www.elsevier.com/locate/foodres

Food Research International 38 (2005) 937–942

Characterization of in vitro anthocyanin-producing sourcherry (Prunus cerasus L.) callus cultures

F. Blando a,*, A.P. Scardino a, L. De Bellis b, I. Nicoletti c, G. Giovinazzo a

a Istituto di Scienze delle Produzioni Alimentari, CNR, Via Monteroni, 73100 Lecce, Italyb DiSTeBA, Universita di Lecce, Centro Ecotekne, 73100 Lecce, Italy

c Istituto di Metodologie Chimiche-CNR, Area della Ricerca di Roma 1, Via Salaria Km 29,300, 00015 Montelibretti, Roma, Italy

Received 28 September 2004; accepted 27 February 2005

Abstract

Anthocyanin-producing callus cultures from in vitro sour cherry (Prunus cerasus L.) leaf tissues were established. As in the parentleaf tissues, the calli extracts showed the synthesis of a prevalent anthocyanin, cyanidin 3-glucoside. When the dark grown calluscultures were exposed to the light, cyanidin 3-glucoside content was increased from 0.1 to 4.5 mg 100 g�1 fresh weight. Thus, ofthe available strategies for the enhancement of pigment production light resulted the triggering factor in this cell system. The addi-tion of 50 lM jasmonic acid to the culture medium stimulated cyanidin 3-glucoside synthesis which resulted in an earlier appearanceof pigment on the calli.� 2005 Elsevier Ltd. All rights reserved.

Keywords: Anthocyanin; Prunus cerasus; Sour cherry; Callus cultures; Jasmonic acid

1. Introduction

Anthocyanins from sour cherry fruit have beenshown to possess antioxidant and anti-inflammatoryproperties (Wang et al., 1999) and to inhibit tumourdevelopment in animal and human cell lines (Kang, See-ram, Nair, & Bourquin, 2003).

The production of anthocyanins from sour cherrycell cultures is attractive since it allows both continu-ous yield from cells biomass and the control of antho-cyanin forms. Anthocyanins from plant tissue cultureshave been successfully obtained in a number of plantspecies, such as carrot (Ozeki & Komamine, 1981),Euphorbia milli (Yamamoto, Kinoshita, Watanabe, &Yamada, 1989) strawberry (Mori, Sakurai, Shigeta,Yoshida, & Konda, 1993), cranberry (Madhavi,Smith, & Berber-Jimenez, 1995), Ajuga reptans (Calle-

0963-9969/$ - see front matter � 2005 Elsevier Ltd. All rights reserved.doi:10.1016/j.foodres.2005.02.014

* Corresponding author. Tel.: +39 0832 422617.E-mail address: federica.blando@ispa.cnr.it (F. Blando).

baut, Terahara, de Haan, & Decleire, 1997), ohelo-berry (Smith & Pepin, 1999), and sweet potato(Konczac-Islam, Yoshinaga, Nakatani, Terahara, &Yamakawa, 2000). In fruit tree species, the productionof anthocyanins from in vitro cells has been reportedfrom Vitis sp. (Decendit & Merillon, 1996; Yama-kawa, Kato, Ishida, Kodama, & Minoda, 1983). Woo-dy species are considered quite recalcitrant in allbiotechnological manipulations including the in vitroproduction of secondary metabolites. Therefore, thedevelopment of an efficient in vitro system to enhancethe production of anthocyanins requires an integratedapproach.

Anthocyanins belong to the flavonoid family whichhas a well-studied pathway. Both biotic and abiotic elic-itors enhance the synthesis of flavonoids in tissue culturesystems (Zhang & Furusaki, 1999). Enhanced anthocya-nin accumulation in response to jasmonates has beenobserved in other plant cell cultures such as Vacciniumpahalae (Fang, Smith, & Pepin, 1999) or Vitis vinifera

938 F. Blando et al. / Food Research International 38 (2005) 937–942

(Zhang, Curtin, Kikuchi, & Franco, 2002). Moreover,jasmonic acid (JA) significantly alters the accumulationof major anthocyanins (Curtin, Zhang, & Franco, 2003;Fang et al., 1999; Plata et al., 2003; Zhang et al., 2002),resulting in a shift towards more methylated and aci-lated anthocyanins.

Here, we report the preliminary results on the estab-lishment of an anthocyanin-producing callus system ob-tained from sour cherry leaf. We studied the growth ofcallus cultures and their anthocyanin production on var-ious solid media in the dark or under light. The influenceof JA on anthocyanin production in this cell system isalso reported.

2. Material and methods

2.1. Plant materials and callus induction

In vitro plants of Prunus cerasus L., (cv AmarenaMattarello) were grown on a Plant MultiplicationMedium (PMM) (Table 1). Callus cultures from theleaf explants were induced on a Callus InductionMedium (A1M0) (Table 1). The pH of all mediawas adjusted to 5.7 with KOH 1 N. The explants werethen incubated in a growth chamber at 25 ± 2 �C inthe dark and the obtained calli were subcultivatedevery 3–4 weeks.

2.2. Biosynthesis induction of anthocyanins

Callus cultures at the end of their growth cycle weretransferred onto different Anthocyanin Induction Media(AIM) (Table 1) under light (Philips TLD/83, 125 lmolm�2 s�1), with a 16-h-light photoperiod. Jasmonic acid(Duchefa, Haarlem, The Nederland) was dissolved as re-

Table 1Composition of the media used in the experiments

Media Salts and vitamins Sucrose (g L�1) Gro

NAA

PMM MSa 30 –AIM0 MS 30 1AIM1 MS 50 1AIM2 MS 70 1AIM3 MS (NH4NO3 free) 30 1AIM4 MS (NH4NO3 free) 50 1AIM5 MS (NH4NO3 free) 70 1AIM6 MS (50% NH4NO3 and KNO3) 30 1AIM7 MS (50% NH4NO3 and KNO3) 50 1AIM8 MS (50% NH4NO3 and KNO3) 70 1AIM9 MS 30 1

a MS basal medium (Murashige and Skoog, 1962) supplemented with.b Naftalen-acetic Acid (NAA).c 6-Benzylaminopurine (6-BAP).d Indol-butirric acid (IBA).e Jasmonic acid (JA).

ported by Zhang et al., 2002 and added to the mediumat day 0. All growth regulators were filter sterilizedand then added to the autoclaved media.

2.3. HPLC analysis of total anthocyanins

Anthocyanins were extracted from calli according tothe sample extraction procedure previously published(Blando, Gerardi, & Nicoletti, 2004). Chromatographicanalyses were performed on an HPLC system consistingof an SCL-10AVP system controller, two LC-10ADVP

solvent delivery units; an on-line DGU-14A vacuummembrane degasser, a CTO-AS10VP column oven, anSPD-10AVP UV–VIS photodiode array detector, con-nected to an LC workstation Class VP 5.3 (all from Shi-madzu, Milan, Italy). The column employed was aPolaris C18A (150 · 2.0 mm I.D.; 5 lm) from Varian(Palo Alto, CA, USA) equipped with a C18 guard col-umn. The samples were introduced onto the columnvia a model 9125 Rheodyne injection valve with a5 lL loop (Cotati, CA, USA). All samples were analysedusing a gradient method as described by (Blando et al.,2004). The anthocyanin were identified by comparingtheir retention times and UV–VIS spectra (in the range230–600 nm), with those of authentic compounds. Peakpurity was checked to exclude any contribution frominterfering peaks.

3. Results and discussion

3.1. Callus culture establishment and anthocyanin

induction by light exposure

Leaf tissue from axenic cultured cherry plantlets wasanalyzed for its anthocyanins content before starting

wth regulators

b (mg L�1) IBAc (mg L�1) BAPd (mg L�1) JAe (lM)

0.1 0.5 –– 0.1 –– 0.1 –– 0.1 –– 0.1 –– 0.1 –– 0.1 –– 0.1 –– 0.1 –– 0.1 –– 0.1 50

0

2

4

6

8

0 10 20 30 40 50days of culture

wei

ght (

g FW

)

light dark

Fig. 2. Comparison of growth curves of sour cherry callus culturesmaintained in the dark and under light on the same medium (AIM0),FW: fresh weight. Vertical bars represent standard deviation (n = 3).

F. Blando et al. / Food Research International 38 (2005) 937–942 939

callus culture. The anthocyanin content in mature leavesof cherry plantlets (cv Amarena Mattarello) was deter-mined by HPLC analysis of methanolic extracts andcompared with the anthocyanin content of fruit tissuesof the same cultivar (Fig. 1). As shown in Fig. 1, themain anthocyanin detected in leaves, cyanidin-3-gluco-side, was not significantly synthesised in the mature fruittissues, cyanidin-3-glucosylrutinoside (2) and cyanidin-3-rutinoside (4) being the main anthocyanin forms. Thisfinding indicates that the end products of anthocyaninpathway in leaf and fruit are significantly different. Thismay reflect their physiological roles in the various or-gans of the plant.

Leaf explants cultured in the dark on A1M0 medium(see M.M.) produced an abundant, friable and whitecallus from nearly 100% of the explants within 3 weeks.When these newly established cherry callus cultures wereanalyzed for anthocyanin content, they revealed anaverage anthocyanin content similar to that measuredfrom the starting leaf tissue. As shown in the curve ofFig. 2, these calli reached the plateau phase in 30 days.

In order to investigate nutritional conditions whichaffect the yield of anthocyanin, cherry calli were subcul-tured in the dark on media, containing various combina-tions of growth regulators, sucrose concentration, saltsand vitamins (Table 1). No significant differences werefound either in pigment production or in cell growth(data not shown).

Since anthocyanins synthesis in higher plants is stim-ulated by light (Sato, Nakayama, & Shigeta, 1996; Tak-eda, 1990), cherry callus cultures were transferred underlight. The results obtained indicate that cell pigmenta-tion was clearly stimulated by the light. Pigmentation

Minutes

0 2 4 6 8 10

mA

U

010

020

030

040

050

060

0

1 A

B

Fig. 1. HPLC analysis of methanolic extracts from sour cherry fruit (P. ceraswere identified on the basis of the retention time of the authentic standaglucosylrutinoside, (3) cyanidin-3-glucoside, (4) cyanidin-3-rutinoside.

appeared on the surface of the calli, 4–5 days after expo-sure and reached a maximum spreading in 10–15 dayswith no significant differences linked to media composi-tion (data not shown). Anthocyanin accumulation dueto the light did not have any dramatic effect on calligrowth as shown in Fig. 2.

Therefore our results suggest that in cherry calli, un-der our experimental conditions, the induction of antho-cyanin biosynthesis is triggered by light exposure ratherthan by media composition.

Similar results have been already reported in carrotand grape cell cultures (Takeda, 1990; Zhang et al.,2002). Light irradiation was shown to induce theexpression of key enzymes of the phenylpropanoidmetabolic pathway, such as phenylalanine ammonia-lyase (PAL) and chalcone synthase (CHS), at the

12 14 16 18 20

0

100

200

300

400

500

600

2

3

4

3

mA

U

us L.) (A) and leaf tissues (B) recorded at 518 nm. The following peaksrds: Identification peaks: (1) cyanydin-3-sophoroside, (2) cyanidin-3-

940 F. Blando et al. / Food Research International 38 (2005) 937–942

transcription level in the carrot cell culture system(Takeda, 1990). To verify the trigger of the synthesisof CHS in cherry calli, we carried out western blotanalysis on total protein extracts from light exposedcalli (0–7 days) using a specific CHS antibody. Thisanalysis revealed that CHS content progressively in-creased under light (data not shown). This in agree-ment with other reports showing that secondarymetabolism in several plant species is controlled bythe photosensory/photoregulation system (Zhanget al., 2002).

3.2. Effects of jasmonic acid on anthocyanin biosynthesis

in cherry calli

It is well known that JA and its derivatives enhancethe synthesis of defence compounds in plants (Gund-lach, Muller, Kutchan, & Zenk, 1992; Tamari, Boroc-

Fig. 3. Sour cherry callus cultures grown on AIM0 medium additioned withdays.

Mi

5 10 15 20

mA

U

0

200

4

00

1

2

3

4

5

6

A

B

Fig. 4. Chromatographic profile of HPLC separation of anthocyanins fromanthocyanins were compared with a mix of standard (B). In figure are ranthocyanin on the total amount.

hov, Atzorn, & Weiss, 1995). Moreover, it has beenreported that the combined action of JA and light im-proves the anthocyanin production in grape cell cultures(Zhang et al., 2002).

To verify the effect of JA on anthocyanin synthesisand accumulation in P. cerasus cultures, different con-centrations of JA (10, 20, 50 and 100 lM) were addedto the medium (A1M0) on day 0. Similar levels of cellgrowth and pigment accumulation were observedusing JA concentrations ranging from 10 to 50 lM.At a higher concentration (100 lM) an inhibitory ef-fect on anthocyanin synthesis was observed (datanot shown). At 50 lM, JA was able to more rapidlyinduce the synthesis of anthocyanin. The typical redspots appeared in 48 h, and the red pigmentationwas complete in 7 days (Fig. 3). However, the calluscultures grown in the presence of JA and light,reached the plateau phase earlier than those grown

50 lM JA, at different times of light exposure: (left) 2 days, (right) 7

nutes

25 30 35 40

7

Anthocyanins Content(%)

1 Cyanidin 3,5-diglucoside 1.4 2 Cyanidin 3-glucoside 86 3 Cyanidin 3-rutinoside 6.6 4 Delphidin - 5 Peonodin 3-glucoside 4.56 Malvidin 3-glucoside 1.4 7 Cyanidin -

15-day-old cherry callus grown under light (A). The retention time ofeported the number of identification peaks and the percent of each

Table 2Amount of cyanidin 3-glucoside produced by cherry leaf tissues and 15days-old cherry calli maintained in different condition of induction.The anthocyanin was determined by HPLC, the content of pigment indark callus and leaf tissue is similar. Values are the means ± standarderror of at least three experiments

Sources Cyanidin 3-glucoside (mg 100 g�1 FW)

Cherry leaf 0.10 ± 0.001Dark callus 0.12 ± 0.001Light callus 4.50 ± 0.08Light + JA callus 5.30 ± 0.07

F. Blando et al. / Food Research International 38 (2005) 937–942 941

without this elicitor (about 15–20 days, data notshown).

3.3. Analysis of anthocyanins in cherry callus cultures

We observed a clearly increased reddish-purpleanthocyanin pigmentation in calli grown under lightcompared with calli maintained in the dark. In orderto compare the anthocyanins produced by cherry calligrown under different conditions (darkness, light, lightplus JA) we carried out a qualitative and quantitativeanalysis by HPLC. No significant differences were foundin the anthocyanin composition synthesized by leaf andcalli grown under the different conditions (Figs. 1 and4), cyanidin 3-glucoside (3) being the major anthocyanin(86% of total anthocyanin) (Fig. 1). A quantitative anal-ysis, revealed that cherry calli exposed to light for 15days, accumulated about 40-fold higher levels of cyani-din-3-glucoside than calli and leaf tissue grown in thedark (4.5 mg 100 g�1 FW versus 0.1 mg 100 g�1 FW,see Table 2). A slight increase (5.3 mg 100 g�1 FW) inthe synthesis of this antocyanin was measured in meth-anolic extract from red calli at fifteen days of cultivationin the presence of 50 lM JA under light (Table 2). Fur-ther analysis to define the time course of anthocyaninproduction during callus growth in this conditions arein progress.

4. Conclusions

We attempted to establish a high pigment-accumulat-ing sour cherry callus culture to set up a suitable andreliable induction system of anthocyanin production ina cell suspension culture. The occurrence of only onemajor anthocyanin compound in cherry calli cultures re-veals the ability of the in vitro cell system to produce aspecific metabolite with interesting biological functionswhich reflects the typical metabolic pattern of the start-ing tissue. It has been reported that although the meta-bolic flux of in vitro systems is often simplified it couldbe steered towards the accumulation of specific com-pounds with interesting characteristics (Caretto et al.,

2004; Plata et al., 2003). Thus providing a useful, naturalfood colorant and/or a functional food ingredient.

Acknowledgements

The authors are grateful toDr.Angelo Santino for crit-ical reading of the manuscript and helpful suggestions.We thank Sig. Leone D�Amico for technical support.

References

Blando, F., Gerardi, C., & Nicoletti, I. (2004). Sour cherry (Prunuscerasus L.) anthocyanins as ingredients for functional foods.Journal of Biomedicine and Biotechnology, 5, 253–258.

Callebaut, A., Terahara, N., de Haan, M., & Decleire, M. (1997).Stability of anthocyanin composition inAjuga reptans callus and cellsuspensioncultures.PlantCell TissueandOrganCulture, 50, 195–201.

Caretto, S., Speth, B., Fachechi, C., Gala, R., Zacheo, G., &Giovinazzo, G. (2004). Bray Enhancement of vitamin E productionin sunflower cell cultures. Plant Cell Reports, 23, 174–179.

Curtin, C., Zhang, W., & Franco, C. (2003). Manipulating anthocy-anin composition in Vitis vinifera suspension cultures by elicitationwith jasmonic acid and light irradiation. Biotechnology Letters, 25,1131–1135.

Decendit, A., & Merillon, J. M. (1996). Condensed tannin andanthocyanin production in Vitis vinifera cell suspension cultures.Plant Cell Reports, 15, 762–765.

Fang, Y., Smith, M. A. L., & Pepin, M-F. (1999). The effects ofexogenous methyl jasmonate in elicited anthocyanin-producing cellcultures of ohelo (Vaccinium pahalae). In Vitro Cellular and

Development Biology – Plant, 35, 106–113.Gundlach, H., Muller, M. J., Kutchan, T. M., & Zenk, M. H. (1992).

Jasmonic acid is a signal trasducer in elicitor-induced plant cellcultures. Proceedings of the National Academy of Sciences of the

United States of America, 89, 2389–2393.Kang, S. Y., Seeram, N. P., Nair, M. G., & Bourquin, L. D. (2003).

Tart cherry anthocyanins inhibit tumor development in Apcmin

mice and reduce proliferation of human colon cancer cells. CancerLetters, 194, 13–19.

Konczac-Islam, I., Yoshinaga, M., Nakatani, M., Terahara, N., &Yamakawa, O. (2000). Establishment and characteristics of ananthocyanin-producing cell line from sweet potato storage root.Plant Cell Reports, 19, 472–477.

Madhavi, D. L., Smith, M. A. L., & Berber-Jimenez, M. D. (1995).Expression of anthocyanins in callus cultures of cranberry (Vac-cinum macrocarpa Ait.). Journal of Food Science, 60, 351–355.

Mori, T., Sakurai, M., Shigeta, J., Yoshida, K., & Konda, T. (1993).Formation of anthocyanins from cell cultured from different partsof strawberry plants. Journal of Food Science, 58, 788–792.

Murashige, T., & Skoog, F. (1962). Revised medium for rapid growthand bioassays with tobacco tissue cultures. Physiologia Plantarum,

15, 473–497.Ozeki, Y., & Komamine, A. (1981). Induction of anthocyanin

synthesis in relation to embryogenesis in a carrot suspensionculture: correlation of metabolic differentiation with morphologicaldifferentiation. Physiologia Plantarum, 53, 570–577.

Plata, N., Konczak-Islam, I., Jayram, S., McClelland, K., Woolford,T., & Franks, P. (2003). Effect of methyl jasmonate and p-coumaricacid on anthocyanin composition in a sweet potato cell suspensionculture. Biochemical Engineering Journal, 14, 171–177.

Sato, K., Nakayama, M., & Shigeta, J. (1996). Culturing conditionsaffecting the production of anthocyanin in suspended cell culturesof strawberry. Plant Science, 113, 91–98.

942 F. Blando et al. / Food Research International 38 (2005) 937–942

Smith, M. A. L., & Pepin, M-F. (1999). Stimulation of bioactiveflavonoid production in suspension and bioreactor-based cellcultures. In A. Altman et al. (Eds.), Plant biotechnology and in

vitro biology in the 21st century (pp. 333–336). Kluwer AcademicPublishers.

Takeda, J. (1990). Light-induced synthesis of anthocyanin in carrotcells in suspension-II. Effects of light and 2,4-D on induction andreduction of enzyme activities related to anthocyanin synthesis.Journal of Experimental Botany, 41, 749–755.

Tamari, G., Borochov, A., Atzorn, R., & Weiss, D. (1995). Methyljasmonate induces pigmentation and flavonoid gene expression inpetunia corollas: a possible role in wound response. PhysiologiaPlantarum, 94, 45–50.

Wang, H., Nair, M. G., Strasburg, G. M., Chang, Y. C., Booren, A.M., Gray, J. I., et al. (1999). Antioxidant and antiinflammatory

activity of anthocyanins and their aglycon, cyanidin, from tartcherries. Journal of Natural Products, 62, 294–296.

Yamakawa, T., Kato, S., Ishida, K., Kodama, T., & Minoda, Y.(1983). Production of anthocyanin by Vitis cells in suspensionculture. Agricultural and Biological Chemistry, 47, 2185–2191.

Yamamoto, Y., Kinoshita, Y., Watanabe, S., & Yamada, Y. (1989).Anthocyanin production in suspension cultures of high-producingcells of Euphorbia milli. Agricultural and Biological Chemistry, 53,417–423.

Zhang, W., & Furusaki, S. (1999). Production of anthocyanins by plantcell cultures. Biotechnology Bioprocess Engeneering, 4, 231–252.

Zhang, W., Curtin, C., Kikuchi, M., & Franco, C. (2002). Integrationof jasmonic acid and light irradiation for enhancement of antho-cyanin biosynthesis in Vitis vinifera suspension cultures. Plant

Science, 162, 459–468.