JPH07184679A - Production of anthocyanin-based pigment and medium to be used for the production - Google Patents

Abstract

PURPOSE:To obtain the subject pigment such as cyanidin-3-glucoside having been difficult to produce purely in large quantities and at low cost by culturing pigment-producible rose cells in a medium to produce the anthocyanin-based pigment in the cultured product. CONSTITUTION:The stems and leaves of rose of Cardinal species are trimmed, and their surfaces are sterilized and then washed with water to produce cells to be induced to the callus, and the cells are then cultured in a medium containing indole-3-acetate and gelatin, etc., to induce callus. The callus is then put to subculture at 25 deg.C under 3000lux light irradiation, and only the deep red portion of the callus is selected as anthocyane-based pigment-producible cell, which is then cultured in a modified Murashige-Skoog medium containing auxin and cytokining as phytohormones to produce an anthocyanin-based pigment such as cyanidin-3-glucoside in the cultured product, and this pigment is collected, thus obtaining the objecting pigment such as cyanidin-3-glucoside having been difficult to produce purely in large quantities and at low cost.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method for producing anthocyanin pigments, particularly cyanidin-3-glucoside, using rose pigment-producing cells, and a medium used in the method.

[0002]

2. Description of the Related Art Secondary metabolites of higher plants have been deeply involved in our lives as medicines, luxury items, dyes, etc. since ancient times. However, it is generally difficult to obtain such secondary metabolites from plants in a large amount regardless of the season.

Due to the recent progress in plant cell culture technology,
Large-scale production of useful secondary metabolites using tissue culture has been attempted. And, for example, shikonin, berberine,
Japanese Akane extract etc. are already in the stage of commercial production. By the way, the pigment anthocyanin is a flavonoid pigment and the red color of the petals, fruits, roots, etc. of plants except Dianthus.
It is known as a blue or purple pigment and has been used as a coloring agent in various foods since ancient times and has a high practical value.

Nevertheless, at present, the production of the anthocyanin pigment is based on a so-called extraction method of extracting the anthocyanin pigment from a plant that originally contains anthocyanins. Furthermore, since microorganisms usually do not have the ability to produce anthocyanin pigments,
The anthocyanin dye is suitable as a research target in plant cell culture.

[0005]

Therefore, the problem to be solved by the present invention is to provide a means for producing the above-mentioned anthocyanin dye, particularly cyanidin-3-glucoside, which has been difficult to obtain as a pure product. is there.

[0006]

Means for Solving the Problems The present inventor has conducted extensive studies to solve the above problems. As a result, callus that produces anthocyanin pigment was obtained by inducing callus from rose-derived cells. Then, the inventors have found a means for producing the anthocyanin-based dye, particularly the cyanidin-3-glucoside, in large quantities and at low cost by using the callus, and completed the present invention.

That is, the present invention has the following matters. (1) A method for producing an anthocyanin pigment, which comprises culturing rose pigment-producing cells in a medium to produce an anthocyanin pigment in the culture. (2) The rose pigment-producing cells are rose pigment-producing cells selected from among them by culturing rose stalks or leaves in a medium to produce callus, and the anthocyanin pigment is cyanidin-3-glucoside. Characterized by the above
(1) The method for producing an anthocyanin dye as described above.

(3) The above (1) or (2), which comprises a modified medium of Murashige-Skoog medium characterized by the following component composition:
Described anthocyanin pigment production medium: Phosphoric acid 0.1 to 0.5 mM, saccharose 45 to 180 mM, and a weight mixing ratio of ammonia nitrogen and nitrate nitrogen is 1 :.
2 to 1: 6, and the total amount of both nitrogens is 40 to 80 mM.

(4) A medium for producing anthocyanin-based pigment, which comprises adding auxin and cytokinin as plant hormones to the medium for producing anthocyanin-based pigment described in (3) above. (5) The modified medium of Murashige-Skoog medium, wherein the magnesium content is 1.5 to 6.0 mM, and the medium for producing anthocyanin pigment according to (3) or (4) above.

(6) The anthocyanin system according to any one of (3) to (5) above, wherein the calcium content in the modified Murashige-Skoog medium is 1.0 to 6.0 mM. Medium for producing dye. Hereinafter, the present invention will be described in detail. The method for producing an anthocyanin dye of the present invention is characterized by culturing rose pigment-producing cells in a medium.

The variety of the rose plant which is a raw material for the rose pigment-producing cells is not particularly limited as long as it contains cells producing a desired anthocyanin pigment intended for production in the plant. That is, the variety can be appropriately selected according to the type of the desired anthocyanin dye. Specific examples include cardinal species and curl red species. In addition, the site of the rose plant that selects cells for tissue culture can be appropriately changed depending on the kind of the desired anthocyanin dye.

[0012] For example, when the production of cyanidin-3-glucoside, which is an anthocyanin dye, is intended, cells of leaf or leaf of cardinal species can be selected as callus-inducing cells. Hereinafter, a method for producing cyanidin-3-glucoside, which is an anthocyanin-based pigment, using a cardinal species rose plant, which is a typical embodiment of the method for producing an anthocyanin-based pigment of the present invention, will be described.

Cyanidine which is an anthocyanin dye
As cells for producing 3-glucoside (hereinafter, referred to as pigment-producing cells), it is effective to select cells of rose plant leaves or leaves as described above, in order to efficiently produce cyanidin-3-.
It is preferable in that glucoside can be produced. The rose variety is also cyanidin-3.
-It is preferable to use rose of cardinal species in that it is possible to extremely efficiently produce cyanidin-3-glucoside by the production method of the present invention.

As a method for securing the pigment-producing cells, a generally known method can be adopted. That is, for example, a method of trimming the plow or leaf, sterilizing the surface with, for example, a surfactant, and then washing with distilled water can be used. The tissue piece thus prepared is used as a pigment-producing cell in the following callus induction step.

Induction of callus can also be performed by using a generally known method. That is, for example, the above-mentioned tissue pieces can be transferred to an induction medium containing agar or a liquid suitable growth regulator (plant hormone) to induce desired callus. The medium that is the base of the callus induction medium is, for example, Nitch medium or Murashig-Skoog.
e-Skoog) medium (hereinafter referred to as MS medium) and the like. Examples of growth regulators include indole-3-acetic acid (IAA) and α-naphthalene acetic acid (NA).
A), auxins such as 2,4-dichlorophenoxyacetic acid (2,4-D) and indole-3-butyric acid (IBA); zeatin, kinetin, 6-benzylaminopurine (BA), 6- (3-
Mention may be made of cytokinins such as methyl-2-butenylamino) -purine (2iP). The amount of auxin added is 10 ^-8 to 10 ^-5 M, and the amount of cytokinin added is 10 ^-7 to 10 ^-5 M.

The illuminance in callus induction is 1500 to 30.
It is about 00lux, the temperature is about 25 to 30 ° C., and the number of passages can be from once a month for about 3 years to twice a month for about 2 years. Then, the desired cyanidin-3-glucoside is produced from the thus-produced cells of cyanidin-3-glucoside in callus.

Such production can be carried out by culturing the above-mentioned callus in an anthocyanin dye-producing medium. At this time, selecting cyanidin-3-glucoside-producing cells from the callus and culturing the pigment-producing cells in the anthocyanin-based pigment-producing medium can efficiently produce a desired cyanidin-3-glucoside. It is preferable in that it is possible.

The selection method is not particularly limited, and for example, it is possible to visually select and select only a very dark red portion from the callus. MS having a generally known composition as the above-mentioned medium used in this production process
It is possible to use a medium (liquid medium). Further, it is possible to more efficiently produce cyanidin-3-glucoside by using a modified medium in which the constituent components and constituent ratio of the MS medium are modified.

The basic composition of this MS medium is described below.

[0020]

[Table 1]

Further, by adjusting the phosphate, saccharose, ammonia nitrogen and nitrate nitrogen, and magnesium and calcium contents of the above MS medium, and further adding auxin and cytokinin as growth regulators, anthocyanin pigments can be obtained. It becomes possible to more efficiently produce cyanidin-3-glucoside.

Specifically, the phosphoric acid content in the MS medium is 0.1 to 0.5 mM, saccharose is 45 to 180 mM, and the weight ratio of ammonia nitrogen and nitrate nitrogen is 1: 2.
It is preferable to adjust the total amount of both nitrogens to be 1: 6 and to be 40 to 80 mM in consideration of the above-mentioned object that it is possible to more efficiently produce cyanidin-3-glucoside. Furthermore, it is preferable to adjust the magnesium content in the modified medium to 1.5 to 6.0 mM; and the calcium content to 1.0 to 6.0 mM for the same reason as the magnesium content adjustment.

By adding auxin and cytokinin as plant hormones to the modified medium and culturing the callus in the modified medium containing the hormone, cyanidin-3-glucoside can be produced more efficiently. . Here, auxins include α-naphthalene acetic acid (NAA) and 2,4-dichlorophenoxyacetic acid (2,4-D).
, Indole-3-butyric acid (IBA) and the like. Moreover, as cytokinins, zeatin, kinetin, 6-benzylaminopurine (BA), 6- (3-
Methyl-2-butenylamino) -purine (2iP) and the like can be mentioned.

The combination of the above various auxins and cytokinins to be added to the production medium must be a combination that promotes the production of anthocyanins as much as possible. For example, with respect to the anthocyanin content, it is not preferable to add NAA and kinetin in combination with 2,4-D and zeatin or kinetin, since this will have an adverse effect. However, 2,4-D and BA or 2iP, I
It is preferable to add BA and zeatin in combination because the anthocyanin content increases. The above auxin and cytokinin are added up to 10 ⁻⁶ M. Further, the preferable range of the addition is 10 ⁻⁸ to 10 ^−7.
M 3 and particularly preferably 10 ⁻⁷ M.

Among the above combinations of auxin and cytokinin, IBA (10 ^-7 M) and zeatin (10 ^-7 M) were used.
The combination of M) is particularly preferred. Further, regarding the total amount of anthocyanins, when NAA or 2,4-D is used as an auxin, it is not preferable to add it in combination with zeatin or kinetin as a cytokinin, since this has an adverse effect. However, it is preferable to add BA or 2iP as a cytokinin in combination with the auxin because the total amount of anthocyanins increases. Further, when IBA is selected as the auxin, it is not preferable to add kinetin or BA in combination as the cytokinin because the total amount of anthocyanins is adversely affected. However, it is preferable to add zeatin or 2iP as a cytokinin in combination to the auxin because it is possible to increase the total amount of anthocyanins. In addition,
The addition of the above auxin and cytokinin is performed with an upper limit of 10 ⁻⁶ M. Further, the preferable range of the addition is 10 ⁻⁸ to 10 ⁻⁷ M, and particularly preferably 10 ⁻⁷ M.

Particularly, a combination of 2,4-D (10 ^-7 M) and BA (10 ^-7 M) is preferable. Furthermore, by adjusting the magnesium concentration or calcium concentration of the MS-modified medium, it becomes possible to more efficiently produce cyanidin-3-glucoside, which is an anthocyanin dye. Specifically, in the modified medium of MS medium, the content of magnesium is adjusted to 1.5 to 6.0 mM; the content of calcium is adjusted to about 1.0 to 6.0 mM, particularly about 2 mM.
-Preferred because it increases the production of glucosides.

The callus culture in the liquid medium is
Generally, the culture temperature is about 25 ° C, the culture time is about 7 days, the illuminance is about 3000 lux, and the air permeability is about 80 rpm rotation culture in a 50 ml liquid medium / 200 ml Erlenmeyer flask, but the conditions are not limited to these. The culture can be performed by appropriately changing the culture conditions. Thus, the cyanidin-3-glucoside produced in the liquid medium can be purified using a generally known method.

That is, the cultured cells are disrupted, the pigment component is extracted therefrom, and the desired cyanidin-3 is extracted from the extract.
-Glucosides are separated and purified. Examples of the crushing means include means for crushing the cultured cells while adding liquid nitrogen with a mortar or pestle. Examples of the method for extracting the dye component include a commonly known extraction method using an extraction solvent. Examples of the separation / purification means for the dye component include a separation / purification means using adsorption column chromatography.

[0029]

EXAMPLES The present invention will be specifically described with reference to the following examples, but the technical scope of the present invention is not limited to the examples. [Example 1] Production of anthocyanin dye Preparation of Anthocyanin Pigment-Producing Cells (1) Preparation of Cardinal Anthocyanin-Producing Callus A rose and a leaf of a cardinal species were trimmed (a stem having a length of 1 cm was vertically divided, and a leaf was trimmed to a 1 cm square). , Surface with 10% ethanol for 1 minute, then 1
The cells were sterilized with 10% antiformin for 10 minutes and then washed three times with distilled water for 3 minutes to give callus-induced cells.

Callus induction includes 3% glucose, 1% agar, 10 ^-7 M indole-3-acetic acid (IAA), 10 ^-4 M zeatin or 10 ^-6 M IAA, 10 ^-5 M zeatin.
/ 2 Nitsch medium planted cardinal flower stalk, 25
It was performed at 3000C under irradiation with 3000 lux. After subculturing the obtained callus every month for 26 months under the above conditions, 3%
Transfer to an MS medium containing sucrose, 1% agar, 10 ^-8 M α-naphthalene acetic acid (NAA) and 10 ^-6 M zeatin, and
Subculture was carried out every month for 11 months under irradiation of 3000 lux at ℃.

(2) Cardinal anthocyanin-producing cells 3 g of cardinal anthocyanin-producing callus at the second week of culture was transferred to a 200 ml Erlenmeyer flask containing 50 ml of MS medium containing 3% saccharose, 10 ^-8 M NAA and 10 ^-6 M zeatin. Shaking culture was carried out at 80 rpm under irradiation of 3000 lux at 25 ° C. The liquid-cultured cells thus obtained were used for Nalgene sterilization filter unit type S (pore size: 0.2 μm)
After suction filtration using 3g, subculture was carried out every 3 weeks under the same conditions as above. In this example,
Liquid culture third generation cells were used.

(3) Selective culture of cell aggregates of anthocyanin pigment-producing callus As anthocyanin pigment-producing cells, only the dark red part of the callus was selected and subcultured under the same conditions as in (2) above. It was As shown in FIG. 1, cell cluster selection was performed 36 times in a year and a half. The amount of anthocyanins in 2 callus was measured for each passage, and changes in the amount of anthocyanins due to selection of cell aggregates were examined. As a result, the anthocyanin content of callus increased about 9 times as much as the initial content. From this result, it was found that the passage after the selection of the cell aggregates was extremely effective in the production of the anthocyanin dye. B. Analysis of anthocyanin pigments (1) Identification of anthocyanins Cardinal callus, liquid cultured cells, leaves and petals,
Crushed under liquid nitrogen using a mortar and pestle until powdered. Thereafter, 1% HCl / MeOH was added and dissolved, and the solution was centrifuged at 39800 xg for 10 minutes to collect the supernatant. The resulting supernatant is MEMBRANE FILTER PITE. (Pore size:
0.2 μm), concentrated to dryness, and 1% HCl / Me
It was dissolved in a OH: 4.5% formic acid = 1: 1 (v / v) solution. The lysate was analyzed under the liquid chromagraphy conditions shown below, and anthocyanin was identified from its retention time.

(1) Column: μBondapak C ₁₈ (3.9φ × 300m
m) (2) Pump: Shimadzu LC-6A (3) Detector: Shimadzu SPD-6AV, 525nm (4) Mobile phase: A 4.5% Formic acid B Acetonitrile (5) Flow rate: 1.5 ml / min (6) Gradient conditions : Shown in Table 2

[0034]

[Table 2] Table 2 ────────────────────── Time (minutes) B concentration (%) ────────────── ───────── 0 10 10 15 18 50 50 22 10 40 End ─────────────────────── Cardinal callus, liquid culture cells, leaves and As a result of identifying the anthocyanins contained in the petals, as shown in FIG. 2, the anthocyanins contained in the callus and the liquid cultured cells were cyanidin-3-glucoside (CYA3G), and the main anthocyanins contained in the petals and the leaves were , Cyanidin-3,5-diglucoside (CYA3,5G).

From these results, the cardinal callus and cultured cells in this Example are bright red pigments,
It was revealed to contain only cyanidin-3-glucoside. This means that in high purity, cyanidin-3-
This leads to the advantage that glucoside can be easily obtained. (2) Measurement of amount of anthocyanin Callus or 1 g of liquid cultured cells after suction filtration was crushed under liquid nitrogen using a mortar and pestle until it became powdery. Thereafter, 9 ml of 1% HCl / MeOH was added and dissolved, and the solution was centrifuged at 24000 xg for 3 minutes to collect the supernatant to obtain an extract. The absorbance at 525 nm of the obtained extract was measured using a Shimadzu spectrophotometer UV-265FW, and the amount of anthocyanin was calculated from the calibration curve of the anthocyanin standard (cyanidine-3-glucoside). [Example 2] Measurement of medium component amount during liquid culture A. Effect of Phosphoric Acid Content in the Medium (1) Test System The MS medium contains 1 mM of dipotassium hydrogen phosphate. So, first of all, 6 kinds of phosphoric acid concentration (0 mM, 0.13 mM, 0.2
200 ml containing 50 ml of 5 mM, 0.5 mM, 1 mM, 2 mM) MS medium
10 Erlenmeyer flasks were prepared. Then, 3 g of cultured cells derived from the same callus were transferred to each and cultured under the same culture conditions as described above. 2 per medium per culture days
Measure the raw cell weight of the flask and the amount of anthocyanins,
The change with time was examined. Then, the culture was carried out in a medium having 6 kinds of phosphoric acid concentrations, and the cell fresh weight and the amount of anthocyanins on the 7th day of the culture were measured in 2 flasks per medium. This was repeated for 4 cultured cells from callus with different passage numbers.

(2) Effect of Phosphate on Anthocyanin Production First, in order to examine what kind of change anthocyanin production shows in each medium having different phosphate concentrations, the cells of A time course of anthocyanin content was taken (Fig. 3). The lower the phosphate concentration, the higher the anthocyanin content of the cells,
On the 7th day of culturing, it was observed that the medium showed the highest value in any medium having any phosphoric acid concentration and tended to settle down. Therefore, it was decided to examine the effect of phosphoric acid on cell growth and anthocyanin production based on the results on the 7th day of culture.

Table 3 shows the cell growth rate using the raw cell weight in media having different phosphate concentrations as an index, and Table 4 shows the cell growth rate of the anthocyanin content (hereinafter referred to as the anthocyanin content rate). Table 5 shows the rate of increase in the amount of anthocyanins in the culture flask (hereinafter referred to as the amount of total anthocyanins) in that case.

[0038]

[Table 3]

[0039]

[Table 4]

[0040]

[Table 5]

The amount of cells increased as the amount of phosphoric acid in the medium increased, while the anthocyanin content and the total amount of total anthocyanins tended to decrease as the concentration of phosphoric acid increased. As a result of the experiment, the highest anthocyanin content was that the phosphoric acid concentration was 0.13 mM.
When the cells were cultivated in the above medium, it was improved to about 3.5 times that at the start of culturing. Further, the total amount of total anthocyanins was highest when the culture was carried out in a medium having a phosphate concentration of 0.25 mM, which was about 7 times that at the start of the culture. However, this value was not significantly different from that when cultured in a medium with a phosphate concentration of 0.13 mM.

From the above results, it is preferable to use a material having a high anthocyanin content as a material for mass production of anthocyanins in the future. Therefore, the phosphate concentration of the medium suitable for anthocyanin production is 0.1 to 0.5 mM, preferably
It was concluded that it was 0.13 mM. B. Effect of sugar amount in medium (1) Test system MS medium contains 30 g / l of saccharose. Therefore, 5 kinds of sucrose concentrations (0g / l, 15g / l, 30g / l, 6
0g / l, 90g / l), 3g of cultured cells derived from the same callus were cultivated in MS medium having a phosphoric acid concentration of 0.13mM, and the cell fresh weight and anthocyanin amount on the 7th day of culture were measured in 2 flasks per medium. . This was repeated for 4 cultured cells from callus with different passage numbers.

(2) Effect of sugar on anthocyanin production Table 6 shows the cell increase rate in the medium having different saccharose concentrations on the 7th day of culture, Table 7 shows the increase rate of the anthocyanin content in that case, and Table 8 shows the case. The rate of increase in the amount of total anthocyanins was shown.

[0044]

[Table 6]

[0045]

[Table 7]

[0046]

[Table 8]

When cultured in a medium having a saccharose concentration of 0 g / l, the cell amount did not change at all and the anthocyanin content was a very low value. There was no significant difference in cell amount between when the cells were cultured in a sucrose concentration of 15 g / l and when they were cultured in a medium of 30 g / l, but the anthocyanin content was when cultured in a medium of 30 g / l. To 1
The value was about 2.5 times that when cultured in a 5 g / l medium.
Then, when cultivated in a medium of 30 g / l, anthocyanin content rate, showing the highest total anthocyanin amount,
The anthocyanin content was improved to about 3.5 times that at the start of culture, and the total amount of anthocyanins was improved to about 7 times that at the start of culture.

Therefore, the saccharose concentration suitable for anthocyanin production in MS medium having a phosphate concentration of 0.13 mM is 20 to 60 g / l, preferably 30 g / l. C. Effect of nitrogen content in medium (1) Test system MS medium is 20 mM potassium nitrate, 20 mM ammonium nitrate
And the concentrations of ammonium and nitric acid are
It is 20mM and 40mM. Therefore, four types of ammonium concentration (0 mM, 10 mM, 20 mM, 40 mM) and four types of nitric acid concentration (20 mM
mM, 40mM, 60mM, 15 kinds of combinations of 80mM) (NH ₄ 20m
(Excluding 40 mM of NO: 40 mM), ₃ g of the same callus-derived cultured cells were cultured in MS medium having a phosphoric acid concentration of 0.13 mM and a saccharose concentration of 30 g / l. Two flasks were measured for each medium. This was repeated for 4 cultured cells obtained from callus having different passage numbers.

(2) Effect of Nitrogen on Anthocyanin Production Table 9 shows the cell increase rate in the medium having different ammonium and nitrate concentrations on the 7th day of culture, Table 10 shows the increase rate of the anthocyanin content in that case, and Table 11 Shows the rate of increase in the amount of total anthocyanins in that case.

[0050]

[Table 9]

[0051]

[Table 10]

[0052]

[Table 11]

The cell amount increased remarkably up to about 3.5 times when cultured in a medium having an ammonium concentration of 0 mM, regardless of the nitrate concentration, but it tended to decrease as the ammonium concentration increased. On the other hand, the anthocyanin content was not significantly affected by the nitric acid concentration, and when cultured in a medium having an ammonium concentration of 10 mM or 20 mM, 0 mM,
The value was significantly higher than that when cultured at 40 mM. Looking at the amount of total anthocyanins, the nitric acid concentration was 40 mM.
When cultured in four kinds of mediums having ammonium concentrations of 10 mM and 20 mM, nitric acid concentration of 60 mM, and ammonium concentrations of 10 mM and 20 mM, all showed high values, about 7 times as high as those at the start of the culture. Among these four types of medium, the medium with the highest total anthocyanin content was the medium with a nitric acid concentration of 60 mM and an ammonium concentration of 10 mM. D. Effects of addition of plant hormones to the medium (1) Test system 3 g of cultured cells derived from the same callus of the third subculture, 3 types of auxin and 4 types of cytokinins at various concentrations (shown in Table 12 below). MS-modified medium containing 0.15 mM phosphate, 30 g / l saccharose, NH ₄ NO ₃ 10 mM, KNO ₃ 50 mM 5
Transfer to a 200ml Erlenmeyer flask containing 0ml, 3000C at 25 ℃
Shaking culture was performed at 80 rpm under ux irradiation. After culturing for 7 days under the above conditions, the liquid cultured cells were suction-filtered, and the content of the anthocyanin dye was measured.

[0054]

[Table 12]

(2) Effect of addition of plant hormone on anthocyanin production Tables 13 to 16 show the results of the rate of increase in the anthocyanin content on the 7th day of culture, and Tables 17 to 17 show the results of the rate of increase in the amount of total anthocyanins. Shown in 22.

[0056]

[Table 13]

[0057]

[Table 14]

[0058]

[Table 15]

[0059]

[Table 16]

[0060]

[Table 17]

[0061]

[Table 18]

[0062]

[Table 19]

[0063]

[Table 20]

[0064]

[Table 21]

[0065]

[Table 22]

From these results, it was found that, with respect to the anthocyanin content, when 2,4-D was used as the auxin, good results were obtained when BA or 2iP was used in combination as the cytokinin. It was also found that when IBA was used as the auxin, good results were obtained when zeatin or 2iP was used in combination as the cytokinin.

Regarding the total amount of anthocyanins, it was found that when NAA or 2,4-D was used as the auxin, good results were obtained when BA or 2iP was used in combination as the cytokinin. It was also found that when IBA was used as the auxin, good results were obtained when zeatin or 2iP was combined as the cytokinin. E. Effect of amount of calcium in the medium (1) Test system The MS medium used calcium as CaCl ₂ · 2H ₂ O 4
In this test, 5 kinds of calcium concentration (0m
M, 1 mM, 2 mM, 4 mM, 8 mM) MS modified medium (phosphate O.13m
M, saccharose 30 g / l, NH ₄ NO ₃ 10 mM, KNO ₃ 50 mM) were cultivated in 3 g of cultured cells derived from the same callus, and the cell fresh weight and the amount of anthocyanins on the 7th day of culture were measured in 2 flasks per medium. This was repeated for 3 cultured cells obtained from callus with different passage numbers.

(2) Effect of Calcium on Production of Anthocyanin Dye The anthocyanin content was low when calcium was not contained at all. However, at 1 mM or higher, the values were almost the same. As a result, the total amount of anthocyanins was strongly influenced by the cell weight (the maximum when the calcium concentration was 2 mM), and was the highest in the medium having a calcium concentration of 2 mM (see Tables 23 to 25).

[0069]

[Table 23]

[0070]

[Table 24]

[0071]

[Table 25]

G. Magnesium content of the impact of the medium (1) Test system MS medium, magnesium as MgSO ₄ · 7H ₂ O
Although it contains 1.5 mM, in this test, 5 types of magnesium concentration (0 mM, 0.75 mM, 1.5 mM, 3 mM, 6 mM) MS modified medium (phosphate 0.13 mM, sucrose 30 g / l, NH ₄ NO ₃ 10 mM, KNO ₃ (50mM)
Cultivate 3 g of cultured cells derived from the same callus in
Cell weight and anthocyanin content on day 2 should be 2 per medium
The flask was measured. This was repeated for 3 cultured cells obtained from callus with different passage numbers.

(2) Effect of Magnesium on Production of Anthocyanin Dye As shown in Table 27, the anthocyanin content was as high as 1.5 to 6.0 mM for magnesium, and was highest at 3 mM. Similarly, the amount of total anthocyanins was as high as 1.5 to 6.0 mM for magnesium, and was maximum at 3 mM (see FIG. 28). The cell increase rate is shown in Table 2.
6 shows.

[0074]

[Table 26]

[0075]

[Table 27]

[0076]

[Table 28]

[Brief description of drawings]

FIG. 1 shows the results of selective culture of cell aggregates of anthocyanin pigment-producing callus.

FIG. 2 shows the types of anthocyanins contained in cardinal callus, liquid cultured cells, leaves and petals.

FIG. 3 is a diagram showing changes in the production of anthocyanin pigments when cultured in media having different phosphoric acid concentrations.

Claims (6)

[Claims] 1. Culturing rose pigment-producing cells in a medium,
A method for producing an anthocyanin dye, which comprises producing an anthocyanin dye in a culture. 2. The rose pigment-producing cell is a rose pigment-producing cell selected from among which rose callus or leaves are cultured in a medium to produce callus, and the anthocyanin pigment is cyanidin-3-glucoside. The method for producing an anthocyanin-based dye according to claim 1, wherein 3. An anthocyanin pigment production medium according to claim 1 or 2, comprising a modified Murashige-Skoog medium characterized by the following composition: 0.1-0.5 mM phosphoric acid, 45-180 mM saccharose. , The mixing ratio by weight of ammonia nitrogen and nitrate nitrogen is 1:
2 to 1: 6, and the total amount of both nitrogens is 40 to 80 mM. 4. A medium for producing anthocyanin-based pigment, which comprises adding auxin and cytokinin as plant hormones to the medium for producing anthocyanin-based pigment according to claim 3. 5. The medium for producing anthocyanin dye according to claim 3, wherein the modified medium of Murashige-Skoog medium has a magnesium content of 1.5 to 6.0 mM. 6. A modified medium of Murashige-Skoog medium, wherein the calcium content is 1.0 to 6.0 mM, and the anthocyanin pigment of any one of claims 3 to 5 is characterized. Manufacturing medium.