JP2001000062A - Method for preparing culture cells having high pigment- synthetic ability from sweet potato bearing anthocyanin pigment - Google Patents

Abstract

PROBLEM TO BE SOLVED: To prepare the cultured cells with a high content of anthocyanin pigment by repeating the selection operation of cell strain that contains anthocyanin pigment in high concentration from sweet potatoes including anthocyanin pigment through a specific method. SOLUTION: Callus is induced from the tuberose roots of sweet potato including anthocyanin pigment as the cultured cells of higher concentration of anthocyanin are prepared from tuberose roots of sweet potatoes incluing anthocyanin pigment from tuberose roots by culturing. Then, a tissue having the formative ability of adventitious embryo is differentiated from the callus to obtain a tissue that is differentiated into just-like adventitious embryos. Further, callus having adventitious embryo-forming ability is induced from the tissue differentiated into just-like adventitious embryos so that the cell strain that contains increased concentration of anthocyanin is selected repetitively. These operation steps are carried out in the dark.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

[0001] The present invention relates to a method for obtaining an anthocyanin dye as a dye material such as an edible dye, and a method for obtaining a research material for biosynthesis of an anthocyanin dye in plants.

[0002]

2. Description of the Related Art Conventionally, various kinds of synthetic dyes and natural dyes have been used for coloring foods, cosmetics, etc., but synthetic dyes have problems such as mutagenicity and carcinogenicity, and are not safe. Therefore, the use of pigments derived from natural products is desired.

[0003] 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 supply them stably. Therefore, production of natural pigments by tissue culture has been attempted. Cultured cells obtained by tissue culture have the advantage that the target dye can be efficiently planned for production in a short time.

[0004] Among natural pigments, red pigments, particularly anthocyanin pigments, have been relatively studied. For example, anthocyanin pigments are contained in various plants such as grape skin, purple cabbage, purple corn, hibiscus, perilla and berry. Conventionally, a general method for producing anthocyanins has been to use various plants containing anthocyanins as materials and obtain anthocyanins using an appropriate separation and purification method. However, in the method of extracting anthocyanins from cultivated plants, the cost of raw materials is increased, and it has not been possible to produce anthocyanins at low cost. In addition, when a cultivated plant is used as a raw material, there is a problem that the growth of the plant is slow, cultivation takes time and labor, and the production efficiency of anthocyanin is poor. Furthermore, the production of anthocyanins depends on the harvest time of the cultivated plant, and there is a problem that it is not possible to produce anthocyanins stably throughout the year.

[0005] Compared with the conventional method for producing anthocyanins from cultivated plants, the production efficiency of anthocyanins is relatively high. A method for culturing anthocyanin-producing cells in large quantities using the cultured cells thus obtained and extracting anthocyanins from the cultured cells has been studied. For example, for the production of anthocyanin-based pigment, as a cultured cell containing an anthocyanin pigment, strawberry, carrot, grape, rose, apple, jerusalem artichoke, buckwheat, starch, rosemary, etc., cultured cells are known. I have. However, conventional cultured cells containing an anthocyanin pigment have a problem that the anthocyanin content is low both as a pigment extraction material and as a research material for pigment biosynthesis.

[0006] Japanese Patent Application Laid-Open No. 63-233993 discloses that
There are reports on obtaining an anthocyanin dye using cultured cells obtained from sweet potato, but all of them require light irradiation to obtain and grow calli producing anthocyanin dyes.

[0007]

As described above, conventional cultured cells containing an anthocyanin dye have a problem that the dye content is low and a problem that light irradiation is required for their production, growth and maintenance. . In view of this problem, an object of the present invention is to provide a method for obtaining cultured cells containing an anthocyanin pigment at a high concentration from sweet potatoes containing an anthocyanin pigment.

[0008]

Means for Solving the Problems As a result of intensive studies, the present inventors have found a method for obtaining cultured cells containing an anthocyanin dye at a high concentration, which solves the above-mentioned problems by using sweet potato. Was.

That is, according to the present invention, a method for producing cultured cells having a high content of anthocyanin pigment by culturing from a tuberous root of a sweet potato containing an anthocyanin pigment comprises inducing callus from a tuber of a sweet potato containing an anthocyanin pigment to form adventitious embryo A cell line containing a high concentration of an anthocyanin dye by performing a step of differentiating a tissue having the ability to obtain a tissue so differentiated into an adventitious embryo, and further inducing a callus having an embryogenic ability from the tissue having an adventitious embryo formation And a step of repeatedly selecting the above, wherein the various steps are performed in a dark place.

In general, light irradiation is required to obtain and grow calli for producing conventional anthocyanin dyes. However, the method of the present invention requires only light in all the steps of the preparation process. In addition, a cell line containing a high concentration of an anthocyanin dye obtained by the method of the present invention can accumulate anthocyanins in the dark, and does not require cost for light irradiation and is economical. Although there have been many reports of callus having the ability to form adventitious embryos from sweet potatoes, most of them have been derived from young leaves. There has been no example of inducing a callus having ability.

The plant used in the method for producing a cultured cell of the present invention is a sweet potato containing an anthocyanin pigment. Among sweet potato varieties, Ayamurasaki is preferred. The sweet potato variety Ayamurasaki is a variety that exhibits a red color up to the inner part of the tuber root, and is particularly suitable for the method of the present invention because the anthocyanin content in the tuber root is high.

For example, it has been reported that an anthocyanin-accumulating cell line can be obtained from sweet potato cultivar Kintoki. Kintoki is a cultivar having a white part in the tuber root, and an anthocyanin-accumulating cell line from the red part of the perimeter of the tuber. The method differs from the method of the present invention in that cells are obtained and callus having no ability to form somatic embryos from tuberous roots is used. In addition, in this example, not only is the cell line obtained by the method of the present invention different from the cell line obtained by the method of the present invention in that light is required for its anthocyanin accumulation, but the amount of the pigment produced is different from the cell line obtained by the method of the present invention. Two orders of magnitude lower.

[0013] The method for preparing a cultured cell having a high anthocyanin pigment content according to the present invention comprises preparing a cultured cell having a high anthocyanin pigment content by culturing in the dark from a tuberous root of a sweet potato variety Ayamurasaki containing an anthocyanin pigment. Features.

According to this method, cultured sweet potato cells having a high anthocyanin pigment content can be prepared by culturing in a dark place. Whereas conventional cultured cells of anthocyanin synthesis require light for pigment biosynthesis, cultured cells obtained by this method are capable of biosynthesizing anthocyanins under dark conditions, which is advantageous in cost. Also,
This method differs from the conventional method of obtaining an anthocyanin-accumulating cell line from sweet potato in that sweet potato cultured cells having a high anthocyanin pigment content are prepared from tuberous roots of sweet potato variety Ayamurasaki.

The composition of the medium used in the method for producing a cultured cell having a high anthocyanin content according to the present invention is MS
It is preferable to add 3% sucrose as a carbon source to the basic medium and 2,4-dichlorophenoxyacetic acid (hereinafter, abbreviated as 2,4-D) as an auxin as a growth regulator. The MS basal medium is a well-known Murasige & Schoog medium, which is composed of vitamins as inorganic and organic components and does not contain a carbon source (generally sucrose).

The cultured cells containing the anthocyanin dye obtained by the method of the present invention can be obtained by adding the anthocyanin dye at a concentration of at least three times that of the root of the sweet potato variety "Ayamurasaki", which is the origin of the cell, under the culture conditions in which the MS medium is modified. It is possible to accumulate. This is at a much higher concentration than any of the previously known cultured cells that perform anthocyanin biosynthesis.

As described above, the method according to the present invention makes it possible to prepare a cultured cell containing a high concentration of an anthocyanin dye. Cultured cells containing an anthocyanin dye prepared by the method of the present invention have a sufficient dye concentration, efficiency and safety to be used as a dye extraction raw material. By using the cultured cells as a dye extraction raw material, a stable supply of the dye raw material for the whole year becomes possible. further,
These cells are also considered to be effective in elucidating the anthocyanin synthesis system.

[0018]

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS Preferred embodiments of the present invention for producing cultured cells having a high anthocyanin dye content will be described below with reference to the drawings. It should be noted that the present invention is not limited to the embodiment described here, and includes a configuration that can be modified and implemented without departing from the gist of the present invention.

FIG. 1 shows an outline of a procedure of a method for preparing a cultured cell containing an anthocyanin dye according to the present invention. The specific procedure will be described below.

(First stage: from the beginning until the adventitious embryo-like tissue is obtained) First, the tuberous root of the sweet potato containing the anthocyanin pigment is sterilized, peeled, and cut into a die having a side of 2 to 3 cm. Prepare tissue pieces. These pieces of tissue
Mu containing 0.5 to 1 mg / L of 2,4-D and 3% of sucrose
It is placed on a solid medium of rashige & Schoog (hereinafter abbreviated as MS) and cultured at 25 to 27 ° C in a dark place. After culturing for 5 to 10 weeks, in addition to the normal dedifferentiated callus,
Tissues having the ability to form somatic embryos are differentiated, and from these, tissues that have differentiated into somatic embryos (somatic embryo-like tissues) are obtained.

(Second Step: Until Callus of Mixed Colors is Obtained) The above adventitious embryo-like tissue is transferred to the same medium as above (MS solid containing 0.5 to 1 mg / L of 2,4-D and 3% of sucrose). Culture medium)
And transplanted under the same culture conditions (25-27 ° C, dark place).
Incubate for ~ 6 weeks to induce calli. Then, a soft and white callus having no adventitious embryo-forming ability and a hard purple callus having adventitious embryo-forming ability are formed.
A callus having adventitious embryo-forming ability is selected from these two types of calli, and a callus having adventitious embryo-forming ability is selected.
The cells are transplanted to an MS solid medium containing 1 to 3 mg / L of 2,4-D and 3% of sucrose, and cultured under the same culture conditions (25 to 27 ° C, dark place) for 3 to 6 weeks. Then, a mixed-color callus in which a white portion and a purple portion are mixed is formed.

(Third step: until a purple callus is obtained on a solid medium) The above-mentioned white part and purple part are separated,
Callus exhibiting purple color, 2,4-D 0.5-1 mg /
L, transplanted to an MS solid medium containing 3% sucrose, cultured under the same culture conditions (25 to 27 ° C, dark place) for 3 to 6 weeks,
The operation of removing the resulting white portion is repeated several times. By this selection, purple calli are obtained on the solid medium.

(Fourth Step: Until Uniform Callus Having Anthocyanin Synthesizing Capability is Obtained) The purple callus selected as described above is obtained by converting 1,4-mg of 2,4-D to 1-3 mg / L and sucrose of 3% to 3%. The medium is cultivated in a liquid medium containing MS at 25 to 27 ° C. in a dark place while stirring the medium. The cell mass suspended in the medium is collected by aseptic filtration at intervals of one month or more, the purple part is selected, and the operation of transplanting the same to a new medium is repeated for about one year to obtain an anthocyanin pigment content. And a stable cell line (homogeneous callus having anthocyanin synthesis ability) can be obtained.

It is difficult to obtain a callus having a high anthocyanin pigment content from a callus having no adventitious embryo-forming ability.

Hereinafter, a preferred embodiment in which the method of the present invention described with reference to FIG. 1 is carried out for a sweet potato variety “Ayamurasaki” will be described with reference to the drawings. Culture conditions are
All were performed in the dark at 25-27 ° C.

FIG. 2 shows the state of callus formation from tubers of sweet potato variety "Ayamurasaki" in the first stage of the method of the present invention. M containing 3% sucrose
In the medium in which 2,4-D is added to S-solid medium at 0.5 to 1 mg / L, sufficient callus formation is observed. In the presence of BA (benzyladenine), only calli without the ability to form somatic embryos are formed. From this result, in the first stage,
The medium composition of the callus induction step was adjusted to 2,4-D 0.5-1.
An MS solid medium containing mg / L and 3% sucrose was used.

FIG. 3 shows the MS containing 3% sucrose of FIG.
Culture in a solid medium supplemented with 0.5 to 1 mg / L of 2,4-D was continued for 10 weeks, and the formed adventitious embryo-like heart-shaped tissue (adult embryo in the first stage) (Like organization). In addition, the differentiation of the adventitious embryo-like tissue was performed in MS solid medium containing 3% sucrose in 2,4-
D is most frequent in a medium supplemented with 0.5 to 1 mg / L, and the formation of adventitious embryo-like tissue is inhibited in the presence of a high concentration of 2,4-D or BA.

From the heart-shaped somatic embryo-like tissue, 2,4-D was added in an amount of 0.5 to 1 mg / L as described in the second step.
When callus is induced in MS solid medium containing 3% sucrose,
A callus having no adventitious embryo-forming ability and a callus having adventitious embryo-forming ability are obtained. A callus having the ability to form somatic embryos was selected.
% Mixed callus is formed when grown on MS solid medium containing 2%.

FIG. 4 shows the mixed color callus obtained in the second step. As shown, in the mixed callus, a purple colored portion and a white portion appear in each cell mass. The purple portion was cut out and 2,4-D was added to 0.
The cells are transplanted onto an MS solid medium containing 5 to 1 mg / L and 3% sucrose, and the selection of the purple portion is repeated.

FIG. 5 shows purple calli obtained as a result of repeating the third stage of selection on a solid medium five times. As shown, on the solid medium, only purple calli are almost completely formed.

FIG. 6 shows that the purple callus of FIG. 5 obtained in the third step is converted to 2,4-
FIG. 3 shows a state in which D was shake-cultured for 1 month in an MS liquid medium containing 1 to 3 mg / L and 3% sucrose. In the liquid medium, more white parts appear,
Except for that, select the dark purple part as shown by the circle in the figure.

FIG. 7 shows the state of callus obtained by repeating the selection operation of the fourth stage for about one year. Even in a liquid medium, a uniform callus (stable cell line) consisting of only cells having almost completely purple anthocyanin dye synthesizing ability can be obtained.

[0033]

EXAMPLE A cell line (Pigmented purple cel) prepared by the method of the present invention using sweet potato variety Ayamurasaki was used.
l Line: hereinafter referred to as PL) was examined for anthocyanin accumulation when suspended and cultured in a dark place.

In the following test examples, in suspension culture of PL, the amount and composition of pigment accumulated under various culture conditions and the proliferation of cells were investigated. PL suspension culture is
It was performed in a dark place in a liquid medium in which 3% sucrose and 2 mg / L 2,4-D were added to the S basic medium, and the medium was replaced every week. For the PL suspension culture, the PL after 7 days of passage
0.1 g of cell mass was removed from the culture, transferred to a 50 ml flask containing 10 ml of each test medium, and culture was started in the dark. After 14 days, samples were collected from the medium. Sampling was performed four times.

(Method of measuring proliferation)
% Sucrose solution to remove water by filtration under reduced pressure,
The weight was measured. The growth index is W (fresh weight after culture) /
Wo (fresh weight before culture).

(Anthocyanin extraction method) A sample from PL was immersed in 50% acetic acid for 1 hour. As a control, samples from tuberous roots were soaked in 50% acetic acid overnight. The amount of the acetic acid solution was 20 times the sample weight. These samples were centrifuged and the supernatant was used for anthocyanin identification and quantitative analysis.

(Method of Identifying and Quantifying Anthocyanins and HPLC Analysis) The above supernatant was diluted 4-fold with McLbank's buffer, adjusted to pH 3, and the absorbance at 530 nm (OD ₅₃₀ ) was measured. The color value (CV) is a well known indicator of total anthocyanins amount was calculated by the following _{equation: CV = 0.1xOD 530 x4x20 / 1gfw} OD 530: absorbance above measurements 4 and 20: in the above buffer and acetic acid Dilution fw = fresh weight of tissue HPLC analysis was performed using a reverse phase column ODS-2 column.

Test Example 1: Plant growth regulator 2,4-D
Effect] The PL was cultured by the above-mentioned method using a medium in which 2,4-D having the following concentration was added to an MS basic medium to which 3% sucrose was added. 2,4-D; 0, 0.5, 1, 2, 4 mg / L

FIG. 8 is a graph showing the effect of adding 2,4-D to the medium on the pigment concentration of the main cultured cells. The presence of 2,4-D in the medium has the effect of reducing anthocyanin accumulation in PL, and the pigment concentration of PL is highest in the medium without 2,4-D, at 4 m
2,4-D at a concentration of g / L or more significantly reduced anthocyanin accumulation. Also, the presence of 2,4-D in the medium inhibited cell growth in PL suspension culture. 1mg
2,4-D at a concentration of / L or more significantly reduced the growth index.

As is clear from this graph, the fact that the cultured cells (PL) according to the present invention do not require 2,4-D, a plant growth regulator, for anthocyanin accumulation and growth is advantageous in terms of cost. Not only that, there is an advantage that a highly safe pigment can be provided, for example, when the produced anthocyanin pigment is used as a food pigment.

[Test Example 2: Effect of Nitrogen Concentration] A) Ammonium nitrate (NH ₄ NO ₃ ⁻ ) A medium in which 3% sucrose was added and the ammonium nitrate concentration was modified as follows was used. PL was cultured by the above method. Another source of nitrogen in MS basal medium was 18.8 mM potassium nitrate. No plant growth regulator was added. NH ₄ NO ₃ ⁻ ; 0, 2.5, 5, 7.5, 10, 15,
Ammonium nitrate concentration in 20 mM MS basic medium is about 20 m
M.

B) Potassium Nitrate (KNO ₃ ⁻ ) PL in a MS basic medium supplemented with 3% sucrose using a potassium nitrate concentration modified as follows:
Was cultured by the above method. No plant growth regulator was added. KNO ₃ ⁻ ; 4.7, 9.4, 18.8, 37.6, 5
The ammonium nitrate concentration in the 6.4 mM MS basal medium was 2.5
mM. Therefore, the final nitrate ion concentration in the above medium was as follows. NO ₃ ⁻ ; 7.2, 11.9, 21.3, 40.1, 5
8.9 mM In the experimental group (NO ₃ ⁻ ; 7.2, 11.9) in which the potassium ion concentration was lower than that of the MS basal medium by this modification, K was adjusted so as to reach the potassium concentration of the MS basal medium.
Cl was added. MS basal medium with 3% sucrose was used as a control.

FIG. 9 is a graph showing the effect of the concentration of ammonium nitrate in the medium on the dye concentration of the main cultured cells. The dye concentration of PL was highest in the medium with the ammonium nitrate concentration of 2.5 mM, and ammonium nitrate at the concentration of 2.5 mM or more reduced the anthocyanin accumulation.
In addition, cell growth in PL suspension culture was highest in a medium having a concentration of 2.5 mM ammonium nitrate, and ammonium nitrate having a concentration of 7.5 mM or more slightly inhibited growth. Therefore, in order to produce a high-concentration dye by PL, the ammonium nitrate concentration in the MS basic medium is set to 0 to
Preferably, it is modified to 2.5 mM.

FIG. 10 shows the main cultured cells and “Ayamurasaki”
4 is a chromatogram showing the results of an analysis (ODS-HPLC analysis) of the anthocyanin dye of the tuber root by high-performance liquid chromatography using a reversed-phase column (ODS-2 column). The top, second, and third stages are respectively 20 mM ammonium nitrate and 2.5 m
M, PL at 0 mM, the bottom row is the chromatogram of the root of “Ayamurasaki”.

As is clear from this chromatogram,
PL produced various anthocyanin pigments, the composition of which was different from that of tuberous roots. Specifically, the peak at the slow retention time was dominant in the tuber chromatogram. In contrast, in the chromatogram of PL in the 20 mM ammonium nitrate group, the peak of the slow retention time that was predominant in the tuberous root was slightly detected but very small, and the peak of the early retention time was dominant. . Further analysis of these early peak anthocyanins identified non-acylated anthocyanins belonging to the cyanidin and paeonidine glycosides.

As is evident from the PL chromatogram, under the condition where the concentration of ammonium nitrate in the medium is high, the anthocyanins which are not retained for a long period of time, ie, which are not retained in the column for a long time, mainly accumulate. The anthocyanins with a slow retention time, ie, strongly retained on the column, mainly accumulated.

It is considered that an anthocyanin showing a peak having a shorter retention time in reverse phase HPLC has a higher hydrophilicity and a lower degree of acylation, and a peak having a later retention time has a lower hydrophilicity and a higher acylation. It is thought to be an anthocyanin with a more complex structure, which is methylated. Thus, anthocyanins displaying a "early" peak that was predominant in PL under high ammonium nitrate conditions are considered to be precursors in the biosynthesis of highly acylated anthocyanins that predominate in tuberous roots.

Further, in the PL chromatogram, some PL-specific peaks not found in the tuber chromatogram were observed, indicating that the biosynthesis of a pigment of high value was possible. It suggests that the regulation mechanism of anthocyanin biosynthesis is different between the differentiated state (tube) and the undifferentiated state (PL). Thus, the PL prepared by the method of the present invention is also useful in the study of pigment biosynthesis.

From the above results, it was found that in PL, it is possible to modify and control the chemical composition of the accumulated dye by changing the concentration of ammonium nitrate in the medium. Further investigation was made on its relevance to control.

FIG. 11 is a graph showing the effect of the nitrate ion concentration of the medium on the dye concentration of the main cultured cells. The dye concentration of PL tended to increase as the nitrate ion concentration decreased. Regarding the anthocyanin composition, there was no significant difference due to the change in nitrate ion concentration. The increase in anthocyanin pigment concentration due to the decrease in nitrate ion concentration was thought to be due to activation of the entire biosynthetic pathway.

From the experimental results on these nitrogen components,
Both nitrate and ammonium ions affect the activity of the anthocyanin biosynthetic pathway in PL,
It was suggested that the composition of the accumulated anthocyanins was controlled by ammonium ions. Therefore, by using PL, it becomes possible to produce a desired anthocyanin dye by modifying the ammonium ion concentration.

[Test Example 3: Effect of sucrose] PL was cultured by the above method in an MS basic medium to which sucrose was added at the following concentrations. Ammonium nitrate concentration in sucrose; 1, 3, 5, 7, 9% MS basal medium was 2.5
mM, and the concentration of potassium nitrate was changed to 4.7 mM. No plant growth regulator was added. MS basal medium with 3% sucrose was used as a control.

FIG. 12 is a graph showing the effect of the sucrose concentration of the medium on the pigment concentration and growth of the main cultured cells. As is clear from this graph, when the sucrose concentration of the medium was in the range of 1% to 5%, the pigment concentration increased as the sucrose concentration increased. The pigment concentration at PL at 5% concentration was about three times that in Ayamurasaki tuber. However, when the sucrose concentration of the medium exceeded 5%, no significant increase in pigment concentration was observed. Further, when the sucrose concentration of the medium exceeded 7%, the growth of PL was inhibited, and a sucrose concentration of 1 to 5% was also suitable for the growth. Therefore, considering both the pigment accumulation and proliferation of PL,
The concentration of sucrose to be added to the medium is preferably 1 to 5%.
% Was most preferred.

The suspension culture of PL in the above experiments was carried out as follows.
All were performed in the dark, but had very high anthocyanin synthesis ability. Cultured cell lines reported to date require continuous light irradiation for anthocyanin biosynthesis, which has been costly,
The PL prepared by the method of the present invention does not require light in the preparation process, and does not require light for dye synthesis, and is very economical in cost.

[Brief description of the drawings]

FIG. 1 is a diagram showing a flow of a method for producing a cell line containing an anthocyanin dye according to the present invention.

FIG. 2 is a diagram showing callus induction from a piece of Ayamurasaki tuber tissue (basal medium: MS solid medium containing 3% sucrose)

FIG. 3 is a view showing an adventitious embryo-like tissue formed in an MS solid medium supplemented with 0.5 to 1 mg / L of 2,4-D.

FIG. 4 is a diagram showing mixed-color calli obtained by selecting and growing callus having the ability to form adventitious embryos from callus derived from adventitious embryo-like tissue

FIG. 5 is a diagram showing purple calli on a solid medium obtained by selecting and growing a purple part from mixed-color calli.

FIG. 6 is a diagram showing callus obtained by growing purple callus in a liquid medium on a solid medium (a strong purple part in a circle in the figure is selected).

FIG. 7 is a diagram showing a callus obtained by repeating selection of a strong purple portion for about one year.

FIG. 8 is a graph showing the effect of addition of 2,4-D to a medium on the pigment concentration of cultured cells prepared according to the present invention.

FIG. 9 is a graph showing the effect of the concentration of ammonium nitrate in a medium on the concentration of dye in cultured cells prepared according to the present invention.

FIG. 10: ODC- of the anthocyanin pigment composition of the cultured cells prepared according to the present invention and the tuber root of “Ayamurasaki”
Diagram showing analysis results by HPLC

FIG. 11 is a graph showing the effect of nitrate ion concentration in a medium on the dye concentration of cultured cells prepared according to the present invention.

FIG. 12 is a graph showing the effect of the sucrose concentration of the medium on the pigment concentration and growth of cultured cells prepared according to the present invention.

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[Submission date] June 21, 1999 (1999.6.2
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 ────────────────────────────────────────────────── ─── Continuing on the front page (72) Inventor Osamu Yamakawa 6500 Nakahara Jukusha, 6500 Yokoichi-cho, Miyakonojo, Miyazaki Pref.

Claims (3)

[Claims] 1. A method for producing cultured cells having a high content of anthocyanin pigment by culturing from a tuberous root of a sweet potato containing an anthocyanin pigment, wherein the callus is induced from the tuberous root of a sweet potato containing an anthocyanin pigment to have adventitious embryogenic ability. Differentiating the tissue to obtain an adventitious embryo-like differentiated tissue, and further inducing callus with somatic embryogenesis ability from the adventitious embryo-like differentiated tissue, and repeatedly selecting cell lines containing high concentrations of anthocyanin dye A method for producing cultured cells having a high anthocyanin pigment content, wherein the steps are performed in a dark place. 2. The method according to claim 1, wherein the sweet potato containing the anthocyanin dye is sweet potato variety Ayamurasaki. 3. A method for producing a cultured cell having a high anthocyanin pigment content, comprising producing a cultured cell of a high anthocyanin pigment content from a root of a sweet potato variety Ayamurasaki containing an anthocyanin pigment by culturing in a dark place.