AU751998B2 - Cultured cells of sweetpotato with high anthocyanin content, and method of establishing/culturing such cells - Google Patents

Description

AUSTRALIA

Patents Act 1990 COMPLETE SPECIFICATION STANDARD PATENT Applicant(s): S~DIRECTOR GENERAL OF KYUSHU NATIONAL AGRICULTURAL EXPERIMENT STATION, THE MINISTRY OF AGRICULTURE, FORESTRY ANiD FISIIERIES OF JAPAMn Re<eCr4.l OrguAni t'O Invention Title: CULTURED CELLS OF SWEETPOTATO WITH HIGH ANTHOCYANIN CONTENT, AND METHOD OF ESTABLISHING/CULTURING SUCH

CELLS

The following statement is a full description of this invention, including the best method of performing it known to me/us: CULTURED CELLS OF SWEETPOTATO WITH HIGH ANTHOCYANIN CONTENT, AND METHOD OF ESTABLISHING/ CULTURING SUCH CELLS BACKGROUND OF THE INVENTION FIELD OF THE INVENTION The present invention relates to a method of obtaining anthocyanin pigment. More particularly, the invention relates to a cultured cell with high anthocyanin pigment content usable as an extraction material to be processed into a pigment material such as a food colorant and a method of establishing/culturing such cultured cell. The invention relates also to a method of obtaining a research material to be used in the study of plant biosynthesis of anthocyanins, DESCRIPTION OF THE RELATED ART For coloring a food product, cosmetics product or the like, a variety of artificial or natural pigments have conventionally been employed. Since artificial pigments have problems in the respects of mutagenicity, carcinogenicity, and so on, there has been a continued demand for pigments of natural origins in consideration of safety However, production of pigments through extraction from natural products is susceptible to the natural environmental factors such as the seasonal, climate and temperature variations, hence hindering stable supply of the pigment. For this reason, there has been an attempt to produce natural pigments through the tissue culture technique. The tissue culture technique provides the advantage of allowing efficient planed production of the target pigment in a short time period.

Among the natural pigments, relatively extensive research efforts have been made for red pigments, in particular, anthocyanins.

Anthocyanins exist naturally in various kinds of plants such as fruit skin of grapes, violet cabbage, violet corn, Hibiscus, Perilla frutescens crispa (beefsteak plant), berries, etc. Conventionally, the production of anthocyanin generally involves using various anthocyanin-containing plants as the raw material and extracting anthocyanin therefrom through certain separation purification method appropriately selected. However, with such conventional method of extracting anthocyanins from a cultivated or field-grown plant, the material cost is high, hence making low-cost ~production of anthocyanin difficult. Moreover, if such cultivated plant is used as the raw material, since the plant grows slowly and the cultivation is time and labor consuming, thus deteriorating production efficiency of anthocyanin. The method suffers still another problem of the anthocyanin product being necessarily dependent on the harvest time of the cultivated (field-grown) plant, thus making the production unstable yearly.

In view of the above-described problems, there has been proposed a method which achieves relatively high production efficiency of anthocyanins as well as more yearly stable mass-scale production thereof as compared 20 with the above-described method, This method involves large-scale culture of anthocyanin-producing cells by using cultured cells induced from a plant body and then extracting anthocyanin from these cultured cells. For use as such cultured cells containing anthocyanin, there are known cultured cells induced from strawberry, carrot, grapes, roses, apples, Jerusalem artichoke, buckwheat, statice, rosemary, etc.

Yet, such conventional anthocyanin containing cultured cells have the problem of low or insufficient anthocyanin content, which makes them unsuitable as a pigment extraction raw material or a research material.

Further, the conventionally proposed anthocyanin containing cultured cells require continuous illumination for their biosynthesis of anthocyanins.

3 This too makes these cultured cells too costly for use as pigment extraction material. These conventional cultured cells suffers another problem relating to the safety of the resultant pigment products, since they require plant growth regulator such as 2,4-D dichlorophenoxyacetic acid) for their maintenance/multiplication.

For example, Japanese laid-open patent gazette: No. Sho. 63-233993 reports some methods of obtaining anthocyanin pigments by using cultured cells derived from sweet potato Ipomoea Batatas). These methods, however, invariably require continuous illumination for obtaining callus for producing anthocyanin pigments as well as for the multiplication of the cultured cells and their biosynthesis of anthocyanin.

15 In summary, the anthocyanin-containing cultured cells proposed by the convention suffer the problems of low pigment content, requirements of continuous illumination and plant growth regulator for the multiplication/maintenance of the cultured cells and the biosynthesis of the pigment.

All references, including any patents or patent applications, cited in this specification are hereby incorporated by reference. No admission is made that any S: reference constitutes prior art. The discussion of the 25 references states what their authors assert, and the applicants reserve the right to challenge the accuracy and pertinency of the cited documents. It will be clearly understood that, although a number of prior art publications are referred to herein, this reference does not constitute an admission that any of these documents forms part of the common general knowledge in the art, in Australia or in any other country.

SUMMARY OF THE INVENTION In view of the above-described state of the art, the present invention provides a method which allows H:\Juanita\Keep\Patents\42555.0o.doc 23/11/01 3a establishment of a cultured cell containing anthocyanins in higher concentrations from sweet potato with high anthocyanin content, the cell requiring no illumintino for its mulitplaction or for its anthocyanin accumulation, the method comprising the steps of: inducing callus from a tuberous root of "Ayamurasaki" sweet potato containing anthocyanin, causing differentiation of a tissue thereof having embryogenic ability, thus obtaining an adventive embryoid differentiated tissue; and further inducing, from said adventive embryoid differentiated tissue, a callus having embryogenic ability, then repeatedly selecting a cell line having a higher concentration of anthocyanin; wherein said steps and are effected through tissue culture in the dark.

The invention further provides a cultured cell with higher concentrations of anthocyanins which does not require continuous illumination or plant growth regulator for its anthocyanin accumulation, to be usable as a raw material from which high-quality anthocyanin pigment can be produced and extracted, and to provide also a method of culturing such cell.

25 For achieving the above-noted provisions, the present inventors have invented an improved method of establishing a cultured cell with a higher H:\KarraR\Keep\speci\Biotech\42555-OO.doc 24/06/02 concentration of anthocyanin from sweet potato.

More particularly, according to a first aspect of the present invention, there is provided a method of establishing a cultured cell with high anthocyanin content, which comprises the following steps: inducing callus from a tuberous root of sweet potato containing anthocyanin, causing differentiation of a tissue thereof having embryogenic ability, thus obtaining an adventive embryoid differentiated tissue; and further inducing, from said adventive embryoid differentiated tissue, a callus having embryogenic ability, then repeatedly selecting a cell line having a higher concentration of anthocyanin; wherein said steps and are effected through tissue culture technique in the dark.

As described hereinbefore, the establishment and multiplication of callus producing anthocyanins conventionally requires continuous illumination. In contrast, the above method proposed by the present invention requires no illumination through the entire process. Moreover, the cell line established by this method with a high concentration of anthocyanin is capable of accumulating anthocyanins in the dark. As the method can eliminate the cost of illumination, the method provides the cost 20 advantage.

Further, although a number of cases have been reported regarding induction of an embryogenic callus from sweet potato, most of them involve induction of the callus from young leaves of the potato. None are known which report direct induction of embryogenic callus from the tuberous or storage root of the potato The plant to be used in the method of establishing a cultured cell rich in anthocyanin content is sweet potato (Ipomoea Batatas). Among the various cultivars of sweet potato, the cultivar referred to as "Ayamurasaki" is particularly preferred. This "Ayamurasaki" cultivar of sweet potato exhibits red color as far as to the inner flesh portion of the tuberous root s thereof. Hence, for its high anthocyanin content in the tuberous root, Ayamurasaki cultivar is particularly suitable for use in the method of the present invention.

On the other hand, it has been reported that an anthocyanin accumulating cell line may be established from a further cultivar of sweet potato known as "Kintoki". However, this cultivar, "Kintoki", exhibits white color in the inner flesh portion of its tuberous root Hence, this conventional method differs from that of the present invention in that the former establishes the anthocyanin accumulating cell line from the periderm portion of the tuberous root which exhibits the red color and also in that it uses the non-embryogenic callus derived from the root.

Furthermore, the cell line established by this conventional method further differs from that of this invention in that it requires continuous illumination ee for its anthocyanin accumulation. More importantly, the production amount of the pigment from the conventional cell line is more than two-digit lower than that available from the cell line established according to the present invention.

According to a second aspect of the present invention, there is 0 provided a method of establishing a cultured cell with high anthocyanin content, which comprises establishing the cultured cell from a tuberous root of "Ayamurasaki" cultivar of sweet potato containing anthocyanin by tissue culture technique in the dark.

With the above method, it is possible to establish a cultured cell of sweet potato having a high anthocyanin content by culturing in the dark.

While the conventional anthocyanin biosynhesizing cultured cell requires illumination for its biosynthesis of the pigment, the cultured cell established by the method of the present invention is capable of biosynthesis of anthocyanin in the darkness, thus providing the cost advantage. This method of the invention differs from the conventional method of establishing an anthocyanin accumulating cell line from sweet potato further in that it establishes the sweet potato cultured cell rich in anthocyanin content from a tuberous root of Ayamurasaki cultivar of sweet potato.

Preferably, the composition of the medium for use in establishing the cultured cell rich in anthocyanin content comprises basal MS medium supplemented with 3% of sucrose as the carbon source and auxin 2,4dichlophenoxyacetic acid (hereinafter, 2,4-D) as a growth regulator. The basal MS medium refers to the well-known Murashige and Skoog basal medium containing various inorganic components and vitamins as organic components, but not containing any carbon source (generally sucrose).

The anthocyanin-rich cultured cell established by the methods according to the first and second aspects of the invention too constitutes a part of this invention.

Further, for fulfilling the above-noted objects, the present inventors employed an anthocyanin-rich sweet potato cultured cell established through the tissue culture technique from an anthocyanin-containing tissue of sweet potato and investigated its culture conditions. As a result, the present inventors have achieved the cultured cell and culture method as originally intended.

20 Namely, the anthocyanin-rich cultured cell according to the third aspect of the present invention established through the tissue culture technique from an anthocyanin-containing sweet potato tissue does not require continuous illumination for its multiplication or anthocyanin accumulation. As described hereinbefore, the conventional method of anthocyanin biosynthesis using a cultured cell requires continuous illumination for the biosynthesis of anthocyanin by the cell. Whereas, the sweet potato cultured cell according to this aspect of the present invention is distinguished over such conventional cultured cell in that it is capable of anthocyanin accumulation in the dark.

Further, the anthocyanin-rich cultured cell according to a fourth -1 aspect of the present invention which is established through tissue culture technique from an anthocyanin-containing sweet potato tissue does not require any plant growth regulator for its multiplication or anthocyanin accumulation. This sweet potato cultured cell according to this aspect of the invention is distinguished over the conventional anthocyanin biosynthesizing cultured cell in that it can multiply well in a medium free from any plant growth regulator and that the anthocyanin accumulation by the cell does not depend on any plant growth regulator, either.

According to a fifth aspect of the present invention, there is provided an anthocyanin-rich cultured cell capable of accumulating and providing various compositions of anthocyanin through modification of the nitrogen condition in the culture medium. With the cultured cell according to this aspect of the present invention, the composition of the anthocyanin may be artificially controlled through modification of the nitrogen condition in the culture medium. In addition, this cultured cell is capable also of biosynthesis of a pigment component not inherent in the tissue of its origin such as the tuberous root of sweet potato, for its subsequent extraction and use. Such modification of nitrogen condition of the culture medium may be done by e.g. varying the ammonium ion concentration in the medium.

20 The plant used as the origin of the cultured cells according to the third through fifth aspects of the invention is sweet potato containing anthocyanin. Among various cultivars available, the "Ayamurasaki" cultivar is particularly preferred.

The sixth aspect of the invention relates to a method of culturing an anthocyanin-rich cultured cell. This method comprises culturing the cultured cell in the dark. As described hereinbefore, the conventional anthocyanin biosynthesizing cultured cell requires continuous illumination for its pigment biosynthesis, hence being incapable of accumulating anthocyanin in high concentrations in the dark culture conditions. On the other hand, the culture method according to this aspect of the invention is capable of causing the cultured cell to biosynthesize anthocyanins in the dark. In this respect, the culture method of the present invention differs from the conventional culture method.

The seventh aspect of the invention relates to a method of culturing an anthocyanin-rich cultured cell which method comprises culturing the cultured cell in a medium free from any plant growth regulator. The conventional medium used for anthocyanin synthesizing cultured cell is caused to contain a plant growth regulator for the multiplication and pigment accumulation of the cell. In contrast, the culture method according to this aspect of the invention is capable of causing the cultured cell to biosynthesize anthocyanins efficiently without any plant growth regulator in the culture medium. Moreover, a pigment product obtained from the cultured cell cultured in such medium free from any artificial plant growth regulator provides the advantage of greater safety when the product is used as a food colorant for instance.

Preferably, the composition of the medium for use in the methods according to the sixth and seventh aspects of the invention for culturing anthocyanin-rich cultured cell comprises basal MS medium supplemented with sucrose as the carbon source and modified to contain ammonium 20 nitrate in the concentration range of 0 to 2.5 mM.

The concentration of sucrose added in the medium ranges preferably between 1% and most preferably Also, although ammonium nitrate may not be added at all, but when it is added, it is preferred that its concentration should be lower than 2.5 mM.

The anthocyanin-containing cultured cell according to the present invention is capable of accumulating anthocyanin in more than threefold concentration of that of its origin, i.e. the tuberous root of sweet potato cultivar "Ayamurasaki" in the medium conditions modified from the basal MS medium. This is much higher than those concentrations available from the known cultured cells having anthocyanin biosynthesis ability.

As described above, according to the present invention, it is possible to provide a cultured cell having a very high concentration of anthocyanin pigment. This anthocyanin-containing cultured cell according to the present invention has sufficient pigment concentration, biosynthesis efficiency and safety suitable for its use as a pigment extraction raw material. Hence, by using this cultured cell as the pigment extraction raw material, the pigment material may be supplied stably throughout the year.

Moreover, this cell is believed to be useful for clarifying the anthocyanin biosynthesis system.

10 Further and other objects, aspects and advantages of the present invention will become apparent from the following detailed description thereof with reference to the accompanying drawings.

For the purposes of this specification it will be clearly understood that the word "comprising" means "including but not limited to", and that the word "comprises" has a corresponding meaning.

S

BRIEF DESCRIPTION OF THE DRAWINGS Fig. 1 is a flow chart illustrating the flow of establishing a cell line (cultured cells) containing anthocyanin pigment relating to the present invention, Fig. 2 shows callus induction from a tissue piece of a tuberous root of "Ayamurasaki" cultivar of sweet potato (basal medium: MS solid medium containing 3% sucrose), Fig. 3 is a view showing an adventive embryoid tissue formed on the MS solid medium supplemented with 2,4-D in the concentration of 0.5 to Img/L, 25 Fig. 4 is a view showing mottled callus obtained by selecting and multiplying embryogenic callus induced from the adventive embryoid tissue, Fig. 5 shows purple-colored callus obtained by selecting/multiplying *purple-colored portions of the mottled callus, Fig. 6 shows callus obtained by multiplying the purple-colored oo** callus of Fig. 5 in a liquid medium, Fig. 7 is a view showing callus obtained by repeating selection of strongly purple-colored portions (encircled in Fig. 6) for one year approximately, i.e. the anthocyanin-rich cultured cell according to the invention, Fig. 8 is a graph showing the effect of supplementation of 2,4-D to the medium on the pigment concentration of the cultured cell according to the present invention, Fig. 9 is a graph showing the effect of ammonium nitrate concentration in the medium on the pigment concentration of the cultured cell according to the present invention, Fig. 10 shows the result of ODS-HPLC analysis of the anthocyanin pigment composition of the cultured cell of the invention in comparison with that of a tuberous root of Ayamurasaki cultivar sweet potato, 15 Fig. 11 is a graph showing the effect of nitrate ion concentration in the medium on the pigment concentration of the cultured cell according to :"the present invention, and Fig. 12 is a graph showing the effect of sucrose concentration in the medium on the pigment concentration and multiplication of the cultured cell according to the present invention.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS Preferred embodiments of the present invention will be described in details hereinafter with reference to the accompanying drawings. It is understood that the present invention is not to be limited to these specific embodiments disclosed, but may be modified by one skilled in the art in any other way without departing from the essential spirit thereof set forth in the appended claims.

First, callus is induced from a tuberous root of sweet potato

II

containing anthocyanin pigment. By repeated selection of cell lines having higher concentrations of anthocyanin, an anthocyanin-rich cultured cell of sweet potato is established.

Fig. 1 schematically illustrates the method according to the invention of establishing a cultured cell of the invention containing a high concentration of anthocyanin. This method will be described more particularly as follows.

(first stage: from the beginning to obtainment of adventive embryoid tissue) First, a tuberous root of sweet potato (Ipomoea Batatas) containing anthocyanin is sterilized and its periderm is removed. This root is then cut into tissue pieces in the form of cubes sized to have each side of 2-3 cm.

These tissue pieces are placed on a solid medium of Murashige Skoog (referred to as "MS" hereinafter) containing 2,4-D in concentration of 0.5 to 1 mg/L and 3% of sucrose. The tissue pieces are cultured (incubated) at to 27°C in the dark for 5 to 10 weeks, when in addition to dedifferentiated callus, there are partially differentiated tissues having embryogenic ability, from which an adventive embryoid differentiated tissue (adventive 20 embryoid tissue) is obtained.

(second stage: obtainment of mottled callus) The adventive embryoid tissue is transferred to the same medium as that described above (MS solid medium supplemented with 2,4-D in the concentration of 0.5 to 1 mg/L and 3% of sucrose). This tissue is cultured (incubated) under the same culture conditions (25 to 27 0 C in the dark) for 3 to 6 weeks to induce callus therefrom.

With the above, both a soft white callus having no embryogenic ability and also a hard purple callus having embryogenic ability are formed.

From these two kinds of calli, the embryogenic callus is selected. Then, this selected callus having embryogenic ability is transferred to the MS solid medium supplemented with 2,4-D in the concentration of 1 to 3 mg/L and 3% of sucrose. This callus is cultured under the same culture conditions (25 to 27°C in the dark) for 3 to 6 weeks.

With the above, a mottled callus having a white portion and a purple portion in a mixed state is obtained.

(third stage: obtainment of purple callus on the solid medium) The white portion and the purple portion are separated from each other, and the callus from the purple portion is transferred to MS solid medium supplemented with 2,4-D in the concentration of 0.5 to 1 mg/L and 3% of sucrose. This callus is cultured in the same culture conditions (25 to 15 27°C in the dark) for 3 to 6 weeks. And, a white portion which has again developed is selectively removed. With repetition of this selecting operation for several times, there is obtained a purple-fleshed callus on the solid medium.

20 (fourth stage: obtainment of homogeneous callus (cultured cells) having anthocyanin biosynthesis ability) The purple callus selected in the manner described above is cultured in MS liquid medium containing 1-3 mg/L of 2,4-D and 3% of sucrose at 25 to 27°C in the dark, with stirring of the medium. With an interval of 1 month approximately, the cell aggregates suspended in the medium are collected through sterilized filtration and the purple portions of the aggregates are selected and transferred to a new similar medium.

With repetition of these operations for a period of about 1 year, there may be established a stable cell line having high anthocyanin content (homogeneous callus (cultured cells) having anthocyanin biosynthesis ability). Incidentally, it is difficult to obtain a callus (cultured cell) with high anthocyanin content from a callus not having embryogenic ability.

Next, the method of the present invention described generally hereinbefore with reference to Fig. 1 will be described more particularly in connection with preferred embodiments thereof the invention carried out for the "Ayamurasaki" cultivar of sweet potato. Through the entire process as follows, the culturing operations were invariably done at 25 to 27°C in the dark.

Fig. 2 shows a condition in which callus was formed from a tuberous root of "Ayamurasaki" cultivar of sweet potato in the first stage of the method of the invention. As shown, sufficient callus formation may be observed on the MS solid medium containing 3% of sucrose and supplemented with 0.5 to 1 mg/L of 2,4-D. In the presence of BA (benzyladenine), non-embryogenic callus alone may be induced. From this result, the MS solid medium containing 0.5 to 1 mg/L of 2,4-D and 3% of sucrose was selected as the composition of the medium for callus induction process in the first stage.

Fig. 3 shows formation of heart-shaped tissue (adventive embryoid tissue in the first stage) which was differentiated into an adventive 20 embryoid as the result of continuation of ten (10) weeks of culture on the MS solid medium of Fig. 2 containing 3% of sucrose and supplemented with to 1 mg/L of 2,4-D.

Incidentally, the differentiation of the adventive embryoid tissue occurs with highest frequency in the case of the medium modified from the MS solid medium containing 3% sucrose by supplementing it with 0.5 to 1 mg/L of 2,4-D. In the presence of higher concentrations of 2,4-D or BA, the formation of adventive embryoid tissue is inhibited disadvantageously.

As described hereinbefore in connection with the second stage of the process, with callus induction from the heart-shaped adventive embryoid tissue on the MS solid medium containing 0.5 to 1 mg/L of 2,4-D and 3% of sucrose, there were obtained a callus having no embryogenic ability (non-embryogenic callus) and a callus having such embryogenic ability (embryogenic callus). From these, the embryogenic callus was selected and multiplied on the MS solid medium containing 1-3 mg/L of 2,4- D and 3% of sucrose, whereby a mottled callus was obtained.

Fig. 4 shows such mottled callus obtained in the second stage. As shown, on this mottled callus, a purple-colored portion and a white-colored portion appear in a mixed state in each cell aggregate.

Then, the purple portion was picked out selectively and, as described hereinbefore in connection with the third stage, this was transferred to a MS solid medium containing 0.5 to lmg/L of 2,4-D and 3% i.:ev: of sucrose. This selecting operation of the purple portion was repeated for several times.

Fig. 5 shows the purple callus obtained after five times of selecting 15 operations on the solid media in the third stage. As shown, the callus on the solid medium appears purple-colored substantially entirely.

Fig. 6 shows a condition after the purple callus of Fig. 5 obtained in the third stage were cultured in a MS liquid medium containing 1 to 3 mg/L of 2,4-D and 3% of sucrose, with shaking of the culture for 1 (one) month, as 20 described hereinbefore in connection with the fourth stage. In the liquid medium, white portions were developed again. Therefore, with elimination of these, the dark-purple portions encircled in the figure were selected.

Fig. 7 shows a condition of the callus established by repeating the selecting operation of the fourth stage for about 1 year. As shown, in the liquid medium too, there were established a homogeneous callus (stable cell line) which consists solely of substantially entirely purple-colored cells having anthocyanin biosynthesis ability.

These cultured cells were established as the result of selecting operations for one year and testing for another year. They are stable in the pigment synthesis ability and other characteristics thereof and contain anthocyanins in very high concentrations.

With this cultured cell according to the present invention, if it is cultured, for example, in the dark in a liquid medium modified from the MS medium by elimination of ammonium nitrate therefrom and added with of sucrose, the cell is capable of accumulating anthocyanin in a very high concentration as high as threefold of that of the tuberous root of "Ayamurasaki" cultivar of sweet potato conventionally used for pigment extraction.

For controlling the amount and quality of anthocyanins accumulated in the cultured cell of the invention, it is very important to obtain understanding about the effects of various components of the medium on the amount and composition of the pigments accumulated in the cultured cell.

Fig. 8 illustrates the effect of addition of 2,4-D to the medium on the pigment concentration in the cultured cell of the present invention.

As shown, the pigment concentration in the cultured cell is highest ~with no addition of 2,4-D. For anthocyanin accumulation in the cultured cell of the invention, no addition of any plant growth regulator is necessary.

Fig.9 shows the effect of nitrogen component (ammonium nitrate) 20 concentration in the medium on the pigment concentration of the cultured cell of the invention.

For optimal pigment accumulation in the cultured cell of the invention, the concentration of ammonium nitrate in the MS medium should be modified to 0 to 2.5 mM.

Fig. 10 shows results of analysis by high performance liquid chromatography (HPLC) using reversed-phase column, on anthocyanin pigments in the cultured cell according to the present invention under the various culture conditions shown in Fig. 9 and that in the tuberous root of "Ayamurasaki" cultivar in comparison.

In the presence of high concentration of ammonium nitrate in the %%0 medium, the cultured cell of the invention accumulates mainly anthocyanins which are not retained for a long time in the reversed-phase column; that is, the cell accumulates mainly anthocyanins having high hydrophilicity. Conversely, in the presence of low concentration of ammonium nitrate, the cell accumulates mainly anthocyanins having low hydrophilicity which was highly acylated or methylated, which anthocyanins are retained firmly in the reversed-phase column.

As described above, with the cultured cell according to the present invention, the chemical composition of the pigment to be accumulated in the cell can be modified/controlled by adjustment of ammonium nitrate concentration in the medium. Further, anthocyanins which are not inherently present in the tuberous root of "Ayamurasaki" cultivar are also accumulated in the cell. Hence, the cell is capable of biosynthesis of pigments having rarity value also.

Fig. 11 illustrates the effect of concentration of nitrogen component (nitrate ion) in the medium on the concentration of pigment in the cultured S. cell of the invention.

In the presence of high concentration of nitrate ion, pigment biosynthesis of the cultured cell is inhibited.

Fig. 12 illustrates the effect of concentration of sucrose in the .i medium on the pigment concentration and multiplication of the cultured cell of the invention.

The pigment concentration of the cultured cell increases in accordance with increase of the sucrose concentration, but its multiplication is inhibited in the presence of sucrose concentration over

EXAMPLES

Investigations were made on the anthocyanin accumulation when the cultured cell line according to the invention established from the \-k "Ayamurasaki" cultivar of sweet potato (Pigmented purple cell line; referred to as hereinafter) was cultured in a suspension culture in the dark.

In particular, detailed investigations were made on the effects of addition of various components (plant growth regulator 2,4-D, nitrogen component, sucrose) to the medium on the amount and composition of accumulated anthocyanin.

In the following experiment examples, investigations were made on the amount and composition of the pigment to be accumulated and on the multiplication of the cells under various culture conditions in the suspension culture of PL.

The PL suspension culture was performed in the dark in a liquid medium comprising the basal MS medium supplemented with 3% of sucrose and modified to contain 2 mg/L of 2,4-D. The medium was changed weekly.

From seven-day-old PL subcultures, 0.1g of aggregates was transferred in 15 50 ml volume flasks containing 10 ml medium of each treatment. The treatments were replicated 4 (four) times. And, the culture was initiated in .the dark. The samples were collected after 14 (fourteen) days of culture.

(determination method of multiplication) The collected samples were washed with 3% sucrose solution and their water contents were removed by filtration under vacuum and these samples were weighed. The growth index was defined as W/Wo, where Wo and W denote fresh weight of the aggregates before and after the culture, respectively.

(extraction method of anthocyanin) The samples from the PL were steeped in 50% acetic acid for 1 (one) hour. As a control, a sample from a tuberous root was steeped in 18 acetic acid overnight. The volume of acetic acid solution was set to be (twenty) fold of the each sample weight. The samples were centrifuged.

The supernatants were used for subsequent anthocyanin identification and quantitative analysis.

(anthocyanin identification and quantitative determination and HPLC analysis) The respective supernatant was diluted fourfold with a. McIlvain' s 10 buffer solution, pH adjusted to 3 and was used for measurement of optical S: density (ODO) at 530 nm. The color value which is the well-known index of the total anthocyanins amount, was calculated using the following formula: CV 0.1 x OD x 4 x where OD,: the spectrophotometric reading described above, o'o 4 and 20: the levels of dilution, and fw fresh weight of the tissue The HPLC analysis was performed by using the reversed-phase column: ODS-2 column.

[experiment example 1: influence of plant growth regulator: 2, 4-D] The PL were cultured by the above-described method by using the medium comprising the basal MS medium supplemented with 3% sucrose and modified to contain the 2,4-D in the following concentrations; namely, 2,4-D: 0, 0.5, 1, 2, 4 mg/L Fig. 8 shows a graph illustrating the effect of the addition of 2,4-D to the medium on the pigment concentration of the cultured cell of the invention.

The presence of 2,4-D in the medium reduces the anthocyanin accumulation in PL. The pigment concentration of PL is highest in the case of medium without addition of 2,4-D at all. The concentrations over 4 mg/L of 2, 4-D significantly reduced anthocyanin accumulation.

The presence of 2,4-D in the medium also inhibited cell growth in the PL suspension culture. The concentrations over 1 mg/L of 2,4-D significantly reduced the growth index.

From this graph, it may be apparent that the cultured cell (PL) according to the invention does not require 2, 4-D, the plant growth regulator, for its anthocyanin accumulation or cell multiplication. Hence, this provides not only the cost advantage, but also the advantage of providing pigment of greater safety when e.g. the biosynthesized anthocyanin pigment is used as a food colorant.

,[experiment example 2: influence of nitrogen component concentration] A) ammonium nitrate (NH 4

NO-)

20 The PL were cultured by the above-described method by using the medium comprising the basal MS medium supplemented with 3% sucrose and modified to contain ammonium nitrate in the following concentrations.

The other nitrogen source of the basal MS medium was 18.8 mM of potassium nitrate. No plant growth regulator was added to the medium.

NH

4 NO3: 0, 2.5, 5, 7.5, 10, 15, 20 mM The basal MS medium has an ammonium nitrate concentration of about 20 mM.

B) potassium nitrate (KNOs) The PL were cultured by the above-described method by using the medium comprising the basal MS medium supplemented with 3% sucrose and modified to contain potassium nitrate in the following concentrations.

No plant growth regulator was added to the medium.

KNO,-: 4.7, 9.4, 18.8, 37.6, 56.4 mM The basal MS medium had been modified to contain an ammonium nitrate concentration of about 2.5 mM.

Therefore, the medium had the final nitrate ion concentrations as follows.

N0 3 7.2, 11.9, 21.3, 40.1, 58.9 mM With the above modification, as for those treatments where the potassium ion concentrations became lower than that of the basal MS 20 medium NO,: 7.2, 11.9 mM), KC1 was added so as to achieve the potassium level of the basal MS medium. A basal MS medium enriched with 3% sucrose was used as control.

Fig. 9 is a graph illustrating the effect of ammonium nitrate in the medium on the pigment concentration in the cultured cell of the invention.

As shown, the pigment concentration of PL was highest in the medium having 2.5 mM of ammonium nitrate. And, the ammonium nitrate concentrations over 2.5 mM reduced the anthocyanin accumulation.

Further, the cell multiplication in the PL suspension culture was highest in the medium having 2.5 mM ammonium nitrate concentration.

The ammonium nitrate concentrations over 7.5 mM slightly inhibited the multiplication.

Accordingly, for higher-concentration pigment production by PL, it is preferred that the ammonium nitrate concentration in the basal MS medium be modified to 0 to 2.5 mM.

Fig. 10 is a chromatogram showing the results of high-performance liquid chromatography analysis (ODS-HPLC analysis) for anthocyanin composition using the reversed-phase column (ODS-2 column) performed on the cultured cell of the invention and the tuberous root of "Ayamurasaki" cultivar. The uppermost, second and third rows comprise the chromatograms of the PL cultured in the media of ammonium nitrate concentrations of 20 mM, 2.5 mM and 0 mM, respectively; and the lowermost row comprises the chromatogram of the "Ayamurasaki" tuberous root.

As may be understood from these chromatograms, the PL produced 15 various anthocyanins with compositions different from that of the tuberous root.

Specifically, in the case of the chromatogram of the tuberous root, the peaks of late retention time were predominant. Whereas, in the case of the chromatogram of PL of the treatment with 20 mM ammonium nitrate, such peaks of late retention time predominant in the tuberous root were slightly detected, but very small in the amount, while the peaks of early retention time were predominant. With further analyses of these anthocyanins having earlier peaks, these early peaks were identified as belonging in cyanidin glycosides and peonidin glycosides which are nonacylated anthocyanins.

As may be apparent from the chromatograms of PL, in the presence of high concentrations of ammonium nitrate in the medium, the PL accumulated mainly anthocyanins with earlier retention times, i.e. those which are not retained long in the column. Conversely, in the presence of lower concentrations of ammonium nitrates, the PL accumulated mainly anthocyanins with late retention times, i.e. those which are firmly retained in the column.

It is believed that those anthocyanins displaying peaks of earlier retention times in the reversed-phase HPLC have higher hydrophilicity and lower level of acylation and those displaying peaks of late retention times have lower hydrophilicity and are highly acylated or methylated complex anthocyanins. It is therefore believed that the anthocyanins predominant in the PL of higher ammonium nitrate concentrations displaying the "earlier" peaks are precursors in the biosynthesis of the highly acylated anthocyanins predominant in the tuberous root.

Further, in the chromatograms of PL, there were observed some peaks specific to PL which were not found in the chromatogram of the tuberous root. This suggests the possibility of biosynthesis of certain pigments having high rarity value and suggests also that the control 15 mechanism of anthocyanin biosynthesis differs between the differentiated condition (tuberous root) and the undifferentiated condition In this way, the PL according to the present invention will prove useful for the study of biosynthesis of pigment.

Based on the above-described results, it was found that the 20 chemical composition of the pigment to be accumulated in PL can be S. modified/controlled by varying the ammonium nitrate concentration of the medium. Further investigations were carried out to find out which of nitrate ion or ammonium ion is related to the control of the pigment composition.

Fig. 11 is a graph showing the effect of nitrate ion concentration in the medium on the pigment concentration of the cultured cell of the present invention.

The pigment concentration of PL tended to increase with decrease in the nitrate ion concentration. As for the anthocyanin composition, there was observed no significant difference due to the variation in the nitrate ion concentration. The increase of the anthocyanin pigment concentration due to reduction of nitrate ion concentration was believed to be attributable to activation of the entire biosynthesis pathway.

From the results of these experiments regarding nitrogen components, it was suggested that both nitrate ion and ammonium ion influence activity of anthocyanin biosynthesis pathway in PL, but that the composition of anthocyanin accumulated in PL is controlled by ammonium ion. Accordingly, by using the PL, a desired anthocyanin can be produced through modification of ammonium ion concentration.

[experiment example 3: effect of sucrose]

S*

PL was cultured using the above-described method by using the basal MS medium enriched with sucrose in the following concentrations: .sucrose: 1, 3, 5, 7, 9% The basal MS medium was modified to 2.5 mM in the ammonium nitrate concentration and to 4.7 mM in the potassium nitrate concentration, 20 respectively. No plant growth regulator was added. Basal MS medium o enriched with 3% sucrose was used as control.

Fig. 12 is a graph showing the effect of sucrose concentration in the medium on the pigment concentration and multiplication of the cultured cell of the invention.

As may be seen from this graph, in the range of 1% to 5% of sucrose concentration in the medium, the higher the sucrose concentration, the higher the pigment concentration. The pigment concentration in the PL with 5% of sucrose was about three times greater than that of the tuberous root of Ayamurasaki. However, when the sucrose concentration in the medium exceeded no significant increase was observed in the pigment concentration.

Further, with the sucrose concentration in the medium over 7%, the multiplication of PL was inhibited. Hence, regarding the cell multiplication too, a sucrose should preferably be 1 to Accordingly, in consideration of both the pigment accumulation and multiplication of PL, the concentration of sucrose to be added to the medium should preferably 1 to most preferably The suspension culture of PL in the above-described various experiments were all performed in the dark. The cultured cell line exhibited very high anthocyanin synthesis ability. As described hereinbefore, those cultured cell lines reported so far suffer the cost problem, since they require continuous illumination for biosynthesis of anthocyanin.

Whereas, the PL according to the invention does not require illumination in the establishing process, or for pigment synthesis, hence very advantageous economically.

The invention may be embodied in other specific forms without departing from the spirit or essential characteristics thereof. The present embodiments are therefore to be considered in all respects as illustrative rirr and not restrictive, the scope of the invention being indicated by the appended claims rather than the foregoing description and all changes which come within the meaning and range of equivalency of the claims are therefore intended to be embraced therein.

Claims (9)

1. A method of establishing a cultured cell with high anthocyanin content, the cell requiring no illumination for its multiplication or for anthocyanin accumulation, the method comprising the steps of: inducing callus from a tuberous root of "Ayamurasaki" sweet potato containing anthocyanin, causing differentiation of a tissue thereof having embryogenic ability, thus obtaining an adventive embryoid differentiated tissue; and further inducing, from said adventive embryoid differentiated tissue, a callus having embryogenic ability, then repeatedly selecting a cell line having a higher concentration of anthocyanin; wherein said steps and are effected through tissue culture in the dark. 20

2. A cultured cell having high anthocyanin content 9* 0 and requiring no illumination for its multiplication or for its anthocyanin accumulation, established by a method according to claim 1. 25

3. A cultured cell according to claim 1 or claim 2, wherein said cultured cell requires no plant growth regulator for its multiplication or for its anthocyanin accumulation. 0*0 V 30

4. A cultured cell according to any one of claims 1 to 3, wherein said cultured cell provides various compositions of anthocyanin accumulated therein through modification of nitrogen condition in the medium.

5. A cultured cell according to claim 4, wherein said nitrogen condition in the medium comprises anmmonium ion concentration. H: \KarraR\Xep\speci\Biotech\4255S-C;0. dcc a12/-07if02 26

6. A method of culturing the cultured cell according to any one of claims 2 to 5, wherein said cultured cell is cultured in the dark.

7. A method according to claim 6, wherein said cultured cell is cultured in medium free from any plant growth regulator.

8. A method according to claim 7, wherein said medium comprises basal MS medium supplemented with sucrose as carbon source and modified to contain ammonium nitrate in a concentration of 0 to 2.5 mM.

9. A method according to claim 8, wherein said medium has a sucrose concentration of A method according to claim 1, substantially as herein described with reference to any of the examples and figures. Dated this 24 th day of June 2002. SINDEPENDENT ADMINISTRATIVE INSTITUTE S. NATIONAL AGRICULTURAL RESEARCH ORGANIZATION By their Patent Attorneys GRIFFITH HACK Fellows Institute of Patent and Trade Mark Attorneys of Australia e e *e O iD H\KarraR\Keep\speci\Biotech\42555-OO.doc 24/06/02