Composition for promoting plant redifferentiation

Abstract

To provide a compound that promotes redifferentiation from plant segments or calli.SOLUTION: A composition for promoting plant redifferentiation includes, as an active ingredient, one or more selected from the group consisting of a compound represented by the following formula, a pharmaceutically acceptable salt thereof, or a solvate thereof. [In the formula, R1 to R11 may be the same or different and each independently represent H or a C1 or C2 alkyl group]. The plant may be a dicot, and may be from the Cannabaceae or Brassicaceae family.SELECTED DRAWING: None

Description

Technical Field

[0001] The present invention relates to a technique for promoting the redifferentiation of plants, particularly plant sections and calli.

Background Art

[0002] As one of the methods for imparting useful traits to plants, there is the production of transformants by genetic recombination. In this method, a useful gene is inserted into the genomic DNA of a plant using bacteria such as Agrobacterium. The target samples for Agrobacterium infection are suitable plant pieces such as stems and leaves, and calli, which are undifferentiated cell masses. Plants into which genes have been introduced are regenerated from these plant pieces and calli. In particular, the process of forming a plant body including shoots (hereinafter referred to as shoots) from calli is called redifferentiation, and this redifferentiation step is almost always involved when producing transformants in rice, tobacco, tomatoes, etc. In recent years, with the advent of genome editing technology, attempts have been made to produce transformants of various plants, but most plants have a very low redifferentiation efficiency, which has become an obstacle in genetic recombination.

[0003] In plant redifferentiation, plant hormones auxin and cytokinin play important roles. By treating callus at an appropriate concentration, shoot formation from callus is induced (Non-Patent Document 1). For example, in the model plant Arabidopsis thaliana, shoot formation is promoted by treating callus in a medium containing a higher concentration of cytokinin compared to auxin (Non-Patent Document 2). There are natural and synthetic types of auxin and cytokinin, and the concentration ratio considering these types has been studied to promote redifferentiation. As an attempt to increase the redifferentiation efficiency, in addition to examining the treatment concentrations of auxin and cytokinin, it has also been reported that overexpressing or deleting specific genes can promote callus formation and shoot formation in Arabidopsis thaliana (Patent Document 1, Patent Document 2). However, in the latter method, since it is necessary to create individuals overexpressing or deleting specific genes for each target plant, it is currently inferior to plant hormone treatment from the viewpoints of convenience and versatility. Also, although redifferentiation efficiency increases by irradiating with gamma rays, a type of radiation (Patent Document 3), it is considered difficult to use because there is a possibility that the original traits may change due to errors generated in genomic DNA. Regarding herbaceous plants, for specific plants such as lavender, a method of culturing in the presence of light and cytokinin to promote callus formation, redifferentiation, and seedling production has been disclosed (Patent Document 4).

Prior Art Documents

Patent Documents

[0004]

Patent Document 1

Patent Document 2

Patent Document 3

Patent Document 4

Non-Patent Documents

[0005] [Non-Patent Document 1] Ikeuchi et al, Annu Rev Plant Biol. 2019 Apr 29 Volume 70 Pages 377-406. [Non-Patent Document 2] Iwase et al, Plant Cell. 2017 Jan; Volume 29 Issue 1 Pages 54-69. [Summary of the Invention] [Problems to be Solved by the Invention]

[0006] An object of the present invention is to provide a compound that promotes redifferentiation from plant sections or callus. [Means for Solving the Problems]

[0007] The present inventor has intensively studied a method for promoting plant redifferentiation from plant sections or callus. As a result, by applying a compound represented by any of the following formulas (I), (II), and (III), more preferably compound A, compound B, or compound C, to plant sections or callus, it has been found that redifferentiation from plant sections or callus to plants is promoted, and the present invention has been completed.

[0008] [Chemical Formula] Formula (I) [In formula I, R1 to R 11 are independently H, CH3, or C2H5. ]

[0009] [Chemical Formula] Formula (II) [In formula II, R 12 ~R 15 and R 18 ~R 26 are independently H, CH3, or C2H5, and R 16 , R 17, R 27 and R 28 is independently H, CH3, C2H5 or C3H7.]

[0010] [Chemical formula] Formula (III) [In formula III, R 29 ~R 39 is independently H, CH3 or C2H5.]

[0011] Structures of compounds A, B and C The structures and IUPAC names of compounds A, B and C are shown below.

[0012] [Chemical formula]

[0013] [Chemical formula]

[0014] [Chemical formula]

[0015] Compound A: (S)-2-((4-chlorophenoxy)methyl)pyrrolidine Compound B: 5-isobutyl-N-(2-(1-isopropylpiperidin-4-yl)ethyl)-4-oxo-4,5-dihydropyrazolo[1,5-a]quinoxaline-2-carboxamide Compound C: (3-(1H-imidazol-1-yl)azetidin-1-yl)(pyridin-2-yl)methanone

[0016] That is, the present invention is as follows. [1] A composition for promoting plant redifferentiation, comprising as an active ingredient one or more selected from the group consisting of a compound of the following formula (I), (II) or (III), or a pharmaceutically acceptable salt thereof or a solvate thereof: [Chemical formula] Formula (I) [In formula I, R1 to R 11 may be the same or different and independently represent H or an alkyl group having 1 or 2 carbon atoms.] [Chemical formula] Formula (II) [In formula II, R 12 ~R 15 and R 18 ~R 26 may be the same or different and independently represent H or an alkyl group having 1 or 2 carbon atoms, and R 16 R 16 R 27 and R 28 may be the same or different and independently represent H or an alkyl group having 1 to 3 carbon atoms.] [Chemical formula] Formula (III) [In formula III, R 29 ~R 39 may be the same or different and independently represent H or an alkyl group having 1 or 2 carbon atoms.]. [2] A composition for promoting plant redifferentiation, comprising as an active ingredient one or more selected from the group consisting of a compound of formula I, II or III, or a pharmaceutically acceptable salt thereof or a solvate thereof, in formula I, R1 to R 11 are H, in formula II, R 12 ~R 15 and R 18 ~R 26 are H, and R 16 R 17 R 27 and R 28is an alkyl group having 1 carbon atom, In formula III, R 29 ~R 39 is H, A composition for promoting the redifferentiation of the plant of [1]. [3] The composition according to [1] or [2], wherein the plant is a dicotyledonous plant. [4] The composition according to [3], wherein the plant is a plant of the family Cannabaceae or Brassicaceae. [5] A compound of formula (I), (II) or (III) of [1]: A method for promoting plant redifferentiation, comprising applying to a plant section or callus a composition for promoting plant redifferentiation containing as an active ingredient one or more selected from the group consisting of a pharmaceutically acceptable salt or solvate thereof. [6] A compound of formula (I), (II) or (III) of [1], In formula (I), R1 to R 11 is H, In formula (II), R 12 ~R 15 and R 18 ~R 26 are H, R 16 , R 17 , R 27 and R 28 is an alkyl group having 1 carbon atom, In formula (III), R 29 ~R 39 is H, A method for promoting plant redifferentiation, comprising applying to a plant section or callus a composition for promoting plant redifferentiation containing as an active ingredient one or more selected from the group consisting of a compound or a pharmaceutically acceptable salt or solvate thereof. [7] The method according to [5] or [6], wherein the plant is a dicotyledonous plant. [8] The method according to [7], wherein the plant is a plant of the family Cannabaceae or Brassicaceae. [9] Applying the composition for promoting plant redifferentiation of the plant of [1] or [2] to a plant section or callus to redifferentiate shoots, somatic embryos, adventitious buds or adventitious roots, and regenerating a plant from the shoots, somatic embryos, adventitious buds or adventitious roots, Method for producing a regenerated plant.

[10] The method according to [9], wherein the plant is a dicotyledonous plant.

[11] The method according to

[10] , wherein the plant is a plant belonging to the family Cannabaceae or Brassicaceae.

Advantages of the Invention

[0017] By applying a composition that promotes the redifferentiation of plants from plant sections or calli to the plant sections or calli, the redifferentiation of plants from the plant sections or calli can be promoted. Furthermore, a plant body that has undergone the redifferentiation process can be produced. In this specification, a plant body that has undergone redifferentiation may be referred to as a regenerated plant.

Brief Description of the Drawings

[0018]

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Mode for Carrying Out the Invention

[0019] The present invention will be described in detail below. The present invention is a composition for promoting the redifferentiation of plants from plant sections or callus. Further, the present invention is a method for promoting the redifferentiation of plants from plant sections or callus.

[0020] In the present invention, redifferentiation means that cells change to have specific forms and functions, that is, cells change to cells that constitute specific tissues and organs. Promoting redifferentiation means causing redifferentiation by applying the composition of the present invention to a plant in which redifferentiation was not originally observed, and also includes both cases where redifferentiation action was observed but redifferentiation is promoted by applying the composition of the present invention. Further, in the present invention, redifferentiation refers to the redifferentiation of shoots (stems and leaves), roots, and adventitious embryos from plant sections or callus. Note that adventitious buds are included in shoots and adventitious roots are included in roots.

[0021] 1. Active ingredient of the composition for promoting the redifferentiation of plants from plant sections or callus The active ingredient of the composition for promoting the redifferentiation of plants from plant sections or calli of the present invention is a compound represented by any of the following formulas (I), (II), and (III), more preferably compound A, compound B, or compound C.

[0022]

Chemical formula

[0023]

Chemical formula

[0024]

Chemical formula

[0025]

Chemical formula

[0026]

Chemical formula

[0027] [Chemical formula]

[0028] Compound A: (S)-2-((4-chlorophenoxy)methyl)pyrrolidine Compound B: 5-isobutyl-N-(2-(1-isopropylpiperidin-4-yl)ethyl)-4-oxo-4,5-dihydropyrazolo[1,5-a]quinoxaline-2-carboxamide Compound C: (3-(1H-imidazol-1-yl)azetidin-1-yl)(pyridin-2-yl)methanone

[0029] The active ingredient of the composition for promoting the redifferentiation of the plants of the present invention also includes a pharmaceutically acceptable salt of the above compound or a solvate thereof.

[0030] In addition, the composition for promoting the redifferentiation of the plants of the present invention may contain one or more compounds selected from the group consisting of the compounds represented by the above formula (I), (II) or (III), or a pharmaceutically acceptable salt thereof or a solvate thereof. Furthermore, the composition for promoting the redifferentiation of the plants of the present invention may contain one or more compounds selected from the group consisting of the compounds of the above Compound A, B or C, or a pharmaceutically acceptable salt thereof or a solvate thereof.

[0031] These compounds can be obtained, for example, by purchasing from Enamine or ChemDiv, or by synthesizing according to the description of the following production examples.

[0032] 2. Target plants In the present invention, the target plants include both angiosperms and gymnosperms, preferably angiosperms. Further, angiosperms include both dicotyledons and monocotyledons. Examples of monocotyledons include Gramineae plants such as rice, corn, barley, wheat, sorghum, etc.; Araceae plants such as taro, konjac, etc.; Amaryllidaceae plants such as onion, leek, etc.; Asparagaceae plants such as asparagus, etc. Among these, Gramineae plants are preferred. Examples of dicotyledons include Malvaceae plants such as flax, hop, paulownia, enoki, etc.; Brassicaceae plants such as cabbage, Chinese cabbage, broccoli, radish, arugula, komatsuna, mizuna, mustard spinach, Arabidopsis thaliana, etc.; Solanaceae plants such as potato (Solanum tuberosum), tobacco, Nicotiana benthamiana, tomato, etc.; Asteraceae plants such as lettuce, artichoke, etc.; Fabaceae plants such as alfalfa, soybean, etc.; Chenopodiaceae plants such as spinach, sugar beet, etc.; Lamiaceae plants such as perilla, basil, etc.; Apiaceae plants such as carrot, Mitsuba, etc.; Cucurbitaceae plants such as melon, watermelon, cucumber, pumpkin, etc.; Malvaceae plants such as cotton, etc. Among these, Malvaceae plants and Brassicaceae plants are preferred.

[0033] 3. Promotion of redifferentiation In the present invention, redifferentiation from plant sections or calli is promoted. Redifferentiation refers to the process by which dedifferentiated cells redifferentiate into cells that each perform their respective functions within the plant body. As a result of redifferentiation, roots, stems, leaves, etc. are generated from plant sections or calli, and a plant body that has undergone redifferentiation is formed. In the present invention, redifferentiation refers to the redifferentiation from plant sections or calli into shoots (stems and leaves), roots, and adventitious embryos.

[0034] Callus refers to a mass of undifferentiated plant cells cultured in a medium, which consists of dedifferentiated cells of plant cells that are not functionally and structurally differentiated and have regained the ability of pluripotency and cell proliferation.

[0035] Dedifferentiation refers to the process by which cells of differentiated tissues and organs return to a highly undifferentiated state and regain pluripotency. Dedifferentiated cells regain the ability to divide. By undergoing dedifferentiation, they enter a state where they have the ability to redifferentiate into various cells.

[0036] In the present invention, a plant section refers to a tissue piece that is a part of a plant tissue regardless of the plant organ, and is obtained by cutting it out from a plant body or a tissue culture seedling. A plant section is also referred to as an explant. For example, a stem fragment, a leaf fragment, a root fragment, etc. may be used as the plant section. Specifically, a cut-off tip of a root can be used. The size of the plant section is not limited, but is usually about 1 to 20 mm on one side, preferably about 1 to 10 mm. For example, when using a root or a stem, a cut-off root or stem of about 1 to 20 mm, preferably about 1 to 10 mm can be used as the plant section.

[0037] Callus can be induced from the above-mentioned plant sections. That is, in the present invention, a plant can be redifferentiated from a plant section or from callus induced from a plant section.

[0038] The callus to be redifferentiated by the composition for promoting redifferentiation of plants of the present invention is induced from a plant section that is a part of a plant body, and may be transformed. Induction of callus from a plant body section can be carried out by a known method. That is, the plant section may be transplanted into a callus-inducing medium (CIM) and cultured.

[0039] "Callus induction medium" means a medium prepared to be able to induce plant pieces into callus. A person skilled in the art can easily prepare it according to the methods described in textbooks such as "Transformation Protocol [Plant Edition], Kagaku Dojin (2012)". The callus induction medium contains plant hormones in addition to the basal medium. Specifically, as the callus induction medium, a medium used for plant tissue culture, for example, Gamborg B5 medium, MS medium (Murashige and Skoog medium), LS medium (Linsmaier and Skoog medium), White medium, Niche medium, Knudson C medium, SB medium, R2 medium, N6 medium, Tuleeke medium, etc. are used as the basal medium, and nutrient sources such as sucrose are added, and plant hormones such as auxins such as 2,4-dichlorophenoxyacetic acid (2,4-D) and cytokinins such as kinetin and benzyladenine are added thereto for preparation. The pH of the callus induction medium is 5 to 8, preferably 5 to 7, and more preferably 5.5 to 6.5. The callus induction medium may be a solid medium or a liquid medium. When using a solid medium, agar, gellan gum, etc. may be used as the gelling agent. The culture conditions for inducing callus may be static or shaking culture at 15 to 35 °C in the presence or absence of light. The culture period is 1 to 2 weeks, preferably 2 to 10 days, and more preferably 5 to 10 days.

[0040] The induced callus may be transplanted to a shoot-inducing medium (SIM) and cultured. The callus maintained in an undifferentiated state can be redifferentiated into a plant body via shoots, roots, and adventitious embryos.

[0041] Shoot induction medium allows for the redifferentiation of shoots and roots from plant sections or callus. A shoot refers to a unit having a stem and leaves, also called a shoot and leaf. Roots include adventitious roots, and shoots include adventitious buds. Shoots grow and branch to form a plant body. The shoot induction medium is also called a redifferentiation medium. As the redifferentiation medium, a known medium can be used, which is a basic medium used for the above plant tissue culture supplemented with sugars, inorganic salts, vitamins, and, if necessary, auxin, cytokinin, amino acids, etc. The pH of the shoot induction medium is 4 - 8, preferably 4 - 7, and more preferably 5.0 - 6.5. The shoot induction medium can be either a solid medium or a liquid medium. When using a solid medium, agar, gellan gum, etc. can be used as the gelling agent. In the presence of light, it is desirable to perform static culture for the solid medium at 15 - 35°C, and shaking culture is preferred for the liquid culture.

[0042] Before transplanting the callus into the shoot induction medium, it may be transplanted into a subculture medium to subculture the callus. The subculture medium is a medium for maintaining and proliferating the callus while keeping it undifferentiated, and it can be any commonly used subculture medium. For example, the subculture medium is a medium obtained by adding sugars, inorganic salts, vitamins, auxin, and, if necessary, cytokinin, amino acids, etc. to the above basic medium, and solid and liquid media are used appropriately depending on the plant species and experimental conditions. For example, it is desirable to use a solid medium for plant species that are sensitive to water stress. The subculture medium for callus may be the same as the callus induction medium. The culture conditions are the same as those for callus induction culture. It is preferable to subculture the callus every 1 - 4 weeks.

[0043] The composition for promoting plant redifferentiation of the present invention is preferably used together with plant hormones commonly used in plant tissue culture. Examples of plant hormones used together with the composition for promoting plant redifferentiation of the present invention include auxin, cytokinin, gibberellin, ethylene, abscisic acid, etc. Among them, auxin and cytokinin are preferred. Examples of auxin include indoleacetic acid (IAA), indolebutyric acid (IBA), naphthaleneacetic acid, naphthoxyacetic acid, phenylacetic acid, 2,4-dichlorophenoxyacetic acid (2,4-D), and 2,4,5-trichlorophenoxyacetic acid (2,4,5-T), etc. Indoleacetic acid or indolebutyric acid is preferred. Examples of cytokinin include zeatin, benzyladenine, and thidiazuron, etc.

[0044] The composition for promoting plant redifferentiation of the present invention promotes the redifferentiation from plant sections or callus to shoots. After the plant sections or callus redifferentiate into shoots, they grow into regenerated plants.

[0045] In addition, the composition for promoting plant redifferentiation of the present invention promotes the redifferentiation from plant sections or callus to adventitious embryos. After the plant sections or callus redifferentiate into adventitious embryos, they grow into regenerated plants. Therefore, by promoting the redifferentiation from plant sections or callus to normal adventitious embryos, a plant body that has finally undergone the redifferentiation process efficiently can be obtained.

[0046] The composition for promoting plant redifferentiation of the present invention promotes redifferentiation into organs. Examples of organ differentiation include redifferentiation into adventitious organs, such as adventitious buds and adventitious roots. The composition for promoting plant redifferentiation of the present invention promotes the redifferentiation from callus into adventitious organs, or the redifferentiation from plant sections into adventitious organs, such as adventitious roots and adventitious buds. When adventitious organs, such as adventitious buds and adventitious roots, redifferentiate from callus, the callus grows into a regenerated plant through the adventitious buds or adventitious roots. When adventitious buds or adventitious roots are redifferentiated from plant sections, a regenerated plant is obtained by the appearance of adventitious buds or adventitious roots from the plant sections.

[0047] The composition for promoting redifferentiation of the present invention can exert its redifferentiation-promoting effect by adding it to the subculture medium or the shoot induction medium during the period from 3 weeks before transferring to the shoot induction medium to 2 weeks after transplantation to the shoot induction medium. More preferably, the differentiation-promoting effect can be exerted by adding it to the subculture medium or the shoot induction medium during the period from 2 weeks before transplantation to the shoot induction medium to 1 week after transplantation.

[0048] The present invention also includes a method for producing a regenerated plant. The composition for promoting redifferentiation of the plant of the present invention can be said to be an auxiliary composition for adding to a medium for producing a regenerated plant.

[0049] 4. Method of application and dosage of the active ingredient of the composition for promoting redifferentiation of plants In the present invention, when redifferentiating shoots, roots, and adventitious embryos from plant sections or calli, the composition for promoting redifferentiation of the plant of the present invention is applied to the plant sections or calli to promote the redifferentiation of shoots, roots, and adventitious embryos from the calli. A plant body is regenerated from the shoots, roots, and adventitious embryos. Here, "applying the composition for promoting redifferentiation of a plant to a plant section or callus" means culturing the plant section or callus in a medium containing the composition for promoting redifferentiation of the plant, dropping a solution containing the composition for promoting redifferentiation of the plant onto the plant section or callus, or immersing the plant section or callus in a solution containing the composition for promoting redifferentiation of the plant to bring them into contact.

[0050] The present invention includes a method for producing a regenerated plant from a plant section or callus, which includes applying and culturing the composition for promoting redifferentiation of the plant of the present invention to the callus. Preferably, in the method for producing a regenerated plant from a callus, the above composition for promoting redifferentiation of the plant can be applied by dropping it one or more times to the callus during the period from 3 weeks before transplanting the callus to the shoot induction medium to 2 weeks after transplantation. More preferably, it may be applied one or more times to the callus during the period from 2 weeks before transplantation to the shoot induction medium to 1 week after transplantation. At this time, a solution containing the composition for promoting redifferentiation of the plant of the present invention may be used.

[0051] When producing a regenerated plant from a plant section, it can be applied by immersing the cut end in a solution containing the composition for promoting plant redifferentiation of the present invention immediately after cutting out the plant section. The composition for promoting plant redifferentiation of the present invention may be used.

[0052] Instead of directly applying the composition for promoting plant redifferentiation of the present invention to a plant section or callus, the plant section or callus may be cultured in a medium supplemented with the composition for promoting plant redifferentiation of the present invention. In this case, the above shoot induction medium (redifferentiation medium) may be used to culture the plant section or callus.

[0053] The composition for promoting plant redifferentiation of the present invention may be included in a subculture medium for callus. It can also be applied by dropping it onto the callus on a solid medium. When the composition for promoting plant redifferentiation is used for a solid medium, it may be included in the liquid medium before solidification in advance, or may be added by coating it on the surface of the solid medium.

[0054] By culturing callus on a subculture medium or shoot induction medium containing the composition for promoting plant redifferentiation of the present invention, or by dropping the composition for promoting plant redifferentiation of the present invention onto the callus, the callus redifferentiates into shoots (stems and leaves), roots, and adventitious embryos, and each of the redifferentiated organs or adventitious embryos grows into a plant.

[0055] The application amount of the composition for promoting plant redifferentiation may be applied at a concentration at which the number of shoots formed on one callus is large by culturing the callus of the plant species to be redifferentiated in a shoot induction medium containing about 0 to 30 μM. For example, it may be contained in the shoot induction medium at 0.5 to 30 μM, preferably 0.5 to 15 μM. Also, the concentration may be changed depending on the compound used. For example, in the case of the compound represented by Formula I or Compound A, it may be contained in the subculture medium or the shoot induction medium at 1 to 15 μM, preferably 2 to 12 μM. In the case of the compound represented by Formula II or Compound B, or the compound represented by Formula III or Compound C, it may be contained in the subculture medium or the shoot induction medium at 0.5 to 15 μM. When directly applying to plant sections or calli, a solution containing the compound at the above concentration may be used.

[0056] 5. Confirmation of redifferentiation promoting effect Whether or not the composition for promoting plant redifferentiation of the present invention has a redifferentiation promoting effect can be evaluated by counting the number of shoots formed on the plant sections or calli to which the composition has been applied. That is, if the number of shoots is larger compared to the case where the composition for promoting redifferentiation of the present invention is not applied, it is understood that there is a redifferentiation promoting effect.

Examples

[0057] The present invention will be specifically described by the following examples, but the present invention is not limited by these examples.

[0058] Production Example 1 Method for preparing Compound A Nuclear magnetic resonance spectrum ( 1 H NMR (400 MHz)) was measured using JNM-ECA-400 manufactured by JEOL Ltd. unless otherwise specified. The 1 H NMR spectra of most samples were measured using deuterated methanol as a solvent. At that time, the center of the quartet, which is the signal of the alcoholic hydroxyl group of methanol present in a trace amount in deuterated methanol, was set to 3.31 ppm. The 1The 1H NMR spectrum was measured using deuterated chloroform as a solvent, and tetramethylsilane (δ 0.00 ppm) was used as an internal standard. The multiplicity was abbreviated as s (singlet), d (doublet), t (triplet), q (quartet), m (multiplet), b (broad).

[0059] Reagents and solvents were used as received unless otherwise specified. The reaction was monitored by thin-layer chromatography using Merck Silica gel 60 F254 (layer thickness 0.25 mm), and the color development by ultraviolet absorption, phosphomolybdic acid coloring reagent, iodine, etc. was used as an indicator. Flash silica gel column chromatography was performed using FL60D manufactured by Fuji Silysia Chemical Ltd.

[0060] Synthesis of N-Boc protected compound [Chemical formula]

[0061] Under an argon atmosphere, 4-chlorophenol (117 μL, 1.17 mmol, 1.2 equiv) and triphenylphosphine (350.6 mg, 1.27 mmol, 1.3 equiv) were sequentially added to a toluene-tetrahydrofuran solution (4:1, 4.75 ml, 0.2 M) of N-(tert-butoxycarbonyl)-L-prolinol (200.2 mg, 0.974 mmol). After cooling the reaction solution to 0 °C, diethyl azodicarboxylate (0.58 ml, 1.27 mmol, 1.3 equiv) was slowly added dropwise, and then the reaction solution was warmed to room temperature and stirred for 24 hours. After completion of the reaction, the solution was concentrated under reduced pressure. The obtained residue was purified by silica gel column chromatography (hexane:ethyl acetate = 7:1) to obtain a colorless oily substance 1 (220.5 mg, 0.707 mmol, yield 72%). 1: 11H NMR (400 MHz, CD3OD) δ 7.21 (2H, d, J = 8.4 Hz), 6.90 (2H, d, J = 8.4 Hz), 4.06 (2H, m), 3.92 (1H, dd, J = 7.6, 9.6 Hz), 3.43 (2H, m), 2.01 (3H, complex), 1.85 (1H, m), 1.43 (9H, s).

[0062] Synthesis of Compound A

Chemical formula

[0063] Hydrogen chloride (10% methanol solution, 2.4 ml) was added dropwise to a methanol solution of 1 under ice-cooling. Then, the mixture was warmed to room temperature and stirred for 31.5 hours. After completion of the reaction, the solution was concentrated under reduced pressure. The obtained residue was washed twice with diethyl ether and dried to obtain a white solid (Compound A, 102.9 mg, 0.415 mmol, yield 69%). Compound A·HCl: 1 1H NMR (400 MHz, CD3OD) δ 7.31 (2H, d, J = 8.0 Hz), 7.01 (2H, d, J = 8.0 Hz), 4.33 (1H, dd, J = 2.8, 10.8 Hz), 4.12 (1H, dd, J = 9.2, 9.6 Hz), 4.03 (1H, ddd, J = 2.8, 7.6, 16 Hz), 3.37 (2H, d, J = 7.2 Hz), 2.28 (1H, m), 2.13 (2H, m), 1.91 (1H, m).

[0064] Production Example 2 Synthesis of Compound A’ Compound A’: (S)-1-(2-((4-chlorophenoxy)methyl)pyrrolidin-1-yl)octan-1-one

Chemical formula

[0065] Under a nitrogen atmosphere, triethylamine (144 μL, 1.311 mmol, 3.0 equiv) and hexanoic anhydride (142 μL, 0.524 mmol, 1.2 equiv) were sequentially added to a dichloromethane solution (2.2 ml, 0.25 M) of compound A (110.6 mg, 0.437 mmol), and the mixture was stirred at room temperature for 1 hour. After completion of the reaction, the solution was concentrated under reduced pressure. The obtained residue was purified by silica gel column chromatography (hexane:ethyl acetate = 4:1) to obtain a colorless oily substance (compound A’, 123.8 mg, 0.400 mmol, 91% yield). Compound A’: 1 H NMR (400 MHz, CDCl3) δ 7.21 (2H, d, J = 9.2 Hz), 6.90 (2H, d, J = 9.2 Hz), 4.42 (1H, m), 4.19 (1H, dd, J = 3.2, 10.0 Hz), 3.96 (1H, dd, J = 7.2, 9.6 Hz), 3.46 (2H, m), 2.26 (2H, dd, J = 7.6, 7.6 Hz), 2.09 (2H, m), 1.97 (2H, m), 1.62 (1H, m), 1.33 (5H, complex), 0.88 (3H, t, J = 7.2 Hz)

[0066] Test Example 1: Examination of Compounds that Promote the Redifferentiation of Arabidopsis thaliana Callus 1. Purpose Regarding the existence of compounds that promote the redifferentiation of plants, a search for novel compounds that promote redifferentiation was conducted using Arabidopsis thaliana as the material.

[0067] 2. Experimental Method (1) Experimental Materials The wild strain of Arabidopsis thaliana (accession: Col-0) was used as the experimental material. The seeds were purchased from Inplanta Innovations Co., Ltd. (https: / / www.inplanta.jp / ).

[0068] (2) Growth Method of Arabidopsis thaliana Arabidopsis thaliana seeds were sterilized with a Kitchin Hitter solution (Kao) diluted to half strength with distilled water for 3 minutes and then washed three times with sterilized distilled water. The washed seeds were sown on an agar medium consisting only of 1 / 2 Murashige & Skoog Medium including Vitamins (Nacalai), 10 g / L sucrose (FUJIFILM Wako), and 10 g / L gellan gum (FUJIFILM Wako), incubated at 4 °C overnight, and then transferred to an incubator (TOMY) to initiate growth. The set conditions of the incubator were a temperature of 22 °C and a photoperiod cycle of 16 hours of light period and 8 hours of dark period. The light conditions were 5000 - 6000 lux with white fluorescent lamps, and the conditions of the incubator including the light conditions were the same for the induction of callus and shoot.

[0069] (3) Callus induction in Arabidopsis thaliana One centimeter from the tip of the root of the plant on the 7th day after sowing was cut with scissors and transplanted onto a callus-inducing medium (CIM). CIM consists only of Gamborg's B5 Medium Salt Mixture (FUJIFILM Wako), 20 g / L glucose (FUJIFILM Wako), 0.5 g / L MES (FUJIFILM Wako), 1 - Gamborg’s vitamin solution (Sigma-Aldrich), 500 μg / L 2,4-D (Sigma-Aldrich), 50 μg / l kinetin (Sigma-Aldrich), and 0.8% (w / v) gellan gum. The pH was adjusted to 5.7 using KOH (FUJIFILM Wako). The CIM with the transplanted root was transferred to an incubator and cultured for 7 days to induce callus formation.

[0070] (4) Shoot induction and evaluation from callus in Arabidopsis thaliana Callus induced from roots using CIM was transplanted onto a shoot-inducing medium (SIM). SIM consists of Gamborg's B5 Medium Salt Mixture, 10 g / L sucrose, 0.5 2-morpholinoethanesulfonic acid (FUJIFILM Wako), 1-Gamborg’s vitamin solution, 2 μg / mL trans-zeatin (FUJIFILM Wako), 0.4 μg / mL indole-3-butyric acid (FUJIFILM Wako), 1 μg / mL D-Biotin (Nacalai), and 8 g / L gellan gum. After adding any one of compound A (manufactured by Enamine, https: / / enamine.net), B (manufactured by ChemDiv, https: / / www.chemdiv.com), and C (manufactured by ChemDiv) to it, the pH was adjusted to 5.7 using KOH. The SIM with transplanted callus was cultured for 2 weeks to induce shoot formation, and the promoting effect of the compound on redifferentiation was evaluated by quantifying the number of shoots formed on one callus.

[0071] 3. Results When compound A was added to SIM, the number of shoots per callus significantly increased in the treatment groups of 5 μM and 10 μM compared to the control. Similarly, when compounds B and C were added to SIM, the number of shoots per callus significantly increased in the treatment groups of 1 μM and 10 μM compared to the control.

[0072] 4. Conclusions Since the number of shoots per callus increased in the medium containing compounds A, B, and C compared to the control, it was found that these compounds promote redifferentiation in Arabidopsis thaliana.

[0073] Test Example 2: Examination of Compounds that Promote Redifferentiation of Hop Callus 1. Objective It was verified whether the promoting effect of compounds A, B, and C on redifferentiation observed in Arabidopsis thaliana could also be confirmed in hops.

[0074] 2. Experimental method (1) Experimental materials Hops (variety: Hallertauer). Hops seedlings grown in the Kirin Brewery Co., Ltd. nursery in Iwate Prefecture were used.

[0075] (2) Preparation and growth method of tissue culture seedlings Tissue culture seedlings of hops required for evaluating the redifferentiation efficiency were prepared. Stem segments containing one node were excised from hops seedlings as explants, sterilized by incubating in 70% ethanol (FUJIFILM Wako) for 1 minute and 1% (v / v) Sodium Hypochlorite Solution for 5 minutes. The explants were washed 3 times with sterile water for 1 minute each time, blotted dry with a paper towel, and then placed on an agar medium containing 1 / 2 Murashige & Skoog Medium including Vitamins, 20 g / L glucose, and 2 g / L gellan gum, and the culture was started in an incubator. The set conditions of the incubator were a temperature of 20 °C and a photoperiod cycle of 16 hours of light period and 8 hours of dark period (these conditions were the same for callus induction and shoot induction). After 2 weeks of culture, axillary buds elongated from the nodes were excised and placed on the above agar medium, and individuals that grew well were used as tissue culture seedlings. The tissue culture seedlings were subcultured by placing the apical buds on a new agar medium once every 1.5 months. The composition of the agar medium used for subculture was 1 / 2 Murashige & Skoog Medium including Vitamins, 20 g / L glucose, and 8 g / L agar (Ina Food Industry Co., Ltd.).

[0076] (3) Callus induction in hops The stems were excised from the tissue culture seedlings 1 to 1.5 months after subculture and transplanted onto the induction medium. The length of the stems was set to approximately 1 cm and they were made to exclude nodes. The composition of the induction medium was as follows: Murashige & Skoog Medium including Vitamins, 30 g / L sucrose, 2 mg / L thidiazuron (FUJIFILM Wako), 0.5 mg / L indole-3-acetic acid (FUJIFILM Wako), 6 g / L agar. The induction medium with the transplanted stems was transferred to an incubator and cultured for 3 weeks to induce callus formation.

[0077] (4) Shoot induction and evaluation from callus in hops When hop stem segments are placed on the induction medium, callus is formed from both ends. After 3 weeks of culture, the callus formed at both ends was excised and transplanted with the cut surface facing the medium side onto the induction medium containing any one of compounds A, B, and C. It was cultured in an incubator for 3 weeks to induce shoot formation from the callus, and the promoting effect of the compound on redifferentiation was evaluated by quantifying the number of callus with one or more shoots formed.

[0078] 3. Results Compared with the control, the proportion of callus with one or more shoots formed in the induction medium containing any one of compounds A, B, and C increased significantly.

[0079] 4. Conclusion When treated with compounds A, B, and C, the number of callus with shoots formed was significantly increased compared to the control, indicating that these compounds promote redifferentiation in hops.

[0080] Test Example 3: Search for the active structure of the novel compound A 1. Purpose For compound A, for which the promoting effect on redifferentiation was confirmed in Arabidopsis thaliana and hops, compound A' with a modified structure of compound A was synthesized to explore the active structure, and the shoot formation rate when compound A' was treated in Arabidopsis thaliana was analyzed. The structure of compound A' is shown in Production Example 2.

[0081] (1) Experimental materials It is the same as in Example 1.

[0082] (2) Method for growing Arabidopsis thaliana It is the same as in Example 1.

[0083] (3) Callus induction in Arabidopsis thaliana It is the same as in Example 1.

[0084] (4) Shoot induction from callus in Arabidopsis thaliana The callus induced from the root using CIM was transplanted onto a shoot-inducing medium (SIM). SIM consists of Gamborg's B5 Medium Salt Mixture, 10 g / L sucrose, 0.5 g / L MES, 1-Gamborg’s vitamin solution, 2 μg / mL trans-zeatin (FUJIFILM Wako), 0.4 μg / mL indole-3-butyric acid (FUJIFILM Wako), 1 μg / mL d-biotin (Nacalai), 8 g / L gellan gum, and Compound A'. The pH was adjusted to 5.7 using KOH. The SIM transplanted with the callus was cultured for 2 weeks to induce shoot formation, and the promoting effect of the compound on redifferentiation was evaluated by quantifying the number of shoots formed on one callus.

[0085] 2. Results When compared with the control, treatment with Compound A' decreased the number of shoots per callus in a concentration-dependent manner.

Industrial Applicability

[0086] By applying a composition for promoting plant redifferentiation to callus or plant sections, plant redifferentiation can be promoted. As a result, by the method of the present invention, regenerated plants can be efficiently produced.

Claims

1. A composition for promoting plant redifferentiation, comprising as an active ingredient one or more selected from the group consisting of a compound of the following formula (I), (II) or (III), or a pharmaceutically acceptable salt thereof or a solvate thereof: 【Chemical 1】 Formula (I) [In Formula I, R 1 ~R 11 may be the same or different and each independently represents H or an alkyl group having 1 or 2 carbon atoms.] 【Chemical Formula 2】 Formula (II) [In Formula II, R 12 ~R 15 and R 18 ~R 26 may be the same or different and each independently represents H or an alkyl group having 1 or 2 carbon atoms, and R 16 , R 17 , R 27 and R 28 may be the same or different and each independently represents H or an alkyl group having 1 to 3 carbon atoms.] [Chemical Formula 3] Formula (III) [In Formula III, R 29 ~R 39 may be the same or different and each independently represents H or an alkyl group having 1 or 2 carbon atoms.].

2. A composition for promoting plant redifferentiation, comprising as an active ingredient one or more selected from the group consisting of a compound of the formula (I), (II) or (III) or a pharmaceutically acceptable salt thereof or a solvate thereof, In formula I, R 1 ~R 11 is H, and In formula II, R 12 ~R 15 and R 18 ~R 26 are H, and R 16 , R 17 , R 27 and R 28 are alkyl groups having 1 carbon atom, In formula III, R 29 ~R 39 is H, which is the composition for promoting plant redifferentiation according to Claim 1.

3. The composition according to Claim 1, wherein the plant is a dicotyledonous plant.

4. The composition according to Claim 3, wherein the plant is a plant of the family Cannabaceae or Brassicaceae.

5. A method for promoting plant redifferentiation, comprising applying one or more selected from the group consisting of the compound according to Claim 1 or 2 or a pharmaceutically acceptable salt thereof or a solvate thereof to a plant section or callus.

6. Applying the composition for promoting plant redifferentiation according to Claim 1 or 2 to a plant section or callus to redifferentiate shoots, roots, and adventitious embryos, and regenerating a plant from the shoots, roots, and adventitious embryos, which comprises a method for producing a regenerated plant.

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