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

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

Plant callus inducer, callus induction method, callus production method and plant body production method

[0001] 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.

[0002] It is known that plants can form a mass of dedifferentiated plant cells (callus) from differentiated plant tissue, and this is used in a wide range of fields, including the production of useful substances and the creation of transformants. Generally, callus induction requires simultaneous treatment of plants 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 affect germination, flowering, and phototropism, and cytokinins such as kinetin and zeatin, which affect lateral bud formation, senescence, and nutritional responses. However, the composition of these plant hormones required for callus induction has had to be examined for each plant species.

[0003] For example, Patent Document 1 discloses a two-step culture method that involves a callus preparation step in which callus is prepared from a plant body, and a plant organ formation step in which plant organs are formed from the callus, whereby plant bodies can be efficiently produced by controlling culture conditions such as light intensity and glucose concentration. Patent Document 2 also discloses a callus inducer containing a 1,4-disubstituted piperazine derivative or a 1-substituted piperazine derivative, specifically fipexide, as its active ingredient, which has a basic structure different from that of conventional callus inducers, and describes its applicability to a wide range of plants. Patent Document 3 also discloses a method for preparing differentiated embryogenic callus (DEC) tissue from 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 find a novel, simpler callus inducer that is applicable to a wide range of plants.

[0004] JP 2019-24375 A International Publication No. 2015 / 186591 Pamphlet Special Publication No. 2019-520820

[0005] Tetrahedron135(2023)133333

[0006] An object of the present invention is to provide a novel callus inducer, a method for inducing callus using the callus inducer, and a method for producing callus.

[0007] The present inventors have previously synthesized aryloxyacetylthiourea derivatives in order to develop inhibitors for the radicle elongation of parasitic weeds (Non-Patent Document 1). They discovered that compounds with specific structures among these derivatives have plant callus-inducing activity, which led to the completion of the present invention.

[0008] That is, the present invention has the following configuration: <1> A compound represented by the following formula (I): (wherein R represents a halogen atom) or a salt thereof. <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 grass family, a cruciferous family, a legume family, a solanaceae family, or a phytolacca family. <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 according to <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 grass family, a cruciferous family, a legume family, a solanaceae family, or a phytolacca family. <7> A method for producing callus, comprising the step of contacting a plant body, plant cell, plant tissue fragment, or plant seed with the callus inducer according to <1>, and inducing and proliferating callus. <8> A method for producing callus according to <7>, wherein the halogen atom of the compound represented by formula (I) or a salt thereof is chlorine or bromine. <9> A method for producing callus according to <7> or <8>, wherein the plant is any one of a grass family, a crucifer family, a legume family, a solanaceae family, or a phytolacca family. <10> Use of the 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 grass family, a crucifer family, a legume family, a solanaceae family, or a phytolacca family. <13> A method for producing a plant, comprising the steps of contacting a plant, a plant cell, a plant tissue fragment, or a plant seed with the callus inducer according to <1> to induce callus, and transplanting the callus obtained by callus induction in the step onto a shoot induction medium to induce shoots. <14> A method for producing a plant according to <13>, wherein the halogen atom of the compound represented by formula (I) or a salt thereof is chlorine or bromine. <15> A method for producing a plant according to <13> or <14>, wherein the plant is any one of a grass family, a cruciferous family, a legume family, a solanaceae family, or a phytolacca family.

[0009] The callus inducer of the present invention can be applied to a wide range of plants, including those for which callus induction has been difficult in the past. Among these, it is particularly significant that the agent can be applied to sorghum, which has high tolerance to drought and soil toxicity.

[0010] 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. Photographs of Arabidopsis shoot-induced after callus induction in a medium containing compound (II) and CIM (Example 2). a. Photographs of Arabidopsis shoot-induced after callus induction in a medium containing compound (II) and CIM (Example 2), viewed from above (Example 2). b. Schematic diagram of shoot induction rate and shoots. Photographs showing the results of GUS staining of Arabidopsis callus induced in a medium containing compound (II) and CIM (Example 2). Photographs of sorghum callus induced in a medium containing compound (II) (Example 3). a. a. Photographs of red clover seedlings grown in a medium containing compound (II), Kinetion, IPA, and zeatin, and in CIM (control) (Example 4). b. Enlarged photographs of IPA and compound (II) in a. Photographs of Arabidopsis callus induced in a medium containing compound (II) and a medium containing FPX (Example 5). Photographs of Sorghum bicolor callus induced in a medium containing compound (II) and a medium containing FPX (Example 6). Schematic diagram showing the steps of explant preparation, callus induction, and shoot induction from Arabidopsis tissue explants (Example 7). Photographs of Arabidopsis root explants induced into callus in a medium containing compound (II) and CIM (Example 7). Photographs of Arabidopsis root explants induced into shoots after callus induction in 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 explants were prepared (Example 8). It is a photograph of shoot, hypocotyl, root, and root tip explants of Nicotiana benthamiana on which callus was induced in a medium containing compound (II) and CIM (Example 8). It is a photograph of Phytolacca americana on which callus was induced in a medium containing compound (II) (Example 9).

[0011] The callus inducer of the present invention has callus induction activity in plants and is characterized by 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):

[0012] (wherein R represents a halogen atom).

[0013] Examples of the halogen atom in the compound represented by (I) include fluorine, chlorine, bromine, and iodine. Among these, chlorine or bromine is preferred, with chlorine being even more preferred. When the halogen atom R is chlorine (Cl), it is shown in formula (II), and when it is bromine (Br), it is shown in formula (III).

[0014]

[0015]

[0016] The compound represented by formula (I) can be synthesized by the method described in "Synthesis of aryloxyacetylthiourea derivatives for the development of radicle elongation inhibitor of parasitic weeds" (Tetrahedron 135 (2023) 133333) by the present inventors (Non-Patent Document 1). The compound represented by 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 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. Salts of the compounds are not particularly limited as long as they have the callus-inducing activity of the present invention.

[0017] The callus inducer of the present invention may contain Compound (I) or a salt thereof as an active ingredient, and may also contain other ingredients in addition to Compound (I) or a salt thereof, provided that the callus induction activity is not inhibited. Examples of other ingredients include known pharmaceutical additives such as excipients, emulsifiers, and humectants. The form of the callus inducer of the present invention is not particularly limited, and any form that allows callus induction may be used. Examples include emulsions, liquids, oils, aqueous solutions, wettable powders, flowables, powders, microgranules, granules, aerosols, pastes, and jellies.

[0018] The plants to which the callus inducer of the present invention is applied are not particularly limited. For example, they may be either angiosperms or gymnosperms, and in the case of angiosperms, they may be either monocotyledonous or dicotyledonous plants. In the case of gymnosperms, examples include, but are not limited to, Cycadaceae (cycads, etc.), Ginkgoaceae (ginkgo, etc.), Pinaceae (Japanese red pine, Japanese black pine, fir, spruce, etc.), Cupressaceae (cedar, etc.), and Taxaceae (yew, etc.).

[0019] 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.), and 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.), Pokeweed (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).

[0020] The plants to which the callus inducer of the present invention is applied are preferably any of the grasses, crucifers, legumes, solanaceae, and phytolacca. In addition to Sorghum bicolor, sorghum to which the callus inducer of the present invention is applied also includes any species of Sorghum, such as Sorghum halepense, Sorghum almum, Sorghum sudanense, and Sorghum propinquum.

[0021] The "callus induction method" of the present invention refers to a method comprising the steps of contacting a plant, plant cell, plant tissue fragment, plant seed, or the like with the callus inducer of the present invention to induce callus. The "callus production method" of the present invention refers to a method comprising the steps of contacting a plant, plant cell, plant tissue fragment, or plant seed with the callus inducer of the present invention to induce callus, and then further growing the induced callus. These methods may include steps such as sterilizing the plant seeds to be induced in advance and germinating them after sterilization.

[0022] In the "callus induction method" or "callus production method" of the present invention, the method for contacting a plant body or the like with the callus inducer of the present invention is not particularly limited, 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 the method by culturing a plant body or the like in a medium containing the callus inducer of the present invention.

[0023] The "plant, plant cell, plant tissue fragment, or plant seed" used in the "callus induction method" or "callus production method" of the present invention may be any plant, as long as it 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 apex, stem, leaf, shoot, embryonic cell, root petal, fruit, or epidermis. These plants are preferably plants grown by aseptic sowing or plants sterilized with 70% ethanol, sodium hypochlorite aqueous solution, or the like to make them sterile.

[0024] 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. One type of compound represented by formula (I) may be added to the callus induction medium, or two or more types may be added in combination.

[0025] The compound represented by formula (I) or its salt may be added to a medium at a concentration that satisfies the callus induction effect of the present invention, depending on the target plant and treatment conditions. The concentration is not particularly limited, but can be, for example, in the range of 0.01 to 100 μM. Preferably, it is 0.1 to 10 μM, and more preferably 0.2 to 5 μM. When combining two or more compounds (e.g., compound (II) and compound (III)), the total amount of these compounds should be adjusted to fall within the above concentration range. The callus induction medium may also contain other components in addition to these compounds or their salts. Examples of such components include sugars, gelling agents, and inorganic salts commonly used in callus induction. Other plant hormones, such as auxin and cytokinin, may also be used in combination, provided they do not interfere with the action of the callus inducer of the present invention. The compound represented by formula (I) or its salt can induce callus alone, without the need for auxin, kinetin, cytokinin, or other components conventionally required for callus induction.

[0026] When culturing a plant or the like in a medium containing the callus inducer of the present invention, the culture for callus induction is preferably carried out under sterile conditions. Furthermore, the temperature during culture is preferably 20-25°C, and the light conditions are preferably between constant light irradiation and constant darkness. Culture can be continued until callus induction occurs, but callus induction is usually observed 2-4 weeks after culture. The "callus production method" of the present invention can also be subcultured to grow the induced callus. Subculture can be performed, for example, by replacing the medium with a callus induction medium containing the callus inducer of the present invention every other month.

[0027] 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 include leaves, roots, and stems. The plant organs formed from the callus can be further cultured under specified conditions to form plants, as needed. The culture conditions for shoot induction and plant organ formation are not particularly limited, and may be conventional culture conditions or culture conditions modified depending on the target plant. The plant organs and plants formed can be used for any purpose, such as food, pharmaceuticals, cosmetics, lumber, construction materials, feed, ornamental plants, biofuels, and research.

[0028] The callus induced by the method of the present invention can be used to produce genetically modified crops. For example, after infecting the callus induced by the present invention with Agrobacterium harboring a plasmid containing a target gene, the callus with the plasmid integrated into the chromosome can be isolated and redifferentiated to produce a genetically modified plant. The present invention will now be described in detail with reference to the following examples, but is not limited to these examples.

[0029] Example 1: Preparation of callus inducers. Compounds (II) and (III) were used in the following tests as substances with callus-inducing activity. Compounds (II) and (III) were synthesized by the present inventors using the method described in "Synthesis of aryloxyacetylthiourea derivatives for the development of radicle elongation inhibitors of parasitic weeds" in Tetrahedron 135 (2023) 133333. Compound (II) is 3-[2-(4-chlorophenoxy)acetyl]-1-(3,4-dichlorophenyl)thiourea, which corresponds to compound "3ab" in the same publication. Compound (III) is 3-[2-(4-bromophenoxy)acetyl]-1-(3,4-dichlorophenyl)thiourea, which corresponds to compound "3ac" in the same publication.

[0030]

[0031]

[0032] Example 2: Evaluation of the callus induction activity of the callus inducer of the present invention in Arabidopsis thaliana 1. Test method Callus induction and shoot induction were performed in Arabidopsis thaliana according to the following steps. (1) Germination treatment: Arabidopsis thaliana seeds were treated with 70% ethanol for 2 minutes and then sterilized with 1% sodium hypochlorite for 10 minutes. The seeds were sown on 1 / 2 MS medium and grown at 25°C under a 16-hour light / 8-hour dark cycle for 4-5 days. (2) Callus induction: Approximately 1 cm from the root tip of the germinated Arabidopsis thaliana was transplanted to 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 roots were also transplanted 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 transplanted to 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 cycle. (4) Callus GUS staining The callus induced Arabidopsis thaliana callus as described above was stained with GUS. GUS staining was carried out according to the following procedure. The plant bodies were immersed (fixed) in 90% acetone for 15 minutes at 4°C, and washed three times for 1 minute each with phosphate buffer. Thereafter, the plants were treated with GUS staining solution in a dark room and allowed to stand under reduced pressure (approximately 800 hPa) for 30 minutes. Thereafter, the plants were allowed to stand overnight in a dark room at 37°C, and then washed with 70% ethanol. The lateral root formation induction sites were observed using an inverted microscope. The composition of the GUS staining solution is shown below.<GUS staining solution (per mL)> 0.2 M phosphate buffer (pH 7.0) 500 μL, 0.5 M EDTA 20 μL, 0.05 M Fe(II) 100 μL, 0.05 M Fe(III) 100 μL, 1% Triton 100 μL, 50 μg / ml X-gluc 20 μL, DW 160 μL.

[0033] 2. Morphological Observation Results The state of Arabidopsis calli on days 14 and 20 after the start of culture is shown in Figure 1. This figure confirms that compounds (II) and (III) of the present invention have callus-inducing activity. In particular, significantly larger calli were formed on day 20 after the start of culture compared with CIM. The state of shoot-induced Arabidopsis plants is shown in Figures 2 and 3. The shoot induction rate for calli induced by CIM was 25% (1 out of 4), while the shoot induction rate for calli induced by compound (II) was 100% (8 out of 8) (Figure 3b). Furthermore, shoots were induced from the calli in contact with the medium (i.e., the lower side of the calli). In contrast, shoots were regenerated from the upper part of the calli induced by CIM (Figures 3a and 3b).

[0034] The results of GUS staining of Arabidopsis callus on days 2, 7, and 10 after the start of culture are shown in Figure 4. As shown in this figure, specific GUS staining was observed in the apical ends of the primary and lateral roots of Arabidopsis callus induced on a medium containing 0.26 μM Compound (II) from day 7 after the start of culture, whereas widespread staining of the lateral root induction site was observed from day 7 after the start of culture in Arabidopsis callus induced on CIM. Similar staining was also observed on day 10 after the start of culture in Arabidopsis callus induced on a medium containing 2.6 μM Compound (II).

[0035] Example 3 Evaluation of the callus induction 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 treated with 70% ethanol for 2 minutes and then sterilized by treatment with 1% sodium hypochlorite for 60 minutes. The seeds were sown on a medium containing Compound (II) (1 / 2 MS medium, 1% sucrose, 2.6 μM Compound (II)). For comparison, seeds were also sown on CIM. After sowing, the seeds were cultured in the dark at 25°C for 4 weeks to induce callus.

[0036] 2. Morphological Observation Results The state of callus of Sorghum bicolor on days 4, 10, and 20 after the start of culture for callus induction is shown in Figure 5. It was confirmed that the callus inducer of the present invention also has callus-inducing activity for sorghum.

[0037] Example 4 Evaluation of the callus induction activity of the callus inducer of the present invention on red clover (Trifolium pratense) Callus induction of red clover (Trifolium pratense) was performed according to the following steps. 1. Callus induction Red clover seeds were treated with 70% ethanol for 2 minutes and then sterilized by treatment 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, seeds were also sown in CIM medium containing 1 ppm kinetin, zeatin, 1 ppm IPA, or 10 ppm IPA instead of Compound (II). After sowing, the seeds were cultured for 4 weeks, and callus induction was performed.

[0038] 2. Morphological Observation Results Figure 6a shows the state of red clover cultured in each medium on the 7th day after the start of culture for callus induction. Of the red clover shown in Figure 6a, Fig. 6b shows enlarged views of a portion of red clover cultured in a medium containing IPA and a portion of red clover cultured in a medium containing Compound (II). It was confirmed that the callus inducer of the present invention also has callus-inducing activity in red clover. On the other hand, in media supplemented with cytokinins such as kinetin, zeanin, and IPA, root elongation was inhibited, but callus-inducing activity was not confirmed.

[0039] Example 5 Comparison of callus induction activity of 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) in Arabidopsis thaliana Callus induction in Arabidopsis thaliana was performed according to the following steps. 1. Callus induction Arabidopsis seeds were sown in medium containing Compound (II) (1 / 2 MS medium, 1% sucrose, 0.1 or 1.0 μM Compound (II)) or medium containing FPX (1 / 2 MS medium, 1% sucrose, 1.0 μM FPX). These were cultured under a 16-hour light / 8-hour dark cycle at 25°C for 4 weeks to induce callus.

[0040] 2. Morphological Observation Results The state of Arabidopsis thaliana plants on days 7 and 28 of culture during callus induction is shown in Figure 7. When cultured in a medium containing 1.0 μM Compound (II), callus formation was confirmed on day 28 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 day 28 of culture.

[0041] Example 6 Comparison of Callus Induction Activity of Compound (II) and FPX on Sorghum bicolor Callus induction in Sorghum bicolor was performed using the following steps. 1. Callus Induction Sorghum bicolor seeds were treated with 70% ethanol for 2 minutes and then sterilized with 1% sodium hypochlorite for 60 minutes. The seeds were sown on a medium containing Compound (II) (1 / 2 MS medium, 1% sucrose, 3.0 μM Compound (II)) or 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.

[0042] 2. Morphological Observation Results The state of sorghum bikollo on the 17th day after the start of culture for callus induction is shown in Figure 8. It was confirmed that sorghum bikollo cultured in a medium containing 3.0 μM compound (II) formed significantly larger calli than sorghum bikollo cultured in a medium containing the same concentration (3.0 μM) of FPX. Furthermore, the callus induction rate when cultured in a medium containing FPX was 20%, while the callus induction rate when cultured in a medium containing compound (II) was 44%.

[0043] 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 and shoot induction were performed on Arabidopsis thaliana root explants according to the following steps. 1. Test Method (1) Germination Treatment: Arabidopsis thaliana seeds were treated with 70% ethanol for 2 minutes and then sterilized by treatment 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-hour light / 8-hour dark cycle. (2) Explant Preparation and Callus Induction: 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)), and explants were prepared as shown in Figure 9. For comparison, explants were also prepared by transplanting them into CIM. These were cultured for 4 weeks at 25°C under a 16-hour light / 8-hour dark cycle to induce callus. (3) Shoot induction: The callus-induced callus was transplanted into shoot induction medium (1 / 2 MS medium, 1.14 μM IAA, 12.3 μM IPA (isopentenyl adenine)) and shoot induction was performed under a 16-hour light / 8-hour dark cycle at 25°C.

[0044] 2. Morphological Observation Results The state of Arabidopsis roots on days 10, 18, and 25 after callus induction is shown in Figure 10. When Arabidopsis roots were cultured with CIM, the callus induction rate was 12% (3 out of 25) on day 18 and 72% (18 out of 25) on day 25. On the other hand, when callus induction was performed using 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 significantly larger calli than those cultured with CIM. The state of calli obtained by callus induction from Arabidopsis roots and further shoot induction is shown in Figure 11. The shoot induction rate of calli induced with CIM was 22% (4 out of 18). On the other hand, the shoot induction rate of callus induced in the medium containing Compound (II) was 56% (14 out of 25). These morphological observation results and those of Example 2 indicate 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.

[0045] Example 8: Evaluation of the callus induction activity of the callus inducer of the present invention on Nicotiana benthamiana explants. Callus induction of Nicotiana benthamiana explants was performed using 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-hour light / 8-hour dark cycle. (2) Explant Preparation and Callus Induction: Nicotiana benthamiana grown for 7 days was cut into tissue fragments of the 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)). Explants were prepared as shown in Figure 12. For comparison, explants transplanted into CIM were also prepared. These were cultured under a 16-hour light / 8-hour dark period at 25°C for 4 weeks to induce callus.

[0046] 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 Figure 13. It was confirmed that the callus inducer of the present invention also has callus-inducing activity in N. benthamiana. The callus inducer of the present invention induced larger calli, particularly in the hypocotyl.

[0047] Example 9: Evaluation of the 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 using the following steps. 1. Germination and callus induction. The seed coats of Phytolacca americana seeds were removed, treated with 70% ethanol for 2 minutes, and then sterilized by treatment with 1% sodium hypochlorite for 45 minutes. The seeds were sown on MS medium and grown at 25°C for 30 days under a 16-hour light / 8-hour dark cycle. After 30 days of growth, the Phytolacca americana plants were cut into leaves and stems approximately 0.5-1.0 cm long. The leaves and stems approximately 0.5-1.0 cm long were then transplanted into a medium containing Compound (II) (MS medium, 3% sucrose, 2.6 μM Compound (II)) to prepare explants. These explants were cultured in the light at 25°C for 4 weeks to induce callus.

[0048] 2. Morphological Observation Results The state of pokeweed on days 0, 15, and 28 after the start of culture for callus induction is shown in Figure 14. After the growth of axillary buds was induced, callus was induced from the cut surface of the explant (circled area). It was confirmed that the callus inducer of the present invention also has callus-inducing activity for pokeweed.

[0049] The callus inducer of the present invention is applicable to a wide range of plants, making it possible to induce callus in plants where callus induction was previously impossible. 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 production, and research.

Claims

1. The following formula (I): (wherein R is a halogen atom) or a salt thereof.

2. 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.

3. The callus inducer according to claim 1 or 2, wherein the plant is any one of a Gramineae plant, a Brassicaceae plant, a Leguminosae plant, a Solanaceae plant, and a Phytolaccaea 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 claim 1 to induce callus.

5. 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.

6. 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.

7. A method for producing callus, comprising the steps of contacting a plant body, a plant cell, a plant tissue fragment or a plant seed with the callus inducer of claim 1 to induce and proliferate callus.

8. A 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.

9. 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.

10. Use of a compound represented by formula (I) or a salt thereof for inducing callus in a plant.

11. 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.

12. 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.

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 claim 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 claim 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 body according to claim 13 or 14, wherein the plant is any one of a Gramineae plant, a Brassicaceae plant, a Leguminosae plant, a Solanaceae plant, and a Phytolaccaea plant.

Images