Plant Cell Reports (2000) 19 : 472477

Q Springer-Verlag 2000

I. Konczak-Islam 7 M. Yoshinaga 7 M. Nakatani N. Terahara 7 O. Yamakawa

Establishment and characteristics of an anthocyanin-producing cell line from sweet potato storage root

Received: 8 January 1999 / Revision received: 2 March 1999 / Accepted: 30 June 1999

Abstract Anthocyanin pigments accumulated in a cell line derived from storage-root explants of sweet potato (Ipomoea batatas L.) cv Ayamurasaki. Somatic proembryos were induced on the explants cultured on Murashige and Skoog medium supplemented with 1 mg/l 2,4-D. The pro-embryo structures produced callus when transferred to MS medium with 0.5 mg/l 2,4-D. A cell line was isolated from this callus which accumulated anthocyanin pigment. The color value of the pigment extracted after 27 days of culture in MS medium with 2 mg/l 2,4-D was 8.2, which was very close to that of a pigment extracted from roots, which was 8.9. Most of the pigments from the cell extract were hydrophilic and appeared on the ODS-column HPLC with a lower retention time than the main anthocyanins of the root tissues. The majority of the pigments were identical with the root anthocyanins. Cell line-specific anthocyanins were detected. Key words Ipomoea batatas 7 Callus culture 7 Anthocyanin composition Abbreviations MS: Murashige and Skoog (1962)

Introduction
The deep-purple color of the flesh of sweet potato (Ipomoea batatas L.) cultivar Ayamurasaki storage root makes it an attractive source of natural food colorants (Yamakawa et al. 1997). The main pigments that accumulate in the storage roots of Ayamurasaki plants are anthocyanins with aromatic acyl groups (Odake et al. 1992; Goda et al. 1997; Terahara et al. 1999). These anthocyanins possess a high thermostability (Odake et al. 1994) and contribute towards antioxidative activity (Furuta et al. 1995) and the hepato-protective activity of purple-colored sweet potato juice (Suda et al. 1997). According to Kamei et al. (1993) anthocyanins inhibit the growth of human cancer cells. Therefore, the addition of natural anthocyanins as food colorants would not only enhance the decorative value of the food but also improve its beneficial properties. Pigment produced by a field-grown storage root is a natural source of food colorants. However, there are some disadvantages, like seasonality, large plantation requirements, extended period of time to produce the source, high transport and labor costs, etc. Colorant production in a factory setting based on an efficient tissue culture system would overcome these disadvantages. Tissue culture techniques have been successfully applied to induce the biosynthesis of anthocyanins in vitro from different plant materials, such as grape (Decendid and Mrillon 1996),Vitis sp. (Yamakawa et al. 1983), carrot (Ozeki and Komamine 1981), strawberry (Mori et al. 1993), Euphorbia milli (Yamamoto et al. 1989), cranberry (Madhavi et al. 1995) and Ajuga reptans (Callebaut et al. 1997). With respect to sweet potato research a few reports have been published on cultures originating from storage-root explants. The initiation of shoots and roots (Carswell and Locy 1984), plantlet regeneration from friable callus induced from storage-root explants (Murata et al. 1989) and shoot regeneration from

Communicated by T. Yoshikawa I. Konczak-Islam 7 M. Yoshinaga 7 M. Nakatani (Y) O. Yamakawa Laboratory of Upland Crop Genetic Resources, Department of Upland Farming, Kyushu National Agricultural Experiment Station, 6644 Yokoichi, Miyakonojo, Miyazaki 885-0091, Japan e-mail: mnakatan6mykz.affrc.go.jp N. Terahara Department of Food Science and Technology, College of Horticulture, Minami-Kyushu University, Takanabe, Miyazaki 884-0003, Japan Present address: I. Konczak-Islam Food Science Australia, 16 Julius Avenue, Riverside Corporate Park, Delhi Road (PO Box 52), North Ryde NSW 2113

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meristematic bud-like centers have been obtained (Hwang et al. 1983). Cell cultures of sweet potato have also been used for studies on the production of primary or secondary metabolites. Sasaki and Kainuma (1984) reported starch regulation and exocellular polysaccharide biosynthesis in sweet potato suspension culture. Nozue et al. (1987) selected an anthocyanin-accumulating cell line from friable callus originating from the storage root of white-fleshed sweet potato cv Kintoki. The selected cell line accumulated pigment only under continuous illumination. The recently developed, purple-fleshed cultivar Ayamurasaki with an outstanding quality to accumulate pigment in the storage root seems to be a suitable plant material for the development of intensively pigmented cultures. Therefore, we attempted to establish a high pigmentaccumulating cell line suitable for the development of an efficient technological procedure for colorant production.

7-day-old subcultures by transferring 0.1 g of callus aggregates into 50-ml Erlenmayer flasks containing 10 ml MM. The samples (four replications) were harvested in 3-day intervals for a period of 30 days (growth period). A growth measurement was obtained by removing the aggregates from the medium and washing them with a 3% sucrose solution. Subsequently, they were separated from the liquid by filtration under vacuum and weighed. The growth index was defined as W/Wo, where Wo and W denote aggregates fresh weight before and after the cultivation, respectively.

Extraction of anthocyanins Fresh cells were ground and steeped in 50% acetic acid for 1 h. Five randomly selected storage roots were chopped up and steeped overnight in 50% acetic acid for the storage root samples. The volume of acetic acid solution was adjusted to an equivalent of 20 times that of the sample weight. The samples were centrifuged at 10000 rpm for 10 min. The supernatants were used for anthocyanin identification and quality analysis.

Materials and methods

Plant material Explants were prepared from the storage roots of sweet potato (Ipomoea batatas L.) cv Ayamurasaki maintained for 6 months after harvest at 13 C. Storage roots were scrubbed clean under running tap-water and submerged in 10% commercial bleach solution for 30 min. After being rinsed three times with sterile distilled water the skin (0.5-cm layer) was peeled off under aseptic conditions; from the remaining tissue small cubes (10!10!5 mm) were prepared. Callus culture Callus was developed from somatic pro-embryo structures induced on the surface of the storage-root explants. For proembryo induction the explants were exposed to MS basal medium enriched with the auxin 2,4-dichlorophenoxyacetic acid (2,4-D) and cytokinin 6-benzylaminopurine (BA) in concentrations ranging from 0 to 4 mg/l. Seven explants were placed in a petri dish (10-cm diameter). At least five plates per treatment were prepared. The experiments were repeated twice. The cultures were incubated at 27 7C in the dark with unmonitored light interruptions during the daily observations. The pro-embryos were subsequently isolated from the explants and exposed to MS basal medium supplemented with 2,4-D in concentrations of 0, 0.5, 1, 2 and 3.0 mg/l for callus induction. Only the compact callus selectively multiplied at 2,4-D concentrations of 0, 1, 2 and 3 mg/l in MS medium were further tested. The cultures were subcultured every 3 weeks. In the process of callus multiplication mottled-white, red and purple aggregates were obtained. These were used for the selection of an intensively pigmented purple cell line (PL). Suspended cell cultures were initiated by transferring about 1 g (fresh weight) of callus to 25 ml of liquid medium in 100-ml flasks. Basal MS medium supplemented with 2 mg/l 2,4-D was used as the multiplication medium (MM). The cultures were incubated on a rotary shaker (120 rpm) at 27 7C in the dark. The medium was changed weekly. Determination of growth In order to obtaining a time course of PL suspension culture growth and anthocyanin accumulation, we initiated cultures from

Anthocyanin identification and high-pressure liquid chromatography (HPLC) analysis The supernatant diluted fourfold with a McIlvaines buffer solution and pH adjusted to 3 was used for the measurement of the optical densities at 530 nm with spectrophotometer CS-9300PC (Shimadzu, Japan). A color value (CV) for the pigment extract, which is a commercial indicator of total anthocyanins, was calculated using the following formula: CVp0.1!OD530!4!20/1gfw, where OD530 is a spectrophotometric reading at 530 nm, 4 and 20 are the levels of dilution, and fw is tissue fresh weight (Shimizu and Nakamura 1993). For HPLC analysis the crude extract was vacuum-dried. The residue was redissolved in 15% acetic acid and filtered through a 0.2-mm filter membrane (DISMIC-13cp, Advantec, Japan). HPLC analysis was performed on an L-6200 Intelligent Pump (Hitachi, Japan), L-4200 UV-VIS Detector, D-2000 Integrator and 655A-52 Column oven (Hitachi, Japan). Analytical HPLC was run on Luna [3 m C18(2), 4.6 id!100 mm Phenomenex, USA] column at 35 7C and monitored at 520 nm. The following solvents in water with a flow rate of 1 ml/min were used: (A) 1.5% phosphoric acid, and (B) 1.5% phosphoric acid, 20% acetic acid, 25% acetonitrile. The elution profile was a linear gradient elution for B15%r35% for 100 min in solvent A. Spectrofluorometric detector Shimadzu RF-10A was used for the spectral analysis of peak YGM-0a. The identification of anthocyanins was carried out by comparing the peaks with standard peaks of purple-fleshed sweet potato YGM anthocyanins: YGM-1a [cyanidin 3-(6,6b-caffeylp-hydroxybenzoylsophoroside)-5-glucoside], YGM-1b [cyanidin 3-(6,6b-dicaffeylsophoroside)-5-glucoside], YGM-2 [cyanidin 3-(6-caffeylsophoroside)-5-glucoside], YGM-3 [cyanidin 3-(6,6b-caffeylferulylsophoroside)-5-glucoside], YGM4b [peonidin 3-(6,6b-dicaffeylsophoroside)-5-glucoside], YGM-5a [peonidin3-(6,6b-caffeylp-hydroxybenzoylsophoroside)-5-glucoside], YGM-5b [cyanidin 3-(6-caffeylsophoroside)-5-glucoside] and YGM-6 [peonidin 3-(6,6b-caffeylferulylsophoroside)-5-glucoside] according to Odake et al. (1992), Goda et al. (1997) and Terahara et al. (1999). The peak YGM-0a was identified by cochromatography with a cyanidin 3-sophoroside-5-glucoside standard isolated from Ajuga reptans (Terahara et al. 1996). For the data presented in Table 2 the HPLC analysis was performed using a crude pigment extract according to Odake et al. (1992) on a Shimazu LC-9A liquid chromatograph equipped with an Inertsil ODS-2 column (250!4.6 mm, GL Sciences). Anthocyanins were separated by the linear gradient elution for 40 min from 25% to 85% solvent B in solvent A and monitored at 530 nm.

474 Table 1 Effect of 2,4-D and BA on callogenesis and somatic pro-embryo development from storage-root explants of Ipomoea batatas L. cv Ayamurasaki in the dark at 270 C (FC friable callus, SP somatic pro-embryo) BA/2.4-D (mg/l) 0 0.5 1
a

0 FC 28.5 a 85.7 85.7 SP 0 0 0

1 FC 100 95.2 95.2 SP 10 10 5

2 FC 71.4 85.7 100 SP 5 2 0

4 FC 61.9 80.9 85.7 SP 0 0 0

Each numerical value represents the percentage of explants that formed callus or somatic pro-embryo structures

Results and discussion

Induction of callus and establishment of an anthocyanin-producing cell line Somatic pro-embryos appeared on the surface of storage-root explants after 5 to 10 weeks of culture on MS medium enriched with 1 mg/l 2,4-D. The frequency of pro-embryo formation was low (Table 1), and increasing 2,4-D or BA concentrations suppressed their development. Hwang et al. (1983) reported the induction of meristematic bud-like centers (MBLC) from storage-root explants of selected sweet potato plants. Similar structures were observed in the sweet potato explants obtained in the present investigation. Pro-embryos subcultured on MS medium supplemented with 0.5 mg/l 2,4-D developed two different types of calli: one was slow-growing, compact and slightly pigmented and the other fast-growing, white and friable. For selective multiplication the compact callus was isolated and exposed to MS medium containing 0, 1, 2, and 3 mg/l 2,4-D. The increase in 2,4D concentration to 3 mg/l suppressed differentiation and enhanced callus growth. At the same time the growth of the white, friable callus was strongly suppressed. The compact callus cultivated on MS medium enriched with 3 mg/l 2,4-D in the dark produced mottled white, red and purple aggregates in 3 months.
Fig. 1A,B Growth of suspension culture (A) and changes in pigment accumulation (B) in the PL cell-line originating from the storage-root of Ipomoea batatas (L.) cv Ayamurasakib, maintained in MS medium with 2 mg/l 2,4-D

After selective subculture for 1 year a cell line consisting of highly pigmented purple aggregates (PL) was established. Growth and pigment accumulation in the PL suspension culture Figure 1 gives the growth rate and pigment accumulation of the PL suspension culture grown in MM in the dark. The culture was analyzed 3 months after initiation. It exhibited a typical growth curve with an induction period of about 9 days and a subsequent exponential phase between days 9 and 27 (Fig. 1A). Cell fresh weight increased about 1.3-fold during the first 9 days and about 3.5-fold during the whole growth period. Pigment accumulation in the culture occurred mainly during the first 2 weeks (Fig. 1B), when the CV increased from 3.9 at the start of the growth period to 7.6 on the 15th day. From the 15th day until the 27th day there were no significant differences in anthocyanin concentration. The highest increase in the anthocyanin concentration in culture was associated with a low growth rate. At the exponential growth phase, the pigment concentration was constant. A similar magnitude of anthocyanin accumulation has been reported for Vitis vinifera cell suspension culture (Decendid and Mrillon 1996). At the end of the growth period the average CV of the pigment extract reached 8.2 and was very close to that of the storage-

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root extract: 8.9 (Yamakawa et al. 1997). This indicates, that the total anthocyanin biosynthesis ability of PL suspension culture was similar to that of storage root. However, the cell line produced anthocyanins while being maintained on the MM medium in the presence of auxin 2,4-D. Ozeki and Komamine (1981) reported that the presence of 2,4-D in the medium suppresses or eliminates anthocyanin accumulation in the tissue. At the end of the growing period the color value varied from 7 to 10, and there were individual cell aggregates of different color intensities. While the cell line used in the present research accumulated anthocyanins basically in the dark, the majority of cell culture systems require continuous illumination for anthrocyanin accumulation (Mori et al. 1993; Matasumo et al. 1973; Yamamoto et al. 1989; Madhavi and Smith 1995; Nozue et al. 1987).

Pigment composition The PL suspension culture produced a complex mixture of anthocyanins that differed in composition from that of the storage root (donor tissue). Figure 2 shows reversed phase-HPLC-chromatograms of PL maintained for 9 days on MM and the storage-root. The major anthocyanins detected in the storage-root extract were YGM-4b, -5a, -5b and -6. These anthocyanins were identified as peonidin 3-sophoroside-5-glucosides acylated with p-hydroxybenzoic and/or caffeic and/or ferulic acid (Odake et al. 1992; Goda et al. 1997; Terahara et al. 1999). YGM-4b and -6 comprised about 72% of the total anthocyanins as calculated by peak area. The second-largest group of anthocyanins observed in the storage-root extract were YGM-1a, -1b, -2 and -3 (cyanidin 3-sophoroside-5-glucosides with the same acylation pattern; Odake et al. 1992; Terahara et al. 1999). YGM-1a an -3 made up about 15% of the total anthocyanins as calculated by peak area. Only a few of YGM-1 and -6 pigments were detected on the chromatogram of the PL extract, and they were present in a very low amount. Among them the cyanidin-based pigments dominated, YGM-1a, -1b, -2 and -3 at amounts of 0.8%, 0.3%, 3.0% and 1.6%, respectively. Of the peonidin-based pigments only YGM-5b and -6 were detected in amounts of 0.9% and 0.4% of the total anthocyanins as calculated by peak area. Contrary to the storage-root chromatogram, the main peaks in the PL chromatogram appeared with earlier retention times, the majority being identical with the early minor peaks of the storage root (YGM-0a,- 0b, -0c, -0d, -0e, -0f, -0g and -0i). The culture accumulated a high amount of an early pigment, YGM-0a. According to the spectral analysis this pigment appeared as an anthocyanin with a maximum absorption at 512 nm (Fig. 3). By co-chromatography with an anthocyanin isolated from Ajuga reptans as a standard YGM-0a was identified as cyanidin 3-sophoroside-5-glucoside.

Fig. 2 HPLC chromatogram of the PL cell-line at 9 days and of the storage-root of sweet potato (Ipomoea batatas L.) cv Ayamurasaki. The characters in the chromatogram are the YGM numbers of sweet potato anthocyanins according to Odake et al. (1992), Goda et al. (1997) and Terahara et al. (1999)

Fig. 3 Absorption spectrum of anthocyanin YGM-0a

The peaks which appeared with early retention time on ODS-column HPLC are more hydrophilic and seemed to have a simpler structure than the highly acylated YGM-1-6, suggesting that the early pigments might be precursors for the biosynthesis of highly acylated YGM-16. Our spectral data indicate that on the PL chromatogram some cell line-specific

476 Table 2 Anthocyanin composition of PL cell line during one growth period on MM medium Peak Percentage of total anthocyanin calculated from the peak area at 530 nm Day 3 YGM0a YGM0a YGM0b YGM0c YGM0d YGM0e YGM0f YGM0f YGM0 g YGM0g YGM0i YGM1a&b YGM 2 YGM 3&3b YGM 5a&b YGM 6 YGM7a YGM7f
a b

Day 9 12.4B0.6 3.8B0.3 5.7B0.8 3.5B0.9 3.8B1.6 1.4B0.2 3.4B0.2 21.3B1.5 13.2B0.6 4.6B0.4 3.8B0.5 T 2.2B0.7 4.5B0.7 2.0B0.4 1.9B0.4 3.4B0.5 3.1B0.5

Day 15 17.7B0.7 5.1B0.7 6.9B0.5 4.9B0.6 6.2B0.6 1.1B0.1 2.6B0.1 18.4B0.9 12.8B1.5 3.4B0.5 3.1B0.5 1.4B0.2 3.1B0.5 3.2B0.4 1.3B0.7 1.2B0.2 2.4B0.6 2.4B0.5

Day 21 16.4B0.7 5.0B0.2 8.1B0.3 4.9B0.2 6.4B0.4 1.3B0.2 2.6B0.1 15.2B0.7 14.4B0.6 3.1B0.4 3.8B0.2 1.5B0.1 3.6B0.4 3.5B0.2 2.2B0.3 1.4B0.1 2.0B0.1 1.2B0.1

Day 27 14.9B2.5 4.8B0.1 7.9B0.2 4.3B0.5 5.4B0.5 1.2B0.1 2.9B0.1 15.4B0.9 15.0B1.5 3.3B0.4 4.2B1.0 1.1B0.1 3.0B0.4 3.4B0.1 2.4B0.3 1.6B0.4 2.7B0.7 T

10.0B0.6 a 3.3B0.5 5.3B0.5 1.3B0.1 3.2B0.8 Tb 3.2B0.3 23.0B1.6 14.6B1.4 5.0B0.3 4.8B0.2 Tb 2.8B0.8 6.2B0.6 1.9B0.2 2.5B0.6 4.4B0.6 3.4B1.4

Standard deviation of three independent determinations T, Peak area less then 1%

anthocyanin peaks appeared YGM-0ab, -0fb, -0h and -3b. The presence of additional peaks in the PL cell-line chromatogram may suggest that the regulatory mechanisms for anthocyanin biosynthesis might be different under differentiated (storage root) and undifferentiated (suspension cultured cells) states. Table 2 shows the evolution in anthocyanin composition of the PL suspension culture grown in the dark during one growth period. The composition appeared to be relatively constant during the whole growth period. The pigments YGM-0a, -0b, -0f and -0g dominated and made up about 50% of the total anthocyanins as calculated by peak area. Small increases in the relative concentrations of YGM-0a, -0b, -0c and -0d were observed during the first 15 days of the growth period. These increases were concomitant with decreases in the relative concentrations of the anthocyanins YGM-0fb, -0gb, -3&3b, -7a and -7f. No quantitative or qualitative differences were observed between the 15th and 27th day of growth period. This result suggests that gradual depletion of a nutrient in the medium did not induce major changes in the anthocyanin composition of the PL suspension culture. Similarly, no changes in the intracellular concentrations of the major anthocyanins during one growth cycle were reported for Ajuga reptans (Callebaut et al. 1997). Further investigations are in progress to regulate and intensify the accumulations of anthocyanins in vitro and to identify new pigments detected in the PL extract.
Acknowledgments Funding of this project through an STA Postdoctoral fellowship to Izabela Konczak-Islam by the Japan International Science and Technology Exchange Center is gratefully acknowledged.

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