Food Chemistry 122 (2010) 475–481

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Food Chemistry

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Rapid Communication

Effect of grape genotype and tissue type on callus growth and productionof resveratrols and their piceids after UV-C irradiation

Wen Liu a,b, Chunyan Liu a, Chunxiang Yang a, Lijun Wang a, Shaohua Li a,c,*

a Institute of Botany, Chinese Academy of Sciences, Wuhan 430074, PR Chinab Graduate School of Chinese Academy of Sciences, Beijing 100049, PR Chinac Key Laboratory of Plant Germplasm Enhancement and Speciality Agriculture, Wuhan Botanical Garden, The Chinese Academy of Sciences, Wuhan 430074, PR China

a r t i c l e i n f o a b s t r a c t

Article history:Received 22 October 2009Received in revised form 25 January 2010Accepted 11 March 2010

Keywords:StilbeneCallusUV-C irradiationGenotypeTissue type

0308-8146/$ - see front matter � 2010 Elsevier Ltd. Adoi:10.1016/j.foodchem.2010.03.055

* Corresponding author at: Institute of Botany, ChWuhan 430074, PR China. Tel.: +86 27 87510599; fax

E-mail address: shhli@wbgcas.cn (S. Li).

Grape calli systems were used to study the relationships between stilbene production and: (1) four grapegenotypes, (2) leaf, berry exocarp and seed tissues and (3) UV-C irradiation. All explants could be de-dif-ferentiated. However, subsequent callus growth depended mainly on genotype and tissue type. Non-embryogenic callus accumulated more resveratrols and piceids and had a higher growth index thanpro-embryogenic and embryogenic calli. UV-C irradiation for 20 min was most efficient in promotingboth the accumulation of resveratrols and piceids and callus growth index. There was dynamic produc-tion of resveratrols and piceids in UV-C-irradiated leaf-derived calli over a 72 h period, with optimumharvest time for the highest total stilbene content at 48 h. Accumulation of stilbenes in UV-C-irradiatedcalli depended upon genetic background and tissue type, with higher stilbene contents in two interspe-cific root stocks and leaf or exocarp explants.

� 2010 Elsevier Ltd. All rights reserved.

1. Introduction

Stilbenes are nonflavonoid phenolics found in the tissues andorgans of plants in several families, including Vitaceae, Arachaceaeand Pinaceae (Gambino, Bondaz, & Gribaudo, 2006). They arethought to play a phytoalexin-role in plants that produce them.One of the most relevant and extensively studied stilbenes istrans-resveratrol (trans-3,5,40-trihydroxystilbene) (Cantos, Tomás-Barberán, Martínez, & Espín, 2003), which has been reported tohave a number of health-beneficial properties, such as antioxidantcapacity, cardiovascular protective effect, antimutagenic proper-ties and estrogenic and cancer chemopreventive activity (Hung,Chen, Huang, Lee, & Su, 2000; Sgambato et al., 2001). Trans-resve-ratrol is an important and leading stilbenic compound in manyplants, but its cis-form has not been detected often. In additionto resveratrol, its glucoside (piceid) has also shown beneficial ef-fects on health. For example, trans-piceid can inhibit low-densitylipoprotein oxidation, reduce platelet aggregation, and act as atumour and metastatic carcinoma inhibitor (Kimura & Okuda,2000).

There has been particular interest in grapes, as they encompassa broad germplasm base, are widely planted and consumed and

ll rights reserved.

inese Academy of Sciences,: +86 27 87510251.

have a higher stilbene concentration than have most species (Lau-fenberg, Kunz, & Nystroem, 2003). However, stilbene biosynthesisand accumulation in grape tissues under natural conditions varieswith environment and growth stage of the plant. Plant cell culturepotentially offers uniform secondary-product synthesis by elimi-nating effects of unforeseen climatic conditions and diseases, as oc-curs in field-grown plants (Trejo-Tapia et al., 2008). Thus, callusand cell suspension cultures are attractive for optimising produc-tion of secondary metabolites.

However, low and unstable yields have been major obstacles todeveloping large-scale production systems using plant cells. Manystudies of Vitis cell-cultures have been undertaken to stabilise andincrease stilbene yield, but most attempts have been limited to Vi-tis vinifera cultivars and grape leaf tissues. Studies at the Vitisgermplasm level have shown that resveratrol content depends ongenotype (Li, Wu, Wang, & Li, 2006), with range 0.01–356 lgg�1 fresh weight (Fw) in berry skins and 0.01–35.1 lg g�1 Fw inseeds. All V. vinifera cultivars have low trans-resveratrol concentra-tions, but some species–hybrid rootstocks have much higher trans-resveratrol concentrations in their berry skins than has V. vinifera.Moreover, grape leaves accumulate stilbenes at low levels undernatural conditions, and there are different concentrations oftrans-resveratrol in different tissue types within the same geno-type (Li et al., 2006; Tassoni et al., 2005; Zamboni, Vrhovsek, Kasse-meyer, Mattivi, & Velasco, 2006). Little information is available onthe influence of grape genotype and tissue type on callus growthand stilbene production in vitro. The effect of callus-type on

476 W. Liu et al. / Food Chemistry 122 (2010) 475–481

stilbene accumulation has also not been studied. It would be valu-able to know if stilbene accumulation of callus cultures in vitro isaffected by grape genotype and tissue type.

Many studies on stilbene production have shown that some fac-tors could induce stilbene biosynthesis, regulation and metabolism(Tassoni et al., 2005). For example, accumulation of trans-resvera-trol can be induced by stresses, such as fungal attack, wounding,elicitor treatment and UV light (Cantos, García-Viguera, De Pac-ual-Teresa, & Tomás-Barberán, 2000; Versari, Parpinello, Tornielli,Ferrarini, & Giulivo, 2001). UV-C irradiation is an important factorthat can act as a switch, controlling expression of specific genes in-volved in cell growth and secondary metabolism of plants (Versariet al., 2001). Here, we report the establishment of callus culturesand in vitro production of resveratrols and piceids, induced byUV light, from four grape genotypes and three tissue types of eachgenotype. The genotypes all have significantly different concentra-tions of trans-resveratrol in exocarps and seeds under field condi-tions. The aim of this study was to: (1) investigate the effects ofgenotype and tissue type on callus induction and growth in cul-ture, (2) determine the influences of genotype and tissue type onin vitro biosynthetic capacity for resveratrols and piceids, (3) selectthe most sensitive and high-yielding callus-types and (4) deter-mine the potential usefulness of UV-C irradiation for inducingin vitro production of resveratrols and piceids.

2. Materials and methods

2.1. Plant materials

Four grape genotypes were used: two interspecific rootstocks,‘Zhi 168’ (a hybrid of Vitis monticola �Vitis riparia) and ‘Beta’ (a hy-brid of Vitis labrusca � V. riparia), and two V. vinifera cultivars, ‘Mer-lot’ (wine grape) and ‘Jingxu’ (table grape). All genotypes canaccumulate trans-resveratrol under field conditions, with signifi-cantly different concentrations in grape skins (356, 230, 5.5 and0.7 lg g�1 Fw in ‘Zhi 168’, ‘Beta’, ‘Merlot’ and ‘Jingxu’, respectively)and seeds (35.1, 0.14, 2.09 and 1.26 lg g�1 Fw, respectively) (Liet al., 2006). Moreover, three tissue types (leaf, grape exocarpand seed) from ‘Beta’ were tested to determine the role of tissuetype in the production capacity of resveratrols and their piceids.All explants were collected in field production conditions from ma-ture grapevines. The cultivars were planted in the spring of 1993,and were grown under the same standard management practicesin the vineyard, such as irrigation, fertilisation, soil management,pruning and disease control.

Three fully expanded and tender leaves at the shoot tips wereharvested from the vines in the Germplasm Repository for Grapes,Institute of Botany, Chinese Academy of Sciences, when berrieswere at the pea-size stage. They were submerged for 5 min in 2%(v/v) detergent, followed by rinsing in tap water for at least 0.5 h.The leaves were then surface-sterilised using ethanol 70% (v/v)for 30 s, rinsed three times with sterile distilled water, treated with0.1% HgCl2 for 1 min and again rinsed with sterile distilled water atleast five times. Sterilised leaves were cut into 50 mm2 pieces, and8–10 explants were cultured per 90 mm diameter Petri dish, withthe upper surface placed on the media.

Grape clusters were collected 40–50 d after pollination. Onlysmooth-skinned berries located in the middle of a cluster, withoutany wounds and mildew, were selected. For berry sterilisation, theberries were washed gently by hand in 2% (v/v) detergent for5 min, followed by a rinse in tap water for at least 6 h, and thenthey were surface-sterilised, using 0.5–1.0% sodium hypochloritefor 30 s, and rinsed at least five times with sterile distilled water.Sterilised exocarps were cut into 50 mm2 pieces, and about 5–8pieces were cultured per Petri dish with the external surface ofthe exocarp placed on the media.

Seeds were collected from ripe berries. They were treated with0.1% HgCl2 for 5–8 min and rinsed with sterile distilled water atleast five times. From 10–15 seeds cultured on media, the tip re-gions were removed with a sterile razor blade for nutrientabsorption.

2.2. Culture media and incubation conditions

Leaf explants from the four genotypes and from three tissuetypes of ‘Beta’ grape were inoculated on B5 (Gambourg, Miller, &Ojima, 1968) basic solid medium supplemented with 0.1 mg l�1

of 6-benzyl adenine (6-BA) and different concentrations of 2,4-dichlorophenoxy-acetic acid (2,4-D: 0.1, 0.5 and 1.0 mg l�1), anda-naphthalene-acetic acid (NAA: 2.0, 3.0 and 4.0 mg l�1). The com-binations of 6-BA and 0.1, 1.0 or 2.0 mg l�1 of 2,4-D were denotedas B1, B2 and B3, respectively. Combinations of 6-BA with 2.0, 3.0or 4.0 mg l�1 of NAA were denoted B4, B5 and B6, respectively.After 3–4 weeks, calli from the rims of the explants were trans-ferred to media of the same formulation and transferred againevery 2 weeks during the first 2 months, and every 4 weeks there-after. All explants were kept at 25 ± 2 �C in permanent darkness.

After 3–5 months of culture, leaf calli of ‘Beta’, that had formedlarge non-embryogenic cultures, were transferred to the samemedia, but without plant growth regulators, to induce pro-embryogenic masses (PEM). At each subculture, the calli were se-lected carefully; brown and non-embryogenic calli were discarded,and friable and whitish calli (i.e. PEM) selected for the next culture.

Monitoring of phenotypic calli characteristics, such as colour,friability and rate of proliferation, were assessed every cycle duringthe maintenance stage. Calli were classified as described by Mat-kowski (2004): (I) weak initiation and poor growth, (II) good induc-tion but poor growth, (III) good formation and moderate growth,and (IV) best induction and vigorous growth.

2.3. Determination of induction coefficient and growth index

The relative production of calli was determined as: inductioncoefficient = (total number of induced calli/number of cultured ex-plants) � 100%. To assess growth rate of calli, a 30 d callus culturewas defined as one cycle of growth. Calli were harvested from eachtissue type after 0 and 30 d. A growth index was calculated to com-pare the increase in the fresh weight of the calli. Growth index (GI)was calculated using the formula: (final fresh weight – initial freshweight)/initial fresh weight.

Measurement of induction coefficient was replicated from threerepeated induction tests, and the growth index was measured fromcallus cultures in three different growth cycles.

2.4. Exposure to UV-C irradiation

Fifteen day-old subcultured calli were treated with UV-C irradi-ation. Before irradiation, the calli were dispersed equally onto Petridishes using sterile forceps in order to expose all sides to irradia-tion. A UV-C lamp (Model ZW30S26W, Beijing Lighting ResearchInstitute, China), with a maximum wavelength of 254 nm, wasthe light source. The UV-C light was placed at a distance of30 cm (approximately 6 W m�2) for durations of 10, 20 or30 min. The UV-C irradiation was applied with the covers of Petridishes removed in a sterile cabinet to avoid refraction by glass.During irradiation, the calli were agitated with sterilised forcepsevery 5 min for the purpose of symmetrical irradiation. After irra-diation, Petri dishes were re-covered and the calli incubated at25 �C in darkness until they were sampled. Calli not exposed toUV-C irradiation were the controls. At 1, 6, 12, 24, 36, 48 and72 h after exposure, calli were harvested, weighed, frozen with li-quid nitrogen, and stored at �80 �C for stilbene measurement.

Table 1Effects of genotypes and tissue types on calli induction and growth.

Mediacode

Genotypea Inductioncoefficient(%)b

Growthstatusc

Tissuetyped

Inductioncoefficient(%)

Growthstatus

B1 ‘Zhi 168’ 100a IV‘Beta’ 100a IV Leaf 100a IV‘Merlot’ 92a III Exocarp 33c I‘Jingxu’ 83b III Seed 67b III

B2 ‘Zhi 168’ 100a III‘Beta’ 100a III Leaf 100a III‘Merlot’ 99a II Exocarp 59c II‘Jingxu’ 91b II Seed 75b III

B3 ‘Zhi 168’ 100a II‘Beta’ 100a II Leaf 100a II‘Merlot’ 100a II Exocarp 73b III‘Jingxu’ 100a II Seed 80b III

B4 ‘Zhi 168’ 97a III‘Beta’ 98a III Leaf 98a III‘Merlot’ 91b II Exocarp 79c III‘Jingxu’ 87b II Seed 90b II

B5 ‘Zhi 168’ 98a III‘Beta’ 100a III Leaf 100a III‘Merlot’ 95b II Exocarp 86c III‘Jingxu’ 100a II Seed 91b II

B6 ‘Zhi 168’ 100a II‘Beta’ 100a II Leaf 100a II‘Merlot’ 100a II Exocarp 93b IV‘Jingxu’ 100a II Seed 95b II

The different letters following the mean values indicate significant differencesbetween genotypes or tissue types within a given medium (P < 0.05).

a Leaf explants were used to compare genotypes.b Induction coefficient = (total number of induced calli/number of cultured

explants) � 100%.c Calli growth status on different media were classified as described by Mat-

kowski (2004): (I) weak initiation and poor growth, (II) good induction but poorgrowth, (III) good formation and moderate growth, and (IV) best induction andvigorous growth.

d ‘Beta’ was used in the comparison of different tissue types.

W. Liu et al. / Food Chemistry 122 (2010) 475–481 477

Three callus-types (non-embryogenic callus, pro-embryogenicmasses and embryogenic callus), used in analysing stilbene con-tents (Section 3.2), were not subjected to UV-C irradiation in orderto select the optimal callus-type. All irradiated calli in four geno-types and three tissue types were non-embryogenic and aged for15 d. Irradiated and control calli at optimal harvest time (Section3.5) were collected at the same time. Irradiation experiments wererepeated three times and all values were the means of threereplicates ± SE.

2.5. Quantification of stilbenes

The frozen calli were ground to powder in liquid nitrogen usinga mortar and pestle. Stilbenes were extracted from the cell powderat room temperature overnight after adding 15 ml of ethyl acetateand methanol (1/1, v/v) (Li et al., 2006). Insoluble cell material wasremoved by centrifugation at 12,000g for 10 min. The organic sol-vent supernatant phase was removed and vacuum-dried with arotavapor (N-1001D-WD, EYELA, Tokyo Rikakikai, Japan) at 40 �C,and then diluted in 2 ml of 95% ethanol.

Analysis of stilbene was carried out on a Dionex P680 HPLCsystem (Dionex Corporation, CA, USA) fitted with a Dionex PDA-100 detector. Separation was achieved using a Waters C18 column(250 mm L � 4.6 mm I.D., 5 lm particle size) and a guard columncartridge (Sunchrom C18 cartridge) maintained at 25 �C with aDionex TCC-100 thermostatted column compartment. Separationwas performed at a flow rate of 1.0 ml min�1 with the mobilephase consisting of H2O (A) and acetonitrile (B). The solvent gradi-ent was as follows: 0–45 min 95% solvent A; 45–48 min from 95%to 53% solvent A; 48–50 min from 53% to 95% solvent A. For fluo-rimetric detection, maximum excitation wavelength was mea-sured at 334 nm and emission at 404 nm. The extractionsamples were prepared for HPLC analysis with each sample in-jected three times.

Trans-resveratrol and trans-piceid standards were purchasedfrom Sigma (St. Louis, MO, USA) and the Chinese Standards Re-search Institute, respectively. Cis-resveratrol and cis-piceid stan-dards were obtained after irradiation at a distance of 15 cm for45 min with 6 W m�2 UV light of the mixed solution of trans-resve-ratrol and trans-piceid standards. The conversion coefficients were46% and 44% for trans-resveratrol for trans-piceid, respectively.

2.6. Graphs and data analysis

Graphs of the experimental data were developed using SigmaPlot 10.0 (SPSS Inc., Chicago, USA) for Windows. The concentrationof each compound was plotted over sampling periods from threereplicates, and experimental data were subjected to analysis ofvariance using the SPSS 14.0 programme (SPSS Inc.). Means wereseparated by Duncan’s multiple range tests at P < 0.05.

3. Results and discussion

3.1. Calli induction and growth

Calli induction from leaf tissue occurred with all genotypes andon all tested media (B1–B6), but satisfactory induction and growthvaried (Table 1). All the explants had the ability to de-differentiatewith four kinds of callus initiation and growth status (I, II, III andIV). Induction coefficient was greatly affected by grape genotypewithin most media. Leaf explants of ‘Zhi 168’ and ‘Beta’ gave gen-erally higher induction coefficients (97–100%) than did the othertwo cultivars, and there were significant differences in inductionbetween them in media B1, B2, B4 and B5. Leaf calli initiationand growth status varied with media. Generally, of the six media

tested, B1 gave the best growth for all four cultivars. However,the calli growth status of ‘Zhi 168’ and ‘Beta’ were better thanthose of the two V. vinifera cultivars in media B1. Based on the pre-vious results, media B1 was the best for leaf explants to efficientlyinduce callus with high growth quality. Leaf–callus induction andsomatic embryogenesis have been widely reported in some elitecultivars, but of the cultivars in our study only ‘Merlot’ has previ-ously been reported (Vidal et al., 2009). The two interspecific hy-brids (‘Zhi 168’ and ‘Beta’) had V. riparia male parents, and alsogave the highest induction efficiencies and more high quality callusthan did V. vinifera. The strong genotypic influence suggests thatthe de-differentiation response may be controlled by differentgenes or gene combinations (Özgen, Türen, Altmok, & Sancak,1998). Using Arabidopsis, Kandasamy, Gilliland, McKinney, andMeagher (2001) reported that the ACT7 gene family was requiredfor callus formation, with expression differences within the familyresulting in differing capacities for cell proliferation. The reasonsunderlying these differences are complex, though it seems likelythat the specific genotypic background of the interspecific hybridscontributed certain genes that made the hybrids more sensitive tocallus induction.

Regarding the different tissue types, leaf explants always pro-vided the highest induction coefficient under all the six testedmedia, followed by seed explants. In contrast, exocarp explantsgenerally gave the lowest inductions in the tested media. In fact,many seed and exocarp explants of ‘Beta’ grew slowly, blackened,and even died, perhaps due to the high concentration of polyphe-nolic substances in grape skin and seeds (Liang et al., 2008). In re-

478 W. Liu et al. / Food Chemistry 122 (2010) 475–481

gard to the growth status of calli derived from different explanttypes, only leaf and exocarp explants gave the type-IV calli in B1and B6 media, respectively. Many woody plants display this gen-eral behaviour, where callus initiation occurs easily in tissues withhigh meristematic activity (Guerra & Handro, 1998). The successeson different media were associated with the different explant typesexocarp and seed are more advanced tissues compared with leafexplants, which likely led to complicated de-differentiation re-sponses influenced by the differing concentrations of endogenoushormones and unique physiological status of each.

3.2. Stilbene content in different callus-types

‘Beta’ leaf callus was subcultured on different media to promotethe growth and the presence of different callus-types. After care-fully screening every cycle for 3–12 months, three types of calliwere obtained: non-embryogenic callus (NEC), PEM and embryo-genic callus (EC). Four stilbenes, trans-piceid, cis-piceid, trans-res-veratrol and cis-resveratrol were, respectively, identified in thecalli extract (Fig. 1). In the analysis of the main families of stilbenecompounds after one culture cycle of 30 d, two essential stilbenes,trans-piceid and trans-resveratrol, were observed. Trans-piceid hadthe highest concentration of the four stilbene compounds in all cal-lus-types. However, in all calli, no, or only trace amounts of, cis-forms were detected (Fig. 2). Moreover, stilbene concentration var-ied with callus-type. The highest concentrations of trans-piceid andtrans-resveratrol, as well as of total stilbenes, were in NEC. Therewere no significant differences in trans-resveratrol and trans-piceidconcentrations between PEM and EC. As regards callus growth, asignificantly high growth index of 7.1 was achieved from NEC,while PEM had the lowest value. Therefore, NEC was selected forfurther experiments.

3.3. Optimal UV-C irradiation time for stilbene biosynthesis andaccumulation in leaf calli

The effect of UV-C irradiation, for 10, 20 or 30 min, on callusgrowth and stilbene biosynthesis (samples were selected on thelast day in a culture cycle) are illustrated in Fig. 3. The growth in-

21.0 21.5 22.0 22.5 23.0 23.5 24.0 24.5 25.0 25.5 26.0 26.5 27.0 27.

mAU 107

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50

25

-10

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Cis-piceid

mAU

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100

120

2.0

Fig. 1. Typical chromatogram of the extract from callu

dex of calli was affected by irradiation duration. When leaf calliwere exposed to UV-C irradiation for 10 and 20 min, there was asignificantly higher growth index than for controls, but there wasa significant negative effect of 30 min of UV-C irradiation on callusgrowth. UV-C irradiation for 10–30 min increased stilbene produc-tion. The trans-piceid concentration was 6.5 lg g�1 Fw in controls,and 39.6, 51.2 and 59.4 lg g�1 Fw after 10, 20 and 30 min, respec-tively. The irradiation for 30 min was the most effective in stimu-lating piceid and total stilbenes, suggesting that a longerexposure may be necessary for higher secondary production. How-ever, exposure time did not affect production of the other majorstilbene, i.e. trans-resveratrol at 19.6, 22.5 and 23.3 lg g�1 Fw after10, 20 and 30 min, respectively, consistent with results of Günterand Ovodov (2007). This may be expected because, after culturingirradiated calli for about 15 d in darkness, the trans-resveratrolwould be converted to trans-piceid and cis-forms to trans-forms,which would result in an irradiation effect on total stilbene pro-duction but not on every type.

UV-C irradiation is an important factor that acts as a switch,controlling expression of specific genes involved in cell growthand secondary metabolism of plants (Liu et al., 2006). The influenceof UV-C depends on dose, sensitivity of plant species, and theirability to attenuate the irradiation effect (Lavola, Aphalo, Lahti, &Julkunen-Tiitto, 2003). In the present study, UV-C irradiation for10 and 20 min promoted callus growth, but longer periods of irra-diation reduced the growth index, despite greater stilbene produc-tion. The optimal irradiation time for subsequent study was20 min, chosen by combining the growth index and stilbeneproduction.

3.4. Dynamic production of stilbenes in leaf calli after induction of UV-C irradiation

To determine the best time to collect calli for stilbenes, resvera-trol (trans- and cis-) and piceid (trans- and cis-) production by non-embryogenic ‘Beta’ calli was measured at regular intervals up to72 h after the 20 min UV-C irradiation (Fig. 4). The trans-piceidcontent rose steadily from 4.5 lg g�1 Fw at 0 h after induction to9.8 lg g�1 Fw by 6 h later. This production lasted up to 24 h post-

5 28.0 28.5 29.0 29.5 30.0 30.5 31.0 31.5 32.0 32.5 33.0 33.5 34.0

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Trans-resveratrol Cis-resveratrol

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s of leaf pro-embryogenic masses of ‘Beta’ grape.

Stilb

enes

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g·g-1

·Fw

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Trans-resveratrol Trans-piceid Cis-resveratrol Cis-piceid Total stilbenes

Growth index

a

b b

a

b b

b b

a a a a a a

a a

c

b

Fig. 2. The concentrations of stilbenes and the growth index in different calli types of 30 d-old ‘Beta’ leaf calli. The values of stilbenes and growth index are represented byhistogram and line, respectively. Different letters above the same kind of bar-chart and the line indicate, respectively, significant differences (P < 0.05) in the stilbeniccompound concentrations and growth index between tested calli types.

Stilb

enes

con

cent

rati

on (

µg·g

-1·F

w)

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wth

Ind

ex

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Growth index

a

a

b

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d

b

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a a a

b a a a b

a b c d

a

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Fig. 3. The concentrations of stilbenes and the growth index of 30 d-old ‘Beta’ leaf calli, 15 d after UV-C irradiation for 0, 10, 20 and 30 min. The values of stilbenes and growthindex are represented by histogram and line, respectively. Different letters above the same kind of bar-chart and the line indicate, respectively, significant differences(P < 0.05) in the stilbenic compound concentrations and growth index between tested calli types.

W. Liu et al. / Food Chemistry 122 (2010) 475–481 479

treatment, then decreased slightly by 48 h and remained stable upto 72 h. Trans-resveratrol had accumulated in calli by 1 h afterirradiation, whereas no cis-resveratrol was detected at thattime. Accumulation of trans-resveratrol peaked at about32.5 lg g�1 Fw at 48 h after UV-C irradiation. Cis-piceid andcis-resveratrol appeared at 1 and 6 h after irradiation, respectively,and remained at very low levels thereafter. The optimal harvesttime was 48 h after UV-C irradiation, when there were maximalstilbenes in calli.

Vitis spp. and genotypes have different potentials for stilbeneaccumulation, but the dynamic productions over about 72 h afterUV-C irradiation were not significantly different in other genotypes(Douillet-Breuil, Jeandet, Adrian, & Bessis, 1999; Sbaghi, Jeandet,Faivre, Bessis, & Fournioux, 1995). Dynamic production by ‘Beta’was evident after UV-C irradiation. Trans-resveratrol productionpeaked at 48 h, consistent with expression profiles with other elic-itors (Tassoni et al., 2005; Zamboni et al., 2006). It has previouslybeen suggested that trans-resveratrol is synthesised in the free

0 20 40 60 80

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50 Trans-piceid

Trans-resveratrol

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Cis-resveratrol

Stilb

enes

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cent

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g·g-1

·Fw

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Hours after UV-C irradiation (h)

Fig. 4. Changes of stilbene concentrations in 72 h of ‘Beta’ leaf calli, 15 d after UV-Cirradiation for 20 min. Values represent the means ± SE of three replicates.

480 W. Liu et al. / Food Chemistry 122 (2010) 475–481

form and then rapidly glycosylated to trans-piceid (Jeandet et al.,1997), trans-piceid is the stored and transported form of trans-res-veratrol during the response to infection or stresses (Belhadj et al.,2008). In our study, trans-piceid accumulated heavily within 24 h,confirming that trans-resveratrol was produced at high levels.However, possibly because trans-resveratrol also acts as the pre-cursor of many other stilbenic components (e.g. astringinin, picea-tannol and viniferin) and may be converted to cis-forms, theproduction of trans-piceid was steady after 24 h.

3.5. Effect of genotypic variation and tissue type on stilbeneaccumulation in calli after induction of UV-C irradiation

Genotype and tissue type responses to UV-C, using 15-d-old cal-li irradiated for 20 min, were compared. There were differences inthe levels of total stilbenes, resveratrols and piceids among the cal-li from the different grape genotypes and tissue types (Table 2) at48 h after irradiation. In all genotypes, UV-C irradiation evidentlyincreased all the components of stilbenes at 48 h after irradiation,with trans-piceid as the predominant component. Irradiated callifrom ‘Zhi 168’ had the highest trans-piceid concentration, followedby ‘Beta’ (50.2 and 36.8 lg g�1 Fw, respectively), with ‘Jingxu’ and‘Merlot’ the lowest. Trans-resveratrol showed the same response toUV-C irradiation in all genotypes. The cis-piceid and cis-resveratrolconcentrations were very low, although there were significant dif-ferences between the four genotypes.

Stilbene synthesis capacity depends largely on geneticbackground, although it can be activated by many exogenous

Table 2Effects of genotype and tissue type on stilbene contents (lg g�1 Fw) at 48 h after 20 min

Trans-resveratrol Trans-piceid Cis-resve

UV-C Control UV-C Control UV-CGenotypea

‘Zhi 168’ 38.6a 3.9a 50.2a 6.1a 1.4a‘Beta’ 32.5a 2.8b 36.8b 5.2b 1.2b‘Merlot’ 16.0b 2.4b 22.8c 4.7b 1.1ab‘Jingxu’ 12.0c 0.3c 20.7c 2.9c 0.8c

Tissue Typesb

Leaf 32.5a 2.8a 36.8a 5.2a 1.2aExocarp 29.7a 3.5a 40.6a 5.8a 1.7aSeed 11.7b 3.2a 15.5b 6.2a 0.8b

Means followed by different letters within each column indicate significant differencesa Leaf explants were used in the comparison of different genotypes.b ‘Beta’ was used in the comparison of different tissue types.

stress factors. Our results also indicated that the capacity to pro-duce high levels of secondary metabolites does not degenerateafter de-differentiation from plant tissue in vivo to tissue culture,since ‘Zhi 168’ and ‘Beta’ were strong accumulators in vitro as wellas under field conditions. The V. vinifera cultivars produced lowerstilbene quantities, consistent with results for field-grown plantmaterial (Li et al., 2006). When de-differentiation occurs, genes re-lated to the synthesis of secondary metabolites may be shut downor depressed to some extent (Kandasamy et al., 2001). However,when they are subjected to elicitors, such as UV-C, the genesmay be expressed with stilbene production genetically deter-mined. Therefore, it is important to choose genotypes for culturewhich can produce high levels of stilbenes before being subjectedto de-differentiation and UV-C elicitation treatment.

In the UV-C-irradiated calli cultures, trans-piceid and trans-res-veratrol were the major stilbenes in calli of all tissue types; how-ever, the amount of these compounds varied with tissue origin.Leaf and exocarp-derived calli produced almost the same quantityof stilbenes and there were few differences in individual or totalstilbenes. However, the stilbene concentration was clearly lowestin seed-derived calli (<50% of that in leaf and exocarp-derived cal-li). Zheng, Li, Wu, Wang, and Li (2009) proposed that grape seedsshould have low capacity to synthesise trans-resveratrol, and thatmost of the seed trans-resveratrol may result from transport fromleaves. There are various opinions about the tissue-specific expres-sion abilities after de-differentiation. Some workers have thoughtthat the synthesis potential can be retained to some extent in cellsof different origins (Mischenko et al., 1999); however, others foundthat tissue-specific differences were much less (Matkowski, 2004)or were expressed when cells had mature organelles (Eckes, Schell,& Willmitzer, 1985). Although the biosynthesis of stilbenes ingrape cell-cultures has been studied extensively, little has been re-ported about the dependence of stilbene formation on the sourceof explants. Our results show that the stilbene synthesis potentialof de-differentiated cells resulted mainly from genetic backgroundand less from tissue type; however, seeds were a poor source.Hence, it is most important, for in vitro culture, to use tissues ofgrape genotypes that can synthesise stilbenes at high levels.

4. Conclusion

The present study showed that genotype was more importantfor grape callus growth and stilbene production than was tissuetype. All tested callus-types accumulated stilbenes before elicita-tion, but the NEC was more efficient, with higher growth indexand stilbene production despite UV-C irradiation. UV-C irradiationfor 20 min and 48 h post-harvest were optimal for high productionof resveratrols and piceids. UV-C irradiation induced accumulationof trans-piceid and trans-resveratrol in the calli. UV-C-irradiated

irradiation.

ratrol Cis-piceid Total stilbenes

Control UV-C Control UV-C Control

0.0 0.8b 0.0a 91.0a 10.0a0.0 1.3a 0.1a 71.7b 8.2b0.0 0.7b 0.0a 40.7c 7.1b0.0 0.9b 0.0a 34.3c 3.2c

0.0 1.3b 0.1a 71.7a 8.2a0.0 2.0a 0.3a 73.9a 9.6a0.0 0.9c 0.0a 28.9b 9.4a

(P < 0.05) between genotypes or tissue types.

W. Liu et al. / Food Chemistry 122 (2010) 475–481 481

‘Zhi 168’ and ‘Beta’ produced much more stilbenes than did thetwo V. vinifera cultivars, and leaf- and exocarp-derived calli wereboth suitable tissues for in vitro culture and inducing stilbeneaccumulation.

Acknowledgements

This work was financially supported by the Beijing Natural Sci-ence Foundation of China (Grant No. 5082014), and the CAS/SAFEAInternational Partnership Program for Creative Research Teams(Grant No. KSCX2-YW-G-075-24).

References

Belhadj, A., Telef, N., Saigne, C., Cluzet, S., Barrieu, F., Hamdi, S., et al. (2008). Effect ofmethyl jasmonate in combination with carbohydrates on gene expression of PRproteins, stilbene and anthocyanin accumulation in grapevine cell cultures.Plant Physiology and Biochemistry, 46(4), 493–499.

Cantos, E., García-Viguera, C., De Pacual-Teresa, S., & Tomás-Barberán, F. A. (2000).Effect of postharvest ultraviolet irradiation on resveratrol and other phenolicsof cv. Napoleon table grapes. Journal of Agricultural and Food Chemistry, 48(10),4606–4612.

Cantos, E., Tomás-Barberán, F. A., Martínez, A., & Espín, J. C. (2003). Differentialstilbene induction susceptibility of seven red wine grape varieties upon post-harvest UV-C irradiation. European Food Research and Technology, 217, 253–258.

Douillet-Breuil, A. C., Jeandet, P., Adrian, M., & Bessis, R. (1999). Changes in thephytoalexin content of various Vitis spp. in response to ultraviolet C elicitation.Journal of Agricultural and Food Chemistry, 47, 4456–4461.

Eckes, P., Schell, J., & Willmitzer, L. (1985). Organ-specific expression of three leaf/stem specific cDNAs from potato is regulated by light and correlated withchloroplast development. Molecular Genetics and Genomics, 199, 216–224.

Gambino, G., Bondaz, J., & Gribaudo, I. (2006). Detection and elimination of virusesin callus, somatic embryos and regenerated plantlets of grapevine. EuropeanJournal of Plant Pathology, 114(4), 397–404.

Gambourg, O. L. R., Miller, R. A., & Ojima, K. (1968). Nutrient requirements ofsuspension cultures of soybean root cells. Experimental Cell Research, 50(1),151–156.

Guerra, M. P., & Handro, W. (1998). Somatic embryogenesis and plant regenerationin different organs of Euterpe edulis Mart. (Palmae): Control and structuralfeatures. Journal of Plant Research, 111, 65–71.

Günter, E. A., & Ovodov, Yu. S. (2007). The effect of ultraviolet radiation on thegrowth and polysaccharide composition of a callus culture of Silene vulgaris.Applied Biochemistry and Microbiology, 43(4), 465–472.

Hung, L. M., Chen, J. K., Huang, S. S., Lee, R. S., & Su, M. J. (2000). Cardioprotectiveeffect of resveratrol, a natural antioxidant derived from grapes. CardiovascularResearch, 47(3), 549–555.

Jeandet, P., Breuil, A. C., Adrian, M., Weston, L. A., Debord, S., Meunier, P., et al.(1997). HPLC analysis of grapevine phytoalexins coupling photodiode arraydetection and fluorometry. Analytical Chemistry, 69, 5172–5177.

Kandasamy, M. K., Gilliland, L. U., McKinney, E. C., & Meagher, R. B. (2001). One plantactin isovariant is induced by auxin and required for normal callus formation.The Plant Cell, 13, 1541–1554.

Kimura, Y., & Okuda, H. (2000). Effects of naturally occurring stilbene glucosidesfrom medicinal plants and wine, on tumour growth and lung metastasis in

Lewis lung carcinoma-bearing mice. Journal of Pharmacy and Pharmacology,52(10), 1287–1295.

Laufenberg, G., Kunz, B., & Nystroem, M. (2003). Transformation of vegetable wasteinto value added products: (A) The upgrading concept, (B) practicalimplementations. Bioresource Technology, 87(2), 167–198.

Lavola, A., Aphalo, P. J., Lahti, M., & Julkunen-Tiitto, R. (2003). Nutrient availabilityand the effect of increasing UV-B radiation on Scots pine. Environmental andExperimental Botany, 49, 49–60.

Li, X. D., Wu, B. H., Wang, L. J., & Li, S. H. (2006). Extractable amounts of trans-resveratrol in seed and berry skin in Vitis evaluated at germplasm level. Journalof Agricultural and Food Chemistry, 54(23), 8804–8811.

Liang, Z. C., Wu, B. H., Fan, P. G., Yang, C. X., Duan, W., Zheng, X. B., et al. (2008).Anthocyanin composition and content in grape berry skin in Vitis germplasm.Food Chemistry, 111, 837–844.

Liu, Z., Qi, J., Chen, L., Ming, S., Wang, X., & Pang, Y. (2006). Effect of light on geneexpression and shikonin formation in cultures Onosma paniculatum cells. PlantCell, Tissue and Organ Culture, 84, 38–48.

Matkowski, M. (2004). In vitro isoflavonoid production in callus from differentorgans of Pueratia lobata (Wild.) Ohwi. Journal of Plant Physiology, 161, 343–346.

Mischenko, N. P., Fedoreyev, S. A., Glazunov, V. P., Chernoded, G. K., Bulgakov, V. P.,& Zheravlev, Y. N. (1999). Anthraquinone production by callus cultures of Rubiacordifolia. Fitoterapia, 70, 552–557.

Özgen, M., Türen, M., Altmok, S., & Sancak, C. (1998). Efficient callus induction andplant regeneration from mature embryo culture of winter wheat (Triticumaestivum L.) genotypes. Plant Cell Reports, 18, 331–335.

Sbaghi, M., Jeandet, P., Faivre, B., Bessis, R., & Fournioux, J. C. (1995). Development ofmethods using phytoalexin (resveratrol) assessment as a selection criterion toscreen grapevine in vitro cultures for resistance to grey mould (Botrytis cinerea).Euphytica, 86, 41–47.

Sgambato, A., Ardito, R., Faraglia, B., Boninsegma, A., Wolf, F. I., & Cittadini, A. (2001).Resveratrol, a natural phenolic compound, inhibits cell proliferation andprevents oxidative DNA damage. Mutation Research – Genetic Toxicology andEnvironmental Mutagenesis, 496(1–2), 171–180.

Tassoni, A., Fornalè, S., Franceschetti, M., Musiani, F., Micheal, A. J., Perry, B., et al.(2005). Jasmonates and Na-orthovanadate promote resveratrol production inVitis vinifera cv. Barbera cell cultures. New Phytologist, 166(3), 895–905.

Trejo-Tapia, G., Balcazar-Aguilar, J. B., Martínez-Bonfil, B., Salcedo-Morales, G.,Jaramillo-Flores, M., Arenas-Ocampo, M. L., et al. (2008). Effect of screening andsubculture on the production of betaxanthins in Beta vulgaris L. var. ‘DarkDetroit’ callus culture. Innovative Food Science and Emerging Technologies, 9,32–36.

Versari, A., Parpinello, G. P., Tornielli, G. B., Ferrarini, R., & Giulivo, C. (2001). Stilbenecompounds and stilbene synthase expression during ripening, wilting and UVtreatment in grape cv. Corvina. Journal of Agricultural and Food Chemistry,49(11), 5531–5536.

Vidal, J. R., Rama, J., Taboada, L., Marin, C., Ibañez, M., Sefura, A., et al. (2009).Improved somatic embryogenesis of grapevine (Vitis vinifera) with focus oninduction parameters and efficient plant regeneration. Plant Cell, Tissue andOrgan Culture, 96, 85–94.

Zamboni, A., Vrhovsek, U., Kassemeyer, H. H., Mattivi, F., & Velasco, R. (2006).Elicitor-induced resveratrol production in cell cultures of different grapegenotypes (Vitis spp.). Vitis, 45(2), 63–68.

Zheng, X. B., Li, X. D., Wu, B. H., Wang, L. J., & Li, S. H. (2009). Effects of UV-Cirradiation on resveratrol and its glycosides content in leaves and the berries ofneighboring clusters of potted ‘Beifeng’ grape vines (Vitis thunbergii � V.vinifera). Journal of Fruit Science, 26(4), 461–465.