JP3125007B2 - Production method of red pigment by tissue culture of rosemary - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a rosemary (Rosmar
inus officinalis L.), and a method for producing a red pigment, particularly an anthocyanin pigment used for coloring foods and cosmetics, by tissue culture. In the present specification, "callus" refers to amorphous cells obtained by culturing a piece of tissue cut from a plant in a solid medium for tissue culture, and "anthocyanin" refers to a flower or pericarp of a plant or the like. Glycosides that show red, blue, purple, etc., which are often present in parts having beautiful colors.

[0002]

2. Description of the Related Art For coloring foods and cosmetics, various synthetic pigments and natural pigments have been used. However, synthetic pigments have problems such as mutagenicity, carcinogenicity, etc. It is desired to use dyes derived from natural products. However, the production of pigments by extraction from natural products is susceptible to the natural environment such as season, climate, and temperature, and it is difficult to provide a stable supply. Therefore, production of natural pigments by tissue culture has been attempted. The tissue culture method has an advantage that a target dye can be produced in a short time, and a planned production can be efficiently performed on an industrial scale.

As methods for producing anthocyanin-based pigments by tissue culture of plants (callus), callus of Akmegashiwa (JP-A-49-94897) and callus of eye plant belonging to Polygonum (JP-A-53-840) have been used.
26), Callus derived from the leaves of Delis
11281), a callus of a grape genus plant (JP-A-55-1)
18392), Euphorpia hanakirin (Japanese Unexamined Patent Publication No.
7-2696), Roselle callus (JP-A-64-30)
594) are known. Further, in tissue culture of perilla plants, tissue culture under blue light irradiation at a wavelength of 190 to 500 nm, or tissue culture under red light irradiation at a wavelength of 600 to 900 nm, after culturing red pigment-producing callus, respectively. A method for producing a red dye from a culture has been proposed (JP-A-61-195688, JP-A-63-195688).
156865). The above-mentioned perilla plant is a so-called reddish violet usually containing red anthocyanin pigments in its leaves.

[0004]

SUMMARY OF THE INVENTION The present inventor can produce a significant amount of anthocyanin pigments by cultivating callus derived from evergreen rosemary plant tissue belonging to the Labiatae family, which is different from magenta red ginseng. The inventors have found that the present invention has been made based on this finding. An object of the present invention is to provide a method for economically producing an anthocyanin-based dye in high yield by tissue culture of rosemary.

[0005]

Means for Solving the Problems In order to achieve the above object, a method for producing a red pigment according to the present invention comprises a method for producing reddish purple from callus derived from a plant tissue of a rosemary plant of the Labiatae family.
Callus, a rosemary pigment-producing cell mass,
This is a method of culturing the selected calli in a medium containing auxin or cytokinin.

Hereinafter, the present invention will be described in detail. Examples of the plant material used in the present invention include leaves, stems, flowers, roots, seeds, and other tissues of rosemary of a Labiatae plant. First, such a rosemary plant is sterilized to a sterile state. Sterilization can be accomplished, for example, by thoroughly washing the plant material with deionized water, then immersing it in 70% ethyl alcohol for several tens of seconds,
It is carried out by immersing for 10 minutes to sterilize various germs attached to the surface, and washing and removing the residual germicide with sterilized water.
Next, the sterilized plant material is cut into small pieces of an appropriate size using a sterile scalpel, and the cut pieces are placed on a medium containing auxin or cytokinin. After placing, a light place under a constant temperature condition of 20 to 30 ° C, preferably 24 to 26 ° C, preferably 100 lux or more, more preferably 3,000 to
Culture is performed under irradiation of 100,000 lux of daylight light. As the light source, sunlight, a fluorescent lamp, an incandescent lamp, a mercury lamp, or the like can be used. Without light irradiation, no dye is produced. The cultivation period is about 2 to 4 weeks.

[0007] As a medium for inducing callus of the plant and a medium for growing the same, an ordinary inorganic synthetic agar medium for tissue culture is used. For example, Murashige-Skoog medium, Gamborg-
A B5 (Gamborg-B5) medium, a White medium, a Heller medium, a modified medium thereof, and the like are used. In such a medium, a carbon source, a nitrogen source, inorganic salts,
It may contain organic substances, plant hormones and the like. As the carbon source, sugars such as sucrose, glucose, maltose and fructose, starch and the like are used. As the nitrogen source, nitrates, ammonium salts, glycine, urea, and the like are used. As the inorganic salts, those containing elements such as phosphorus, potassium, calcium, magnesium, manganese, copper, zinc, molybdenum, boron, iron, and cobalt are used. As organic substances, inositol, nicotinic acid, pyridoxine hydrochloride, thiamine hydrochloride, calcium pantothenate, p-aminobenzoic acid, folic acid, biotin, riboflavin, ascorbic acid, citric acid, malic acid, coconut milk, yeast extract and the like are used.

[0008] As plant hormones, 2,4-dichlorophenoxyacetic acid (hereinafter abbreviated as 2,4-D), α-
Auxins such as naphthalene acetic acid (hereinafter abbreviated as NAA), indole acetic acid (hereinafter abbreviated as IAA), indole butyric acid (hereinafter abbreviated as IBA), N
Cytokinins such as ⁶ -furfurylaminopurine (hereinafter abbreviated as kinetin) and N ⁶ -benzyladenine (hereinafter abbreviated as BA) can be used. From the viewpoint of callus growth and pigment productivity, auxin is preferably 2,4-D and cytokinin is preferably kinetin. The concentration of auxin used is 10 ^-7 to 10 ^-4 M, preferably 10 ^{-5 to} 5 × 10 ^-5.
M, and the concentration of cytokinin used is 10 ^-7 to 10 ^-5.
M, preferably 10 ^{−6 to} 5 × 10 ⁻⁶ M. If this concentration is too high, callus will not grow,
Conversely, if it is too low, it is not preferable because it is greened or does not grow.

[0009] After a culture period of about 2 to 4 weeks, calli are induced from each of the cut pieces, and callus, which is a red-purple dark rosemary pigment-producing cell mass, is selected from the calli cultured for induction. This is transplanted onto a fresh agar medium having an appropriate composition, and the culture is continued at a constant temperature of 20 to 30 ° C, preferably 24 to 26 ° C. In addition, culture in a liquid medium can be performed. When the callus is subcultured and propagated, the yield of red pigment can be improved. After completion of the growth culture, 0.1 to 1.
After the pigment is extracted with 0% by weight hydrochloric acid-methanol, the solvent is removed by filtration or the like to obtain a red pigment.

[0010]

As described above, according to the present invention, callus derived from the plant tissue of the Labiatae plant rosemary
Mosquito, which is a deep red-purple rosemary pigment-producing cell mass from
By culturing the selected calli in a specific medium, it is possible to produce an anthocyanin-based pigment, which is a natural red pigment suitable as a coloring agent for foods and cosmetics, in a pure form. It can be produced economically with a very high yield as compared with the case of production from plants.

[0011]

Embodiments of the present invention will be described below. The present invention is not limited to the following examples. First, wash the rosemary leaves with running water, and add 3% to 70% ethyl alcohol.
After immersion for 0 second, sterilization was performed by immersion for 15 minutes in an aqueous solution of sodium hypochlorite (effective chlorine concentration: 1.5%), and then washed three times with sterilized water. The sterilized leaves thus sterilized were cut into small pieces having an area of about 1 cm ² using a sterile scalpel. The cut pieces were 2 ppm of 2,4-D, 0.2 ppm of kinetin, 3% of sucrose, and 0.8% of agar.
% Was placed on a Murashige-Skoog medium adjusted to pH 5.8. 25 ° C after placing
At 3,000 lux under a white light irradiation with a fluorescent lamp.

Three weeks after the implantation, callus tissue was induced from each of the cut pieces, and a reddish purple portion (rosemary pigment-forming cell mass) was confirmed in the tissue. Of the rosemary pigment producing cell mass, select a particularly dark portion,
Transplanted into a solid medium of pH 5.8 having the composition shown in Table 1,
C. for 3 weeks under white light irradiation with a fluorescent lamp at 3,000 lux at 3,000.degree. By this culture, calli were obtained, which were entirely colored purple-red. The callus was immersed in 1% hydrochloric acid-methanol at 5 ° C. for 24 hours to extract a red pigment, and a red pigment solution was obtained by suction filtration. This extract had a maximum absorption at 530 nm in the visible region. (Hereafter, this page margin)

[0013]

[Table 1]

──────────────────────────────────────────────────続 き Continued on the front page (58) Fields surveyed (Int. Cl. ⁷ , DB name) C12P 19/00-19/64 C12N 1/00-5/28 BIOSIS (DIALOG) JICST file (JOIS) MEDLINE ( STN) WPI (DIALOG)

Claims (5)

(57) [Claims] 1. A deep purple-red rosemary pigment from callus derived from a plant tissue of a rosemary plant of the Labiatae family
A method for producing a red pigment by tissue culture of rosemary in which callus, which is an adult cell mass, is selected and the selected callus is cultured in a medium containing auxin or cytokinin. 2. An auxin in a medium of 10 ^-7 to 10 ^-4 M.
2. The method for producing a red pigment according to claim 1, wherein the red pigment is contained at a concentration of: 3. The method for producing a red pigment according to claim 2, wherein the auxin is 2,4-dichlorophenoxyacetic acid. 4. The method according to claim 1, wherein the cytokinin is contained in the medium at 10 ^-7 to 10.
^The method for producing a red pigment according to claim 1, which is contained at a concentration of ^-5M . 5. The method according to claim 4, wherein the cytokinin is kinetin.