JP2000093168A - Induction of pigment cell of curcuma plant, and production of pigment from the pigment cell - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method for inducing a pigment cell producing a pigment of a plant belonging to the genus Curcuma, and further to provide a method for producing the pigment from the pigment cell. SOLUTION: This method for inducing a pigment cell of a plant belonging to the genus Curcuma comprises using an amorphous tissue at a middle stage of the undifferentiated cell induced from the callus of the plant belonging to the genus Curcuma and the differentiated tissue in the pigment-producing tissue cultivation of the plant belonging to the genus Curcuma. The method for inducing the pigment cell in the cultured tissue of the plant belonging to the genus Curcuma comprises (1) a step for inducing the callus by culturing a section of the plant belonging to the genus Curcuma by using a medium containing auxins and cytokinins, (2) a step for inducing the amorphous tissue by culturing the obtained callus by using the medium containing the auxins and the cytokinins, and (3) a step for inducing the pigment cell in the obtained amorphous tissue.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to the genus Curcuma ( Cu
The present invention relates to a method for inducing pigment cells that produce pigments of a plant by tissue culture, and a method for collecting pigments from the pigment cells to efficiently produce pigments of a Curcuma plant. More specifically, the present invention uses a non-differentiated cell derived from the callus of a Curcuma plant and an amorphous tissue at an intermediate stage of a differentiated tissue to induce and form pigment cells in a cultured tissue of the Curcuma plant. The present invention relates to a method and a method for mass-producing a dye using the dye cells.

[0002]

2. Description of the Related Art In recent years, there has been a remarkable increase in demand for natural products with higher safety from synthetic products, and the development of environmentally friendly techniques for utilizing natural materials has been activated. Under these circumstances, there has been increasing interest in plant pigments, which are natural pigments.Therefore, techniques for producing plant pigments using plant tissue cultures have attracted attention,
So far, various plants have been studied, and various techniques have been reported (BIO INDUSTRY, V).
ol. 7, No. 10, p. 5-11 (199
0)). In general, there are various merits of producing plant pigments using plant culture tissues, but mainly the following points: 1) There is no variation among varieties because a specific plant tissue is used; 2) As in the case of cultivated plants, there is no risk of disease during cultivation, poor growth due to bad weather, etc. 3) Naturally, plants produce pigments in specific organs such as roots and leaves. Except for the production tissue, it becomes an unnecessary part and the production efficiency of the dye is poor. However, such inefficiency can be improved by using a cultured tissue.

[0003] In such a technical background, regarding a method of producing a plant pigment, as a conventional technique, for example, a technique of producing a pigment by culturing a plant tissue in the state of undifferentiated cells such as callus, On the contrary, a technique of producing a pigment by culturing in a differentiated tissue state such as an adventitious embryo is known as a typical method. Some specific examples thereof include a method for producing a dye from undifferentiated cells such as callus, for example, a method for producing a red dye from a cell mass obtained by tissue culture of T. murasaki (Japanese Patent Laid-Open No. 166396/1986). ), A method for producing a red-red pigment from cells derived and proliferated from a flower of a red flower (JP-A-62-179392), and a method for producing a red pigment from callus induced by tissue-cultivating sweet potato (JP-A-63) Medium used for producing lavender blue pigment by tissue culture of lavender cells (JP-A-3-206881), cultivation method of safflower yellow pigment-producing tissue (Japanese Patent Publication No. 6-4027), genus Lactuca For collecting and producing a yellow pigment from a culture obtained by tissue culture of a plant belonging to the group (JP-A-7-1551)
90), and so on. Examples of a method for producing a pigment from a differentiated tissue such as an adventitious embryo include, for example, a method for producing a purple pigment from an embryoid differentiated from a plant of the genus Oenocera (Japanese Patent Laid-Open No. 62-21092), And a method of differentiating petals to produce a red pigment (Japanese Patent Application Laid-Open No. Hei 2-276581).

However, irrespective of the above-mentioned prior art, a method for inducing pigment cells of a curcuma genus plant such as turmeric according to the present invention has so far been carried out from the callus of a curcuma plant to an intermediate stage between undifferentiated cells and differentiated tissues. After forming a certain amorphous tissue, there is no known technique for inducing pigment cell differentiation in the amorphous tissue. The conditions under which the cultured tissue of a plant forms pigment cells vary greatly depending on the type of plant. For example, in the case of turmeric, pigment cells are not formed even if culture is continued in a callus state.

[0005]

Under such circumstances, the present inventor has proposed that among various plants, the curcuma plant,
Primarily using turmeric ( Curcuma longa ) as a material and conducting intensive studies with the aim of developing a technique for producing a yellow pigment from the plant by tissue culture, undifferentiated plants derived from the callus of Curcuma plants The use of amorphous tissue at the intermediate stage between cells and differentiated tissue, especially the addition of certain plant hormones (auxin and cytokinin) to the culture medium to allow the tissue at the intermediate stage between undifferentiated cells and differentiated tissue (hereinafter After the formation of pigment cells, a pigment-producing pigment cell is formed in the cultured tissue by trying to induce pigment cell differentiation preferably in a medium to which auxin is not added. After further research, the present invention was completed. That is, an object of the present invention is to provide a method for inducing and forming a pigment-producing pigment cell in a cultured tissue of a Curcuma plant. Also, the present invention
It is an object of the present invention to provide a method for collecting and mass-producing a dye from the induced pigment cells.

[0006]

The present invention for solving the above-mentioned problems comprises the following technical means. (1) Inducing pigment cells of a genus Curcuma, which comprises using an undifferentiated cell derived from the callus of the genus Curcuma and an amorphous tissue at an intermediate stage of the differentiated tissue in the culture of the pigment-producing tissue of the Curcuma genus Method. (2) A method for inducing pigment cells in a cultured tissue of a Curcuma genus plant, comprising the following steps: a. A step of culturing a tissue piece of a Curcuma plant using a medium containing auxins and cytokinins to induce callus; b. Culturing the obtained callus using a medium containing auxins and cytokinins to induce an amorphous tissue, c. A method for inducing pigment cells in a Curcuma plant, which comprises the step of inducing pigment cells in the obtained amorphous tissue. (3) Callus obtained by tissue culture from a Curcuma genus plant is cultured using a medium containing auxins and cytokinins to induce an amorphous tissue, and then induces pigment cells in the amorphous tissue. A method for inducing a pigment cell of a Curcuma plant, comprising the steps of: (4) The above-mentioned (2) or (3), wherein the amorphous tissue is cultured using a medium containing no auxins to induce pigment cells.
The guidance method described in 1. (5) The induction method according to (1), (2) or (3), wherein the Curcuma plant is turmeric. (6) A method for producing a pigment of a genus Curcuma, wherein the pigment is collected from a pigment cell induced by the method according to any one of (1) to (5).

[0007]

Next, the present invention will be described in more detail. Curcuma plants In the present invention, curcuma plants are targeted. Examples of the plants belonging to the genus Curcuma include turmeric ( Curcuma longa ) and gejutsu ( Cu
rcuma zedoaria ), Kyoto ( Cur )
cuma aromatica ) and Curcuma petiolata are typical examples, but all plants belonging to the genus are considered.

Callus Induction Step The present invention relates to the formation of an irregular-shaped tissue (FIGS. 3, 5 and 8) intermediate between an undifferentiated cell and a differentiated tissue derived from a callus of a Curcuma plant in a pigment-producing tissue culture of the Curcuma plant.
), And inducing and forming pigment cells that produce a pigment in the amorphous tissue. The method for inducing the callus and the amorphous tissue may be any method. Well, there is no particular limitation. In the present invention, for example, callus can be suitably induced by placing a tissue piece of a Curcuma plant on a solid medium containing auxins and cytokinins and culturing it. In this case, as a tissue piece of a plant, a tissue piece of an appropriate organ of a normal plant is sterilized, or a sterile seedling, a bud of a plant that has grown the sterile seedling,
Roots, rhizomes, leaf sections and the like are preferably used. Among these, a tissue piece of a bud is particularly preferable. The medium used in the present invention may be a medium generally used for plant tissue culture. Specifically, for example, MS (Murash
image & Skoog) medium, LS (Linsmai)
er & Skoog) medium, B5 medium, Nitsch
& Nitsch medium, Gamburg medium, White
A culture medium, a Tuleke medium and a modified medium thereof can be used.

[0009] Of the auxins and cytokinins used to induce callus, auxins include:
2,4-dichlorophenoxyacetic acid (2,4-D), α-
Naphthyl acetic acid (NAA), indole acetic acid (IAA),
And indolebutyric acid (IBA). Examples of cytokinins include furfurylaminopurine (kinetin), benzyladenine (BA), and isopentenylaminopurine (2iP). The concentration of auxins required for callus induction ranges from 0.1 to 1
0 mg / l is preferred, and the concentration of cytokinins is preferably 0.1 to 50 mg / l.

In the above-mentioned medium, saccharides generally used for tissue culture, such as sucrose, glucose and fructose, can be used as a carbon source in addition to plant hormones. The concentration of the saccharide is preferably 1 to 10 g / l. It is also preferable to adjust the pH of the medium to about 5 to 6 by appropriately adding an aqueous solution such as potassium hydroxide or hydrochloric acid. As a support material, for example, 0.5 to 10% by weight of agar is added to a medium containing such components,
After sterilization by a conventional method, the mixture is solidified to prepare a callus-derived solid medium. The support material is not limited to agar, and any suitable material such as gellan gum can be used as long as it has the same effect.

[0011] Callus can be induced by placing the above tissue piece of the Curcuma plant on the callus-inducing solid medium and culturing it.
It is preferable to culture at a temperature of about ° C for about 2 to 3 months.
The callus may be induced under either dark conditions or light irradiation conditions. In addition, as the callus used in the present invention, a callus obtained by subculturing callus obtained by culturing a plant tissue piece can be used.

Step of Inducing Amorphous Tissue In the present invention, the callus obtained as described above is cultured in a medium containing auxins and cytokinins, thereby inducing an amorphous tissue. Here, the amorphous tissue is a tissue at an intermediate stage between an undifferentiated cell and a differentiated tissue, and is a tissue that has undergone a certain degree of tissue differentiation, but has not differentiated into a complete tissue such as an embryo. . The medium used here may be a medium commonly used for tissue culture of plants as described above. As the auxins and cytokinins used to induce the amorphous tissue, the same ones as described above can be used.
A suitable concentration of auxins required for inducing amorphous tissue is 0.1 to 10 mg / l, and a concentration of cytokinins is 0.1 to 10 mg / l.
A suitable value is 50 mg / l.

In addition to the plant hormones, the above-mentioned saccharides can be used as a carbon source in the medium. The concentration of the saccharide is preferably 1 to 10 g / l. The medium containing such components can be sterilized by a conventional method to prepare an amorphous tissue induction medium (liquid medium). An amorphous tissue can be induced by adding the callus to the amorphous tissue-inducing medium and culturing the culture under shaking conditions, for example, about 10 to 150 rpm. In this case, an amorphous tissue can be induced by culturing at a temperature of 20 to 30 ° C. for about 2 months. The induction of the amorphous tissue may be performed under either a dark condition or a light irradiation condition. still,
In the induction of amorphous tissue, it is desirable to transfer the culture to a fresh medium once every two to three weeks.
In addition, as the amorphous tissue used for inducing the pigment cells in the present invention, the amorphous tissue obtained as described above, which is further subcultured, can be used.

Pigment Cell Inducing Step Next, the amorphous tissue obtained as described above is cultured, for example, using a liquid medium, so that the pigment that produces the pigment of the genus Curcuma in the amorphous tissue is obtained. Induce cells,
Can be formed. As the medium used for inducing the pigment cells, the same medium as the above-mentioned one for inducing the amorphous tissue can be used. However, in order to induce and form the pigment cells in the amorphous tissue, auxins (particularly 2, 4 -D) is preferably not contained. Although cytokinins need not be contained, they may be contained.

[0015] The above-mentioned amorphous tissue is added to the pigment cell induction medium, and preferably under shaking conditions, for example, 10 to 1
By culturing under shaking conditions of about 50 rpm,
Pigment cells can be induced. In this case, 20-3
By culturing at a temperature of 0 ° C., pigment cells start to form about 6 weeks later. In order to suitably induce pigment cells, it is preferable to culture under dark conditions. In the induction of pigment cells, once every 2-3 weeks
At a certain rate, it is desirable to transfer the amorphous tissue to a fresh medium.

Pigment Collection Step From the pigment cells that produce the pigment of the Curcuma genus plant induced as described above, the pigment can be collected by any appropriate means. The pigment can be efficiently extracted. As an organic solvent for extracting the pigment, for example, acetonitrile, ether, hexane, ethyl acetate, and the like can be used. The dye can be collected by a normal method.
There is no particular limitation. The collected pigment can be widely used as a natural pigment derived from plant tissue and as an active ingredient of a coloring agent in various product fields such as for food, cosmetics, and clothing.

[0017]

Next, the present invention will be specifically described based on examples, but the present invention is not limited to the examples. Example (1) Induction of Callus A portion containing axillary buds of a turmeric ( Curcuma longa ) rhizome is cut into a block, and the block is sterilized with a 5% sodium hypochlorite solution for 10 minutes.
Washed with sterile water. In a clean bench, a site containing a growth point of about 2 to 3 mm square was cut out from the block aseptically and placed on a callus-derived solid medium. here,
As a callus-inducing solid medium, 2,4-D
1 mg / l, 1 mg / l BA, 3% by weight of sucrose, 0.9% by weight of agar were added to adjust the pH to 5.8, and sterilized with an autoclave was used.
The cultivation was performed under the irradiation conditions of a room temperature of 25 ° C., an illuminance of about 3,000 lux and a photoperiod of 16 hours. About 60 days after the start of culture,
A callus having an uneven surface was induced from the site including the growth point. Figure 1 shows the induced callus (photo).
4 and 7. It can be seen from the cross section of the callus that the epidermal structure is not clear (FIG. 7).

(2) Subculture of callus The callus induced in the above (1) is cultured under the same conditions and in the same medium as the above-mentioned callus-derived solid medium, and replaced with a new medium every 21 days. After transplantation, callus was subcultured.

(3) Induction of amorphous tissue In the above (2), the subcultured calli were subjected to shaking culture using an amorphous tissue-inducing liquid medium.
Here, as the amorphous tissue-inducing liquid medium, 1 mg / l of 2,4-D, 1 mg / l of BA, and 3% by weight of sucrose were added to MS medium, adjusted to pH 5.8, and sterilized by an autoclave. Was used. Culture was performed by ingesting 9 g of callus with fresh weight and applying the liquid medium 8 described above.
Into a 300 ml Erlenmeyer flask containing 0 ml,
The irradiation was performed at a room temperature of 25 ° C., an illuminance of about 3,000 lux, and a photoperiod of 16 hours under a condition of a shaking rotation speed of 120 rpm, and the callus was transferred to a flask containing a fresh medium every 14 days. went. About 28 days after the start of the culture in the above liquid medium, an amorphous tissue having a smooth surface with a surface structure consisting of about 3 to 4 cell layers was induced. Induced amorphous tissue (photo)
Are shown in FIGS. 3 to 3
It can be seen that the four cell layers constitute the epidermis (FIG. 8).

(4) Subculture of amorphous tissue The amorphous tissue induced in the above (3) was cultured in the same medium as the above-mentioned amorphous tissue-inducing liquid medium under the same conditions, and was cultured for 14 days. The medium was transferred to a new medium every other time, and subculture of the amorphous tissue was performed.

(5) Induction of Pigment Cells In the above (4), the subcultured amorphous tissue was subjected to shaking culture using a pigment cell-inducing liquid medium. Here, as the pigment cell-inducing liquid medium, a medium adjusted to pH 5.8 by adding 3% by weight of sucrose to the MS medium was used after being sterilized by an autoclave. The culture was performed by taking 3 g of the amorphous tissue with fresh weight, adding it to a 300 ml Erlenmeyer flask containing 40 ml of the above liquid medium, and shaking at 40 rpm at room temperature and 25 ° C. in the dark. The callus was transferred every 14 days to a flask containing a fresh medium. Approximately 42 days after the start of the culture in the above liquid medium, pigment cells that had accumulated yellow pigment in the amorphous tissue were induced. Amorphous tissues (photographs) from which pigment cells have been induced are shown in FIGS. It can be seen from the cross section of the amorphous tissue that pigment cells have been formed (FIG. 9).

(6) Collection of pigment The yellow pigment contained in the pigment cells thus obtained was extracted with acetonitrile and measured by high-performance liquid chromatography. The pigment was found to be contained in the natural turmeric rhizome. Almost the same retention time peaks as those of curcuminoids were observed. This allows
The yellow pigment is presumed to be a curcuminoid derivative.

[0023]

Industrial Applicability As described in detail above, the present invention relates to the use of an indifferentiated cell derived from callus of a Curcuma genus plant and an amorphous tissue at an intermediate stage of a differentiated tissue in a curcuma plant culture tissue. The present invention relates to a method for inducing and forming a pigment cell that produces a pigment of a Curcuma plant, and a method for collecting and producing a pigment from the pigment cell. According to the present invention, 1) the pigment of a Curcuma plant is A pigment cell to be produced can be induced and formed in a cultured tissue. 2) A pigment can be efficiently collected and produced using the above pigment cell. 3) A cultured cell of a Curcuma genus plant can be efficiently pigmented. And 4) a method for differentiating and inducing pigment cells by new tissue culture using an amorphous tissue at an intermediate stage between an undifferentiated cell and a differentiated tissue. Special effect that is exhibited.

[Brief description of the drawings]

FIG. 1 shows callus (tissue photograph) derived from turmeric cultured tissue.

FIG. 2 shows an amorphous tissue (photograph) induced by culturing the callus.

FIG. 3 shows an amorphous tissue (photograph) in which pigment cells are induced by culturing the amorphous tissue.

FIG. 4 shows an uneven shape (a photograph of a structure) on the surface of the callus.

FIG. 5 shows a smooth surface (a photograph of the structure) of the surface of the irregular structure.

FIG. 6 shows a surface (a photograph of a tissue) of an amorphous tissue from which the above-mentioned pigment cells were induced.

FIG. 7 shows a cross section (photograph of cells) of the callus.

FIG. 8 shows a cross section (photograph of cells) of the amorphous tissue.

FIG. 9 shows a pigment cell (photograph) in a cross section of an amorphous tissue from which the pigment cell has been induced.

Claims (6)

[Claims] 1. A pigment cell of a genus Curcuma, which comprises using an undifferentiated cell derived from a callus of a genus Curcuma and an amorphous tissue at an intermediate stage of a differentiated tissue in the culture of a pigment-producing tissue of a Curcuma genus plant. Induction method. 2. A method for inducing pigment cells in a cultured tissue of a Curcuma genus plant, comprising the following steps: (1) using a medium containing an auxin and a cytokinin as a tissue fragment of the Curcuma plant; (2) culturing the obtained callus using a medium containing auxins and cytokinins to induce an amorphous tissue, and (3) culturing the callus. A method for inducing pigment cells of a genus Curcuma, comprising the step of inducing pigment cells in a fixed tissue. 3. A callus obtained by tissue culture from a Curcuma genus plant is cultured in a medium containing auxins and cytokinins to induce an amorphous tissue, and then, pigment cells are added to the amorphous tissue. A method for inducing pigment cells of a Curcuma genus plant, comprising inducing. 4. The method according to claim 2, wherein the amorphous tissue is cultured in a medium containing no auxins to induce pigment cells. 5. The induction method according to claim 1, wherein the Curcuma plant is turmeric. 6. A method for producing a pigment of a Curcuma plant, wherein the pigment is collected from a pigment cell induced by the method according to any one of claims 1 to 5.