JP4817397B2 - Cell or organ differentiation regulator and method of regulating morphogenesis using the same - Google Patents

Description

  The present invention relates to a method for regulating / controlling cell or organ differentiation, and more particularly, to a method for regulating cell or organ differentiation using a component capable of controlling the redox state of a cell and such component. The present invention relates to a regulator of differentiation of cells or organs contained therein.

  Every living individual is constructed by differentiation and morphogenesis of cells and organs. For example, in a multicellular organism, cells gather to form a tissue or organ, and furthermore, an individual is constructed by a complex combination of tissues and organs. In order for each tissue or organ to perform its own function, the cells must be differentiated and organized in a manner specific to the individual tissue or organ.

  In such cell and organ differentiation, as well as morphogenesis, cells are thought to run their genetic programs under the influence of regulators such as hormones they receive. In amphibians such as newts, it is known that the concentration gradient of regulators at the morphogenesis stage such as development determines organ differentiation. In Drosophila, related genes have been identified through the isolation of mutants that have mutations in their regulatory mechanisms. In the case of plants, it is known that morphogenesis is regulated or controlled by a regulator called a plant hormone. In this way, a unique regulatory substance is mediated by the morphogenesis of an individual organism.

However, in recent years, attention has been paid to the similarity among genes that work in genetic programs related to morphogenesis among a wide range of organisms.
In addition, it is known that all living organisms are greatly affected by the kind of stress (eg, light, temperature, chemical substance, etc.) that is the generation and morphogenesis.

The high similarity and stress common elements (light, temperature, chemical substances, etc.) that make up the genetic program related to morphogenesis among organisms are related to the information transmission mechanism that leads to the genetic program. This suggests the commonality of intervening substances between organisms. However, there are no reports of such substances being isolated and identified.
If it is possible to obtain substances that can control the differentiation of cells and organs in the whole organism world or between a wide range of organisms, it is considered that it can greatly contribute to the development of research and industry.

The present invention aims to provide a means for regulating the differentiation of cells and organs.
Another object of the present invention is to establish a method for controlling, for example, the flowering time of plants and individual regeneration from callus using such a regulator.

Among the stress-related factors that affect development and morphogenesis, the present inventors have found that a substance that regulates the redox state of cells, which is a common factor among organisms, is responsible for cell and organ differentiation, as well as morphogenesis. I found out that it could have an impact and came to complete the present invention,
That is, the present invention provides a cell or organ differentiation regulator containing a cell redox state regulator.

  In the present specification, the phrase “differentiation of cells or organs” is used in the meaning generally known in the field of biology, and usually cells and organs are specialized structurally and functionally. Refers to that. As specific examples of “differentiation of cells or organs”, in the case of plants, embryos including flower bud formation, individual formation at germination, fallen leaves including delamination, winter bud formation, dormancy breaking, pollen germination, fertilization process Examples include generation, regeneration from callus, tubular element formation, root hair formation, trichome formation, and the like. In the case of animals, examples include neuronal differentiation or dendrite formation, epithelial cell or tissue formation, reproductive body formation, egg development including fertilization process, blood cell differentiation, hair matrix formation, and the like. In the case of the fungal kingdom, sporulation or perforated hyphal formation; in the case of the protozoan kingdom, ontogeny, flagellum formation or differentiation of cell organelles; in the case of the bacterial kingdom, colony formation, flagellum formation or spore formation may be mentioned, It is not limited to these.

  Further included is the differentiation and development of organelles such as plastids and mitochondria.

  In the context of the present invention, “morphogenesis” refers to the process by which an individual is formed from the units (molecules, cells, etc.) that make up an organism, and means the formation at the molecular and cellular levels.

  Therefore, the individual organisms subject to “regulation of cell and organ differentiation” or “regulation of morphogenesis” include all multicellular and unicellular organisms with morphogenesis (formation from anatomical structural units). Is included. That is, the main object of the present invention is the plant kingdom including flowering plants, lichens and moss, and the animal kingdom including echinoderms and arthropods to mammals, but in terms of form, it also includes single-celled organisms. To do.

As used herein, the term “cell redox state” is a state determined based on the following indicators. Ease of generating active oxygen, absolute amount of glutathione, ratio of glutathione (reduced form) to its oxidized form (GSH / GSSG), absolute amount of reduced form of nicotinamide adenine dinucleotide phosphate (NAD (P) H) , NADPH / NADP ⁺ ratio, cytochrome c oxidized / reduced ratio, and oxidation / reduction ratio of components of electron transport chains such as plastoquinone and ubiquinone.
However, substances involved in redox in vivo are known in the art and are not limited to the above.

  The term “cell redox state-regulating substance” means a substance that changes the above-mentioned value, a substance that affects the synthesis or amount of glutathione, a substance that promotes or inhibits the production of active oxygen, and oxidizes a compound. Examples thereof include substances that promote or inhibit the change to either the type or the reduced type.

The “cell redox state-regulating substance” used in the present invention may be any substance exhibiting the above-mentioned action. Specific examples thereof include reactive oxygens such as hydrogen peroxide and superoxide anion radical, and superoxide. Antioxidants such as dismutase, catalase, ascorbate peroxidase, dehydroascorbate reductase, monoascorbate reductase, glutathione reductase and glutathione peroxidase, glutathione synthesis inhibitors such as butionine sulfoximine and methionine sulfoximine, reduced form glutathione and oxidized glutathione, more homo glutathione and carboxypropyl glutathione, glutathione derivatives such as di-carboxyethyl glutathione, respiration inhibitors such as paraquat or KCN or NaN _3, up Reshin and spermine, polyamines such as spermidine, but more include the starting materials or inducer allowed to rise to these substances in the cell, but is not limited thereto.

  The cell or organ differentiation regulator of the present invention (hereinafter referred to as differentiation regulator) comprises at least one redox state regulator or contains it together with a suitable carrier and excipient. . Such a preparation can be produced by a known method by appropriately blending a known pharmaceutically acceptable carrier component, a preparation auxiliary agent and the like in each field. The dosage form can be arbitrarily selected from a liquid agent, an emulsion, a solid agent and the like according to the application object and application method.

  In some cases, it is easy to arbitrarily control the speed and state of morphogenesis by using two or more redox state-regulating substances as active ingredients in combination. Such a combination can be selected based on acting in one and the same metabolic system, for example, substances having mutually opposite activities. For example, in the examples described later, the flowering time of plants is controlled by a combination of glutathione and BSO which is a glutathione inhibitor. However, it may be a combination that acts on other metabolic systems.

  When the differentiation regulator of the present invention contains two or more kinds of redox state regulators, these active ingredients may be present in one preparation, but they are formulated separately and simultaneously or at different times. Can also be applied.

  The present invention also provides a method for regulating morphogenesis, characterized by applying the above-mentioned differentiation regulator to an organism individual having altered sensitivity to the regulator, and the organism individual thus obtained. Is. Here, the “individual organism whose sensitivity has changed” means that the specific sensitivity has changed, as exemplified by the mutant fca whose sensitivity to BSO is changed compared to the wild-type plant Ler. Means an individual. Such individuals can be expressed naturally or artificially.

  The differentiation regulator of the present invention can be applied to all kinds of organisms that are sensitive to cellular redox state-regulating substances that are active ingredients. In the case of plants, dicotyledonous and monocotyledonous angiosperms. It is effective against both plants.

  When the target of use of a differentiation regulator comprising the cell redox state-regulating substance of the present invention as an active ingredient is a plant, when artificially regulating flower regeneration, individual regeneration from callus, tubular element differentiation, germination, etc. However, it is considered that the desired adjustment is possible by adjusting the oxidation-reduction state of the cells, particularly the production level of active oxygen, the synthesis of glutathione and the oxidation / reduction ratio. In addition, it is considered that the morphology of plants such as root hairs and trichomes can also be regulated by substances that can regulate the redox state of cells as described above.

  Therefore, according to the method of the present invention, it becomes possible to widely control the morphogenesis of plants. Optimum conditions for the regulation of morphogenesis are not constant and depend on the type of plant, the type of morphology, etc., and the purpose.

  Plants to which the differentiation regulator of the present invention can be applied are exemplified below. Examples of dicotyledonous plants include Asagao genus plants (Asagao), Convolvulus genus plants (Convolvulus, Coleoptera serrata), Sweet potato genus plants (Gumby convolvulus, Sweet potato), Nigiri quail plants (Nenka quail, Legumes), Nadesico plants (carnations, etc.), Jacobe plants, Acanthaceae plants, Euglena plants, Citrus plants, Nominotsuri plants, Oyafusuma plants, Wagaiso plants, Hamakusa plants, Otsumusa plants, Shiotsumesa plants, Mandema, Senno, Fusigro, Nanbanjako, Anemoneaceae, Pomegranate, Plumaceae, Pepperaceae, Pleurotus, Yanagibe, Yamamata, Walnut plant, bag Mushroom plant, Buna family plant, Bungaceae plant, Nectaceae plant, Iranaceae plant, Dendrobaceae plant, Yamagashi family plant, Shabby mushroom plant, Bakada family plant, Yadogi family plant , Horse suzusaku plant, scabbard family plant, echidaceae plant, scallop plant, red plant plant, hiyari plant plant, white-spotted plant plant, Yamatobusa plant plant, Yamagoboshi plant plant, vine Plant, slippery plant, creek family, mountain bear plant, wig family plant, crocodile plant, pine family plant, cyperaceae plant, aceae plant, forage plant, tsuji fuji Family plant, waxy family plant, duckweed family plant, poppy plant, daffodil plant, oilseed family plant, another genus plant family, urchinaceae plant, beetle family plant, cynoaceae plant, Laveraceae plant, Mansaceae Suzukake family, rose family plant, legume plant, beetle family plant, durumaceae plant, linaceae plant, habaceae plant, mandarin family plant, ginger family plant, genus family plant , Himehagi, Todaigusa, Agaricaceae, Boxwood, Ganoderma, Dokugaku, Plant, Urushi, Mochinoki, Nishiki, Mitsuba Plant, Kurodaki Kazura family plant, Maple family plant, Tochino family plant, Muroraceae plant, Amaranthaceae plant, Azalea family plant, Kurome Mado family plant, Vine family plant, Horonodaceae family Plants, Shinanoceae plants, Aoiaceae plants, Papilioceae plants, Monkeysaceae plants, Camellia plants, Fairy solitary plants, Mizohakobe plant, Gyoryaceae plants, Violetae plants, Iriaceae plants, Combaceae, Tokeisou , Plant, scabaceae, urchinaceae, gummy plant, misoaceae plant, pomegranate plant, asteraceae plant, cucurbitaceae plant, papaveraceae plant, rhinoceros plant , Akabana family, Arinoto Usagi family plant, Suginomo family plant, Urchinaceae plant, Rariaceae plant, Mizuki family plant, Iwume family plant, Ryoaceae plant, Ichikuza family plant, Azalea family Plants, Bambooceae Plants, Sakurasaceae Plants, Isomatsuae Plants, Oyster Family Plants, Oyster Family Plants, Erynoceae Plants, Mosaceae Plants, Fuji next Family Plants, Glyceae Plants , Asteraceae plant, garaceae plant, peony plant, murasaceae plant, kumazatsu family plant, solatry plant, eggplant family plant, sesame plant family plant, sesame plant family plant, sesame family plant , Hama cruciferous plant, Iwa Tobacco, Tanukimata, Kitsunegogaku, Hamanchou, Tobacco, Tobacco, Tobacco, Tobacco, Tobacco, Tobacco Examples thereof include pine family plants, cucurbitaceae plants, asteraceae plants, asteraceae plants, and the like.

  Examples of monocotyledonous plants include duckweed plants (duckweeds) and duckweed plants (duckweeds, hinokimo), duckweed plants, cattleya plants, cymbidium plants, dendrobium plants, phalaenopsis plants, banda plants , Including Paphiopedilum plants, Oncidium plants, etc., Rubiaceae plants, Rabbitaceae plants, Ruriaceae plants, Rather family plants, Thorny family plants, Butterberry family plants, Main family plants, and Chigamine family plant, Japanese genus family plant, rice family plant, Papaver family plant, palm family plant, sweet potato family plant, Japanese family plant family, Japanese family plant family, Mizuaoi family plant, rush family plant, Glycaceae plant, Lily family plant, Higanbana family plant, Yamano potato family plant, Ayame family plant, Ganoderma plant, Ginger family plant, Kan Family plant can be exemplified Hinanoshakujou Plants like.

  As described above, in the present invention, the “redox state” of a certain organism depends on factors widely shared in the living world. For example, glutathione, which is an important factor for determining the redox state, is ubiquitous in living organisms, and its synthesis system and protein factors controlled by them exhibit high homology. Furthermore, enzymes related to active oxygen have high homology across biological species. Therefore, the redox state-regulating substance of the present invention is also effective as a regulator of morphogenesis in organisms other than plants, such as animals.

  Such modulators include, for example, parthenogenesis, induction of differentiation of cells and tissues such as nerves and epidermis, repair of differentiated tissues such as neural dendrites, differentiation of immunocompetent cells, zygote or spore formation, It is useful for controlling flagellum formation and the like, but is not limited thereto.

  In the animal kingdom, the organisms exemplified in the above case are, in the animal kingdom, vertebrate, echinoderm, protozoa, coelenterate, mollusc, arthropoda, annelid, sponge, flatfish In addition to fungi such as phylums, basidiomycetes and ascomycetes that form spores, as well as microorganisms such as actinomycetes that form spores, germination and zygotes Although yeast etc. are also included, it is not limited to this.

  Examples of these applications include pharmaceuticals such as regulators for skin repair and burn damage repair by burns, etc., culture control of salmon, uniform quality due to parthenogenesis of fish, prevention of decline in freshness of flowers, etc. Examples include prevention of infection by suppressing the formation of an attachment device, but the present invention is not limited thereto.

  The differentiation regulator of the present invention is used for various living individuals or organs, tissues, or cells by a method according to the dosage form. For example, in the case of solutions and emulsions, in the case of an organism that differentiates cells or organs, for example, in the case of plants, it is sprayed or dripped not only on the growth point of the plant but also on part or the whole of the plant body including stems and leaves Can be applied. Moreover, in the case of a solid agent or a powder agent, for example, it is absorbed into the roots from the ground. In addition, when the plant for cell or organ differentiation is aquatic plants such as duckweed, it can be absorbed from the root as a bottom floor additive, or a solid agent can be gradually dissolved in water. The present invention can be applied by any method and is not limited to the above-described embodiment.

When the differentiation regulator is a bottom floor additive or a solid agent, carrier components are generally talc, clay, vermiculite, diatomaceous earth, kaolin, calcium carbonate, calcium hydroxide, white clay, silica gel and other inorganic substances such as wheat flour and starch. A solid support is exemplified. In the case of a liquid agent, water, aromatic hydrocarbons such as xylene, alcohols such as ethanol and ethylene glycol, ketones such as acetone, ethers such as dioxane and tetrahydrofuran, dimethylformamide, dimethyl sulfoxide, acetonitrile, etc. The liquid carrier is exemplified.
Furthermore, other adjuvants can be appropriately blended with the differentiation regulator of the present invention. Examples of such auxiliary agents include anionic surfactants such as alkyl sulfates, alkyl sulfonates, alkyl aryl sulfonates and dialkyl sulfosuccinates, and cationic surfactants such as salts of higher aliphatic amines, Nonionic surfactants such as polyoxyethylene glycol alkyl ether, polyoxyethylene glycol acyl ester, polyoxyethylene glycol polyhydric alcohol acyl ester, cellulose derivatives, thickeners such as gelatin, casein, and gum arabic, bulking agents, binding Agents and the like.

  If necessary, plant growth regulators such as benzoic acid, nicotinic acid, nicotinic acid amide, pipecolic acid and the like can be incorporated into the preparation as long as the desired effects of the present invention are not impaired.

  In the following, the present invention will be described mainly by taking control and regulation of flowering time as an example, but those skilled in the art will understand that morphogenesis can be similarly controlled in other biological systems according to the description of the specification. I will.

The treatment with a differentiation regulator may be carried out by applying a cell redox state regulator at an appropriate concentration before and / or during the normal cultivation of the seeds or callus of the target plant. it can. Usually, it is effective to apply while performing treatment according to the nature of the target plant (long-day nature, short-day nature, etc.). Such processing is known to those skilled in the art. For example, in the case of relative long-day plants such as eustoma in the examples described later, it is considered effective to use the differentiation regulator of the present invention while performing light irradiation at a certain light intensity or higher.
Therefore, the present invention also includes the use of a differentiation regulator in any plant growing method usually used in the art.

The differentiation regulator may be a redox state regulator alone as an active ingredient, but as described above, a dosage form applicable to each plant, for example, liquid, solid, powder, emulsion, bottom bed addition It is preferably used in a dosage form such as an agent. Such preparations are prepared by using known carrier components, formulation adjuvants, etc. that can be applied in pharmacology in each field, as long as the flowering regulation is not impaired, the redox state of cells that are active ingredients. It can be appropriately blended with a regulator and manufactured by a known method.
EXAMPLES Hereinafter, although an Example demonstrates this invention concretely, this invention is not limited at all by these Examples.

Example 1 Regulation of flowering time (1)
Arabidopsis late-growth mutant fca (deposit number N167) and wild-type plant Ler (deposit number NW20) ordered from the Arabidopsis Stock Center, publicly available at the University of Nottingham, England, for 16 hours with 100 μE / m ² intensity light It was grown at 23 ° C. under the day length condition of the period / 8 hours dark period and used for the experiment. As the medium, soil formed by layering vermiculite (Asahi Kogyo) 2 in the lower layer, Kureha nursery soil (Kureha Chemical) 2 in the middle layer, and vermiculite (Asahi Kogyo) 1 in the upper layer was used.
L-Buthionine sulfoximine (BSO), which is an inhibitor of glutathione synthesis, and glutathione were used as substances that regulate the redox state of cells.

BSO processing:
BSO was dissolved in water to 10 ⁻¹ M to obtain a stock solution. Next, a solution of 10 ⁻³ to 10 ⁻⁷ M was prepared from the solution to prepare a treatment solution (see FIGS. 1 to 3). About 2 to 3 individuals were placed in a pot of about 40 × 50 mm, and the treatment liquid was dropped to the vicinity of the top of the stem so as to be 5 ml / day per pot. The processing was carried out once, 1, 3, 5, and 7 days after the start of the processing, and was treated as one time.

Glutathione treatment:
When performing glutathione treatment, glutathione was added to this BSO solution so that the concentration would be 10 ⁻⁴ M.

The effect of the treatment was evaluated based on the number of rosette leaves and the elongation rate of the flower stalk, and are shown in FIGS. In the figure, 23 cont and 4 cont are the results with and without low-temperature treatment, respectively (control experiment). The low temperature treatment was performed at 4 ° C. in parallel with the drug treatment. In FIG. 2, the BSO concentration used for the treatment is as follows: A, 0 M; B, 10 ⁻⁷ M; C, 10 ⁻⁶ M; D, 10 ⁻⁵ M; E, 10 ⁻⁴ M; F, 10 ^-3 M. Numerical values are expressed as mean ± standard error.

(1) The fca plant on the 17th day after water absorption was treated with a glutathione synthesis inhibitor BSO. The result after one week is shown in FIG. 1C. As a result, flower buds were observed with the naked eye about one week after the end of the treatment, and the number of rosette leaves (rooted leaves) decreased as compared with the water treatment control. In addition, the rate at which the flower stalks grew was examined over about a month after treatment (FIG. 2). From FIG. 2, it can be seen that the speed at which the flower stalk grows was faster in the treated group than in the control group, and flowering was accelerated. These indicate that the reduction in rosette number accelerated the flower bud determination itself as well as the growth after the BSO treatment determined the flower bud.

(2) On the other hand, an fca plant was obtained 12 days after water absorption. The result after one week is shown in FIG. 1A. FIG. 1A shows that the flowering time of fca was delayed by the processing, contrary to the case of (1). On the other hand, by the same experiment, in the case of the wild strain Ler, flowering was significantly accelerated by the start of treatment from 12 days after water absorption (FIG. 1B). When this Ler was grown on an agar medium containing BSO immediately after germination, the extraction of moss was delayed as in the case where fca was treated with a differentiation regulator early (from the 12th day of water absorption).

(3) BSO treatment and glutathione (GSH) treatment were used in combination. Glutathione (concentration: 10 ⁻⁴ M) was added to the BSO solution and used in the same manner as above.
Plants treated at a time when flower bud formation was delayed by BSO were earlier flowered as the GSH concentration increased, and flowered later at 1 mM (FIG. 3B). On the other hand, in the plant treated at the time when flower bud formation was accelerated by BSO, flowering was delayed as the GSH concentration increased (FIG. 3A).
These results indicate that glutathione, glutathione synthesis inhibitors, or combinations thereof can adjust the flowering time and further the flower stem elongation rate. In addition, it became clear that it is necessary to adjust the treatment time appropriately depending on the variety.

Example 2 Adjustment of flowering time (2)
In the same manner as in Example 1, Arabidopsis late-growth mutant fca was grown at 23 ° C. under a day length condition of 16 hours light period / 8 hours dark period with 100 μE / m ² intensity of light. Used for.
As a cell redox state regulator, hydrogen peroxide having the concentration shown in FIG. 4 was used. A hydrogen peroxide aqueous solution was prepared and used immediately before the treatment. About 2 to 3 individuals were placed in a pot of about 40 × 50 mm, and the treatment liquid was dropped from the 17th day of water absorption to the vicinity of the top of the stem so as to be 5 ml / day per pot. The treatment was performed 1, 3, 5, and 7 days after the treatment day, and was treated once. The timing of flower bud formation was compared using individual rosette leaves as an index. The results are shown in FIG. In the figure, 23 cont is water treatment (control experiment). Numerical values are expressed as mean ± standard error.

The time of flower bud formation using the rosette leaf of the individual as an index was earlier in the treated individual than in the case of water treatment (Fig. 4). The elongation rate of flower stems was also accelerated at the optimum concentration (10 ⁻⁵ M) (FIG. 5). In FIG. 5, the horizontal axis represents the number of days after the completion of the drug processing.
The above results indicate that the active oxygen itself and the substance that generates the active oxygen have not only the effect of adjusting the flower bud formation time but also the effect of promoting the elongation of the flower stalk.

Example 3 Control of flowering time 3
In the same manner as in Example 1, Arabidopsis late-growth mutant fca was grown at 23 ° C. under a day length condition of 16 hours light period / 8 hours dark period with 100 μE / m ² intensity of light. It was.
KCN or NaN ₃ was used as a cell redox state regulator. KCN or sodium azide was adjusted immediately before treatment and used at the concentrations described in FIGS. 6 and 7, respectively. About 2 to 3 individuals were placed in a pot of about 40 × 50 mm, and the treatment solution was dropped to the vicinity of the top of the stem so as to be 5 ml / day per pot. The treatment with KCN or NaN ₃ was performed from the 17th day after seed water absorption. On the day of treatment, 1, 3, 5, and 7 days later, the treatment was performed once. The timing of flower bud formation was compared using individual rosette leaves as an index. The experimental results using KCN or NaN ₃ are shown in FIGS. 6 and 7, respectively. In the figure, 23 cont is water treatment (control experiment). Numerical values are expressed as mean ± standard error.

The timing of flower bud formation using rosette leaves as an index is earlier in the treated group than in the water-treated control group at each optimum concentration (KCN: 10 ⁻³ ; NaN ₃ : 10 ⁻⁴ ).
The above results indicate that the substance that generates active oxygen has an effect of adjusting the flower bud formation time.

Example 4 Morphogenesis Regulation 1 at Germination
Place soft paper in a plastic dish (100 x 15 mm), put 15 seeds of wheat (variety: white egret) or rice (variety: Japan fine), and add hydrogen peroxide solution to 15 ml, Germination (light conditions, 50 μE / m ² , 16 h light / 8 h dark cycle; temperature, wheat: 18 ° C., rice: 27 ° C.). The aqueous hydrogen peroxide solution having the concentration shown in FIG. 8 was prepared at the time of use and used for the treatment of these seeds. The length of the cotyledon sheath of wheat treated with each hydrogen peroxide concentration was measured 144 hours (6 days) after water absorption. The results are shown in FIG. In the figure, the numerical value is expressed as an average value ± standard error.
FIG. 8 shows that the cells are elongated in the plant treated with hydrogen peroxide.
The above results indicate that hydrogen peroxide has the effect of regulating morphogenesis related to cell elongation during germination.

Example 5 Morphogenesis control 2 at germination
Place soft paper in a plastic dish (100 x 15 mm), add 15 seeds of zinnia (cv scarlet frame), add hydrogen peroxide solution to 15 ml and let it germinate (light conditions, 50 μE / m ² , 16 h light / 8 h dark cycle; temperature, 27 ° C.). A hydrogen peroxide solution having the concentration shown in FIG. 9 was prepared at the time of use and used for the treatment of these seeds. The germinated individuals were observed 2 days after water absorption (see FIG. 9). In the figure, the numerical value (unit: mM) of the hydrogen peroxide concentration used for the treatment is described at the lower right of each figure. From the figure showing germination in the case of 60 mM hydrogen peroxide treatment, it can be seen that the cotyledon develops faster and the cotyledon comes out from the pericarp before the root. Thus, when the treatment concentration of hydrogen peroxide increases, the treated seeds germinate from the cotyledon rather than from the root, and the development of the cotyledon in the treated group is accelerated compared to the untreated group.
The above results indicate that hydrogen peroxide has an action of accelerating the development time of cotyledons.

上記の結果は、グルタチオン量および酸化還元比を人為的にかえることにより、不定芽形成の調整が可能であることを示している。 Example 6 Regulation of Callus Adventitious Bud Formation The effect of glutathione on adventitious bud formation from callus was examined.
Callus containing tobacco leaves grown aseptically at 1 mg / L benzyladenine (BA) and 1 mg / L naphthaleneacetic acid (NAA) in a basic medium of 1% agar and Murashige / Skoog medium (MS medium) After callus formation on induction medium, the callus is transformed into a 1% agar and Murashige / Skoog medium (MS medium) basic medium with BA 1 mg / L and NAA 0.1 mg / L. Moved. To the adventitious bud induction medium, 0.1 mM BSO, 1 mM GSH, 1 mM GSSG (oxidized glutathione) was added as a redox state adjusting substance. As a control medium, a control without these substances was prepared. After culturing callus on these adventitious bud induction medium and control medium for 3 weeks, the number of adventitious buds was calculated. The results are shown in Table 1.

The above results indicate that adventitious bud formation can be adjusted by artificially changing the amount of glutathione and the redox ratio.

Example 7 Regulation of root hair formation 1
A soft paper was placed in a plastic dish (100 × 15 mm), and Arabidopsis wild-type plant Ler was added, and 15 ml of an aqueous solution with a hydrogen peroxide concentration of 10 ⁻³ M was added to absorb water. Germination was performed at 23 ° C., and after one week from water absorption, root hair formation was observed with a microscope. Root hair formation was observed in water, whereas no root hair formation was observed in the hydrogen peroxide solution.

Example 8 Regulation of root hair formation 2
Soft paper was laid in a plastic dish (100 × 15 mm), black soybean seeds (variety: Tamba black) were added, and 15 ml of an aqueous solution with a hydrogen peroxide concentration of 10 ⁻³ M was added to absorb water. Germination was performed at 23 ° C., and after one week from water absorption, root hair formation was observed with a microscope. Root hair formation was observed in water, whereas no root hair formation was observed in the hydrogen peroxide solution.

Example 9 Adjustment of Tubular Element Formation
According to the method of Sugiyama et al. (Sugiyama M. Fukuda, H. and Komamine, A. (1986) Plant Cell Physiol. 27: 601-606), Zinnia (variety: canary bird) was sown and 14 days after sowing, The mesophyll cells isolated from the first leaf of 3.5-4.5 cm in length are 2.5-4.0 × 106 / ml in D medium containing naphthalene acetic acid (0.1 mg / l) and benzylaminopurine (1 mg / l). And suspended. 2 ml of this culture solution was dispensed into a test tube, and rotated at 25 ° C. in the dark. At the start of the culture, superoxide dismutase (SOD) or catalase was added at the concentration shown in FIG. Sampling was performed after 72 hours of culture, and cells in which stripes due to secondary cell wall thickening were observed were differentiated into tubular elements. In addition, the ratio of tubular element cells in the total number of cells was defined as the differentiation rate. The results are shown in FIG. In FIG. 10, the vertical axis represents the tubular element differentiation rate (TE differentiation) (%). In addition, the concentrations of SOD and catalase added at the start of the culture are described below the column in terms of activity (U / ml, top) and protein amount (μg protein / ml, bottom). Numerical values are expressed as mean ± standard error. In the figure, TE means a tubular element. In the figure, “D” means D medium.

  The present invention has made it possible to control the differentiation of cells and organs regardless of the type of organism.

Regulation of glutathione inhibitor L-Buthionine sulfoximine (BSO) on the flowering time of Arabidopsis late-growth mutant fca (hereinafter referred to as fca) and wild-type plant Ler (hereinafter referred to as Ler) It is a graph which shows an effect. The regulatory effect was evaluated based on the number of rosette leaves. BSO treatment was performed from the 12th day after seed water absorption (FIG. 1A, fca; FIG. 1B, Ler). For fca, BSO treatment was also performed from day 17 (FIG. 1C). 23 cont and 4 cont are the results for low temperature treatment “none” and “yes”, respectively (control experiment). Numerical values are expressed as mean ± standard error. It is a graph which shows the regulation effect of BSO with respect to the elongation rate of the flower stem of fca. BSO treatment was performed from the 12th day after germination and the effect on flower stem elongation was examined. The BSO concentration used for the treatment is as follows. A, 0 M; B, 10 ⁻⁷ M; C, 10 ⁻⁶ M; D, 10 ⁻⁵ M; E, 10 ⁻⁴ M; F, 10 ⁻³ M. Numerical values are expressed as mean ± standard error. It is a graph which shows the influence of glutathione (GSH) with respect to the flowering (flower bud formation) time regulation effect of BSO. BSO treatment was performed from the 12th day after germination. Glutathione was given at the same time as the BSO treatment. In the figure, 23cont and 4cont have the same meaning as in FIG. A is the result of using Ler and B is the result of using fca. It is a graph which shows the control effect of hydrogen peroxide with respect to the flowering time of the late-generation mutant fca. The treatment with hydrogen peroxide was carried out from the 17th day after the seeds were absorbed. 23 ° C is a water treatment (control experiment). Numerical values are expressed as mean ± standard error. It is a graph which shows the control effect of hydrogen peroxide with respect to the elongation rate of the flower stem of fca. Hydrogen peroxide treatment was performed from the 12th day after germination, and the effect on flower stem elongation was examined. The concentration of hydrogen peroxide used in the treatment is indicated on the figure. Numerical values are expressed as mean ± standard error. It is a graph showing the control effect of potassium cyanate (KCN) with respect to the flowering time of the late-generation mutant fca. The treatment with KCN was carried out from the 17th day after seed water absorption. 23 ° C is a water treatment (control experiment). Numerical values are expressed as mean ± standard error. Is a graph showing the regulating effects of NaN ₃ for flowering time of late of mutant fca. Treatment with sodium azide was performed from day 17 after seed water absorption. 23 cont is water treatment (control experiment). Numerical values are expressed as mean ± standard error. It is a graph which shows the regulation effect (morphological regulation 1) with respect to the form change of wheat by hydrogen peroxide. The result of 144 hours (6 days) after water absorption is shown. The length of the cotyledon sheath of wheat treated with each hydrogen peroxide concentration is shown. Numerical values are expressed as mean ± standard error. It is a photograph which shows the control effect (morphological control 2) of hydrogen peroxide with respect to the change of the form of Zinnia. The numerical value (unit: mM) of the hydrogen peroxide concentration used for the treatment is described at the lower right of each figure. The figure shows the plant after 48 hours of water absorption. As the treatment concentration of hydrogen peroxide increases, plants begin to germinate from the development of cotyledons, not roots. From the figure showing the germination in the case of 60 mM treatment, it can be seen that the cotyledon develops early and the cotyledon comes out from the pericarp before the root. Cultivation of mesophyll cells isolated from the first leaf that was 3.5-4.5 cm long 14 days after sowing of Zinnia (variety: Canarybird) in the presence of superoxide dismutase (SOD) or catalase in vitro It is a graph which shows the result of having investigated the adjustment effect of tubular element formation. The concentrations of SOD and catalase added at the start of the culture are shown below the column in terms of activity and protein content. In the figure, TE represents a tubular element. Numerical values are expressed as mean ± standard error.

Claims (4)

It contains one or more redox state regulators of cells selected from the group consisting of hydrogen peroxide, superoxide dismutase, catalase, buthionine sulfoximine, reduced glutathione and oxidized glutathione. A regulator for regulating the differentiation of a plant cell or organ selected from: individual formation at germination, redifferentiation from callus, and tubular element differentiation. Differentiation of cells or organs of the plant is an individual form during germination, modulating agent according to claim 1 redox state regulation substance is hydrogen peroxide. The plant cell or organ differentiation is redifferentiation from callus, and the redox state regulator is one or more selected from the group consisting of buthionine sulfoximine, reduced glutathione and oxidized glutathione. Item 2. The regulator according to Item 1 . Differentiation of cells or organs of the plant is tubular element differentiation, is one or more redox conditioning agent is selected from superoxide dismutase and catalase, modulating agent according to claim 1.

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