノールで２分間処理した後、１％次亜塩素酸ナトリウムで６０分間処理することによって滅菌した。この種子を化合物（II）を含む培地（１／２ＭＳ培地、１％スクロース、３．０μＭ_化合物（II））、ＦＰＸを含む培地（１／２ＭＳ培地、１％スクロース、３．０μＭ_ＦＰＸ）に播種した。播種後暗所、２５℃で４週間培養し、カルス誘導を行った。 Example 6 Comparison of callus induction activity of compound (II) and FPX in Sorghum bicolor Callus induction in Sorghum bicolor was carried out according to the following steps.
1. Callus induction Sorghum bicolor seeds were sterilized by treating with 70% ethanol for 2 minutes, and then with 1% sodium hypochlorite for 60 minutes. The seeds were sown in a medium containing compound (II) (1/2 MS medium, 1% sucrose, 3.0 μM compound (II)) or in a medium containing FPX (1/2 MS medium, 1% sucrose, 3.0 μM FPX). After sowing, the seeds were cultured in the dark at 25° C. for 4 weeks to induce callus.

２．形態観察結果
　カルス誘導における培養開始１７日目のソルガム・ビコロの状態を図８に示した。３．０μＭの化合物（II）を含む培地で培養したソルガム・ビコロは、同濃度（３．０μＭ）のＦＰＸを含む培地で培養したソルガム・ビコロよりも明らかに大きなカルスが形成されていることが確認できる。また、ＦＰＸを含む培地で培養したときのカルス誘導率は２０％であったのに対し、化合物（II）を含む培地で培養したときのカルス誘導率は４４％であった。 2. Morphological Observation Results The state of Sorghum bicolo on the 17th day after the start of culture in callus induction is shown in Figure 8. It can be confirmed that the Sorghum bicolo cultured in the medium containing 3.0 μM compound (II) formed a callus that was obviously larger than that of the Sorghum bicolo cultured in the medium containing the same concentration (3.0 μM) of FPX. In addition, the callus induction rate when cultured in the medium containing FPX was 20%, while the callus induction rate when cultured in the medium containing compound (II) was 44%.

［実施例７］本発明のカルス誘導剤のシロイヌナズナ（Arabidopsis thaliana）の根の外植体に対するカルス誘導活性とシュート誘導に対する影響の評価
　次の工程により、シロイヌナズナ（Arabidopsis thaliana）の根の外植体のカルス誘導を行い、さらにシュート誘導を行った。
１．試験方法
（１）発芽処理
　シロイヌナズナの種子を７０％エタノールで２分間処理した後、１％次亜塩素酸ナトリウムで１０分間処理することによって滅菌した。この種子を１／２ＭＳ培地に播種して１６ｈ明期／８ｈ暗期、２５℃で７日間生育させた。
（２）外植体の調製及びカルス誘導
　前記７日間生育させたシロイヌナズナを切断した組織片を化合物（II）を含む培地（１／２ＭＳ培地、１％スクロース、１．０μＭ_化合物（II））に移植し、図９に示すように外植体を調製した。また、比較としてＣＩＭに移植した外植体も調製した。これらを１６ｈ明期／８ｈ暗期、２５℃で４週間培養し、カルスの誘導を行った。
（３）シュート誘導
　前記カルス誘導をされたカルスをシュート誘導培地（１／２ＭＳ培地、１．１４μＭ_ＩＡＡ、１２．３μＭ_ＩＰＡ(isopentenyl adenine））に移植し、１６ｈ明期／８ｈ暗期、２５℃でシュート誘導を行った。 Example 7 Evaluation of the callus induction activity of the callus inducer of the present invention on Arabidopsis thaliana root explants and its effect on shoot induction Callus induction was performed on Arabidopsis thaliana root explants and shoot induction was then performed according to the following steps.
1. Test method (1) Germination treatment Arabidopsis seeds were sterilized by treating with 70% ethanol for 2 minutes and then with 1% sodium hypochlorite for 10 minutes. The seeds were sown on 1/2 MS medium and grown at 25°C for 7 days under a 16-h light/8-h dark cycle.
(2) Preparation of explants and callus induction The tissue pieces cut from the Arabidopsis thaliana grown for 7 days were transplanted into a medium containing compound (II) (1/2 MS medium, 1% sucrose, 1.0 μM compound (II)) to prepare explants as shown in FIG. 9. For comparison, explants transplanted into CIM were also prepared. These were cultured at 25° C. for 4 weeks under a 16-h light/8-h dark period to induce callus.
(3) Shoot Induction The callus induced as described above was transferred to a shoot induction medium (1/2 MS medium, 1.14 μM IAA, 12.3 μM IPA (isopentenyl adenine)) and shoot induction was carried out at 25° C. under a 16-hour light/8-hour dark period.

２．形態観察結果
　カルス誘導における培養開始１０日目、１８日目及び２５日目のシロイヌナズナの根の状態を図１０に示した。シロイヌナズナの根をＣＩＭによってカルス誘導したときのカルス誘導率は培養開始１８日目で１２％（２５個中３個）、培養開始２５日目で７２％（２５個中１８個）であった。一方で、化合物（II）を含む培地によってカルス誘導したときのカルス誘導率はいずれの培養期間でも１００％（２５個中２５個）であった。また、化合物（II）を含む培地により培養されたシロイヌナズナの根は、ＣＩＭにより培養されたものよりも明らかに大きいカルスを形成していることが確認できる。
　シロイヌナズナの根をカルス誘導して得られたカルスをさらにシュート誘導したものの状態を図１１に示した。ＣＩＭにより誘導されたカルスのシュート誘導率は２２％（１８個中４個）であった。一方で、化合物（II）を含む培地により誘導されたカルスのシュート誘導率は５６％（２５個中１４個）であった。この形態観察結果と実施例２の形態観察結果から、本発明のカルス誘導剤は、従来のカルス誘導培地であるＣＩＭと比較して、カルス誘導して得られるカルスのシュート誘導率を上昇させることがわかった。このことは、本発明のカルス誘導剤を用いることによって高頻度で植物体を再構築できることを示す。 2. Morphological Observation Results The state of Arabidopsis roots on the 10th, 18th and 25th days after the start of culture in callus induction is shown in Figure 10. When Arabidopsis roots were induced to callus by CIM, the callus induction rate was 12% (3 out of 25) on the 18th day after the start of culture, and 72% (18 out of 25) on the 25th day after the start of culture. On the other hand, when callus was induced by a medium containing compound (II), the callus induction rate was 100% (25 out of 25) at both culture periods. It was also confirmed that Arabidopsis roots cultured on a medium containing compound (II) formed calli that were obviously larger than those cultured by CIM.
The state of callus obtained by inducing callus from Arabidopsis roots and then inducing shoots is shown in Figure 11. The shoot induction rate of callus induced by CIM was 22% (4 out of 18). On the other hand, the shoot induction rate of callus induced by the medium containing compound (II) was 56% (14 out of 25). From this morphological observation result and the morphological observation result of Example 2, it was found that the callus inducer of the present invention increases the shoot induction rate of callus obtained by callus induction, compared with the conventional callus induction medium CIM. This indicates that plant bodies can be reconstructed with high frequency by using the callus inducer of the present invention.

［実施例８］本発明のカルス誘導剤のベンサミアナタバコ（Nicotiana benthamiana）の外植体に対するカルス誘導活性の評価
　次の工程により、ベンサミアナタバコ（Nicotiana benthamiana）の外植体のカルス誘導を行った。
１．試験方法
（１）発芽処理
　ベンサミアナタバコの種子を７０％エタノールで２分間処理した後、１％次亜塩素酸ナトリウムで１０分間処理することによって滅菌した。この種子を１／２ＭＳ培地に播種して１６ｈ明期／８ｈ暗期、２５℃で７日間生育させた。
（２）外植体の調製及びカルス誘導
　前記７日間生育させたベンサミアナタバコをシュート、胚軸、根、根端の組織片に切断した後、化合物（II）を含む培地（１／２ＭＳ培地、１％スクロース、１．０μＭ_化合物（II））に移植し、図１２に示すように外植体を調製した。また、比較としてＣＩＭに移植した外植体も調製した。これらを１６ｈ明期／８ｈ暗期、２５℃で４週間培養し、カルスの誘導を行った。 Example 8 Evaluation of callus inducing activity of the callus inducer of the present invention on Nicotiana benthamiana explants Callus induction of Nicotiana benthamiana explants was carried out according to the following steps.
1. Test method (1) Germination treatment Nicotiana benthamiana seeds were sterilized by treating with 70% ethanol for 2 minutes and then with 1% sodium hypochlorite for 10 minutes. The seeds were sown on 1/2 MS medium and grown at 25°C for 7 days under a 16-h light/8-h dark period.
(2) Preparation of explants and callus induction The N. benthamiana grown for 7 days was cut into tissue pieces of shoot, hypocotyl, root, and root tip, and then transplanted into a medium containing compound (II) (1/2 MS medium, 1% sucrose, 1.0 μM compound (II)) to prepare explants as shown in FIG. 12. For comparison, explants transplanted into CIM were also prepared. These were cultured at 25° C. for 4 weeks under a 16-h light/8-h dark period to induce callus.

２．形態観察結果
　カルス誘導における培養開始１３日目のベンサミアナタバコのシュート、胚軸、根及び根端の状態を図１３に示した。本発明のカルス誘導剤はベンサミアナタバコについてもカルス誘導活性を有することを確認した。本発明のカルス誘導剤は特に胚軸においてより大きなカルスを誘導した。 2. Morphological Observation Results The state of the shoot, hypocotyl, root and root tip of N. benthamiana on the 13th day after the start of culture for callus induction is shown in Fig. 13. It was confirmed that the callus inducer of the present invention also has callus-inducing activity for N. benthamiana. The callus inducer of the present invention induced larger calli, especially in the hypocotyl.

［実施例９］本発明のカルス誘導剤のヨウシュヤマゴボウ（Phytolacca americana）の外植体に対するカルス誘導活性の評価
　次の工程により、ヨウシュヤマゴボウ（Phytolacca americana）の外植体のカルス誘導を行った。
１．発芽処理及びカルス誘導
　ヨウシュヤマゴボウの種子の種皮を除去し７０％エタノールで２分間処理した後、１％次亜塩素酸ナトリウムで４５分間処理することによって滅菌した。この種子をＭＳ培地に播種して１６ｈ明期／８ｈ暗期、２５℃で３０日間生育させた。前記３０日間生育させたヨウシュヤマゴボウを葉及び０．５～１．０ｃｍ程度の茎に切断した。さらに、前記葉及び０．５～１．０ｃｍ程度の茎を化合物（II）を含む培地（ＭＳ培地、３％スクロース、２．６μＭ_化合物（II））に移植し、外植体を調製した。これを明所、２５℃で４週間培養し、カルスの誘導を行った。 Example 9 Evaluation of callus inducing activity of the callus inducer of the present invention on explants of Phytolacca americana Callus induction of explants of Phytolacca americana was carried out by the following steps.
1. Germination treatment and callus induction The seed coat of pokeweed seeds was removed, and the seeds were treated with 70% ethanol for 2 minutes, and then sterilized by treating with 1% sodium hypochlorite for 45 minutes. The seeds were sown on MS medium and grown for 30 days at 25°C under a 16-h light/8-h dark period. The pokeweed grown for 30 days was cut into leaves and stems of about 0.5 to 1.0 cm. The leaves and stems of about 0.5 to 1.0 cm were then transplanted into a medium containing compound (II) (MS medium, 3% sucrose, 2.6 μM compound (II)) to prepare explants. The explants were cultured in the light at 25°C for 4 weeks to induce callus.

２．形態観察結果
　カルス誘導における培養開始０日目、１５日目及び２８日目のヨウシュヤマゴボウの状態を図１４に示した。腋芽の成長が誘導されたのち、外植体の切断面からカルスが誘導されたことがわかる（円で囲んだ部分）。本発明のカルス誘導剤はヨウシュヤマゴボウについてもカルス誘導活性を有することを確認した。 2. Morphological Observation Results The state of Phytolacca americana on days 0, 15, and 28 after the start of culture for callus induction is shown in Fig. 14. It can be seen that after the growth of axillary buds was induced, callus was induced from the cut surface of the explant (area surrounded by a circle). It was confirmed that the callus inducer of the present invention also has callus induction activity for Phytolacca americana.

　本発明のカルス誘導剤は適用できる植物の範囲が広く、従来、カルスの誘導が出来なかった植物に対してもカルス誘導が可能となった。特に、干ばつと土壌毒性に対する耐性が高いソルガムのカルスを誘導できる意義は大きい。本発明のカルス誘導剤を使用して得られたカルスは、食品用、医薬品用、化粧品用、木材用、建設資材用、飼料用、鑑賞用、バイオ燃料用、試験研究用等、様々な用途に使用し得る。  The callus inducer of the present invention can be applied to a wide range of plants, making it possible to induce callus in plants where it was previously impossible to induce callus. It is particularly significant that callus can be induced in sorghum, which has high resistance to drought and soil toxicity. Callus obtained using the callus inducer of the present invention can be used for a variety of purposes, including food, pharmaceuticals, cosmetics, lumber, construction materials, feed, ornamental purposes, biofuel, and testing and research.

Claims (15)

次式（Ｉ）：
Figure JPOXMLDOC01-appb-C000001
 （式中、Ｒはハロゲン原子を示す。）
で示される化合物又はその塩を含有する植物のカルス誘導剤。 The following formula (I):
Figure JPOXMLDOC01-appb-C000001
 (In the formula, R represents a halogen atom.)
A plant callus inducer comprising a compound represented by the formula: 式（Ｉ）で示される化合物又はその塩のハロゲン原子が塩素又は臭素である請求項１に記載のカルス誘導剤。 The callus inducer according to claim 1, wherein the halogen atom of the compound represented by formula (I) or its salt is chlorine or bromine. 植物がイネ科植物、アブラナ科植物、マメ科植物、ナス科植物又はヤマゴボウ科植物のいずれかである請求項１又は２に記載のカルス誘導剤。 The callus inducer according to claim 1 or 2, wherein the plant is any one of a Gramineae plant, a Cruciferae plant, a Leguminosae plant, a Solanaceae plant, and a Phytolaccaea plant. 植物体、植物細胞、植物組織片又は植物種子を、請求項１に記載のカルス誘導剤と接触させ、カルス誘導する工程を含む、カルスの誘導方法。 A method for inducing callus, comprising the step of contacting a plant body, a plant cell, a piece of plant tissue, or a plant seed with the callus inducer of claim 1 to induce callus. 式（Ｉ）で示される化合物又はその塩のハロゲン原子が塩素又は臭素である請求項４に記載のカルスの誘導方法。 The method for inducing callus according to claim 4, wherein the halogen atom of the compound represented by formula (I) or its salt is chlorine or bromine. 植物がイネ科植物、アブラナ科植物、マメ科植物、ナス科植物又はヤマゴボウ科植物のいずれかである請求項４又は５に記載のカルスの誘導方法。 The method for inducing callus according to claim 4 or 5, wherein the plant is any one of a Gramineae plant, a Cruciferae plant, a Leguminosae plant, a Solanaceae plant, and a Phytolaccaea plant. 植物体、植物細胞、植物組織片又は植物種子を、請求項１に記載のカルス誘導剤と接触させ、カルス誘導し、増殖する工程を含む、カルスの製造方法。 A method for producing callus, comprising the steps of contacting a plant body, a plant cell, a piece of plant tissue, or a plant seed with the callus inducer of claim 1 to induce and grow callus. 式（Ｉ）で示される化合物又はその塩のハロゲン原子が塩素又は臭素である請求項７に記載のカルスの製造方法。 The method for producing callus according to claim 7, wherein the halogen atom of the compound represented by formula (I) or its salt is chlorine or bromine. 植物がイネ科植物、アブラナ科植物、マメ科植物、ナス科植物又はヤマゴボウ科植物のいずれかである請求項７又は８に記載のカルスの製造方法。 The method for producing callus according to claim 7 or 8, wherein the plant is any one of a Gramineae plant, a Cruciferae plant, a Leguminosae plant, a Solanaceae plant, and a Phytolaccaea plant. 式（Ｉ）で示される化合物又はその塩の植物のカルス誘導のための使用。  Use of a compound represented by formula (I) or a salt thereof for inducing callus in a plant. 式（Ｉ）で示される化合物又はその塩のハロゲン原子が塩素又は臭素である請求項１０に記載の使用。 The use according to claim 10, wherein the halogen atom of the compound represented by formula (I) or a salt thereof is chlorine or bromine. 植物がイネ科植物、アブラナ科植物、マメ科植物、ナス科植物又はヤマゴボウ科植物のいずれかである請求項１０又は１１に記載の使用。 The use according to claim 10 or 11, wherein the plant is any one of a grass family, a crucifer family, a legume family, a solanaceae family, and a phytolacca family. 植物体、植物細胞、植物組織片又は植物種子を、請求項１に記載のカルス誘導剤と接触させ、カルス誘導する工程と、前記工程においてカルス誘導して得られたカルスをシュート誘導培地に移植し、シュート誘導する工程と、を含む、植物体の製造方法。 A method for producing a plant body, comprising the steps of contacting a plant body, a plant cell, a piece of plant tissue, or a plant seed with the callus inducer of claim 1 to induce callus, and transplanting the callus obtained by the callus induction in the above step into a shoot induction medium to induce shoots. 式（Ｉ）で示される化合物又はその塩のハロゲン原子が塩素又は臭素である請求項１３に記載の植物体の製造方法。 The method for producing a plant body according to claim 13, wherein the halogen atom of the compound represented by formula (I) or its salt is chlorine or bromine. 植物がイネ科植物、アブラナ科植物、マメ科植物、ナス科植物又はヤマゴボウ科植物のいずれかである請求項１３又は１４に記載の植物体の製造方法。  The method for producing a plant body according to claim 13 or 14, wherein the plant is any one of a Gramineae plant, a Cruciferae plant, a Leguminosae plant, a Solanaceae plant, and a Phytolaccaea plant.
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