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To explore the effect of pectolytic enzyme application on polysaccharide size and colloidal interactions, rosé wines were prepared from pressed Shiraz grape juice, and red wines were made from the same juice by the addition of skins and seeds from fresh pressings. A pectolytic enzyme preparation containing primarily polygalacturonase and side-activities of arabinase and pectin lyase was used. Enzyme treatment enhanced the contribution of high molecular weight (≈ 200 kDa) polysaccharides rich in mannose and glucose (mannoproteins) and removed arabinose-rich polysaccharides of intermediate (≈ 40 kDa) size. Enzyme application reduced the molecular weight average of a class of smaller polysaccharides of approximately 6 kDa (rhamnogalacturonan II). All wines had similar particle sizes, but enzyme treatment markedly reduced particle concentration in wines, particularly in rosé wines. Wine polysaccharides were purified and when reconstituted in model wine, showed reduced particle concentration in response to enzyme treatment. Aggregation of polysaccharide in the presence of seed tannin was also markedly reduced by enzyme treatment. The results indicated that changes in structure introduced by the enzyme affected wine colloidal properties, potentially increasing polysaccharide solubility.

BACKGROUND: Bioactive compounds, mainly polyphenols, present in berries, are thought to be responsible for the health benefits of these fruit. Therefore, it is worthwhile to define the optimal environmental conditions to maximise their polyphenol content. OBJECTIVE: With the aim to define the optimal conditions for berry cultivation in an innovative environment, red raspberry, wild strawberry and blackberry plants were grown in a traditional greenhouse in comparison with two photovoltaic greenhouses with different shading area. METHODS: Hydroalcoholic extracts of ripe berries were evaluated by HPLC analysis, for their anthocyanins, organic acids and sugar contents. Moreover, phenolic content (by the Folin-Ciocalteu assay) and antioxidant activity (by the Trolox equivalent antioxidant capacity-TEAC assay) were assayed on the same berry extracts. RESULTS: Total anthocyanins, phenols content and antioxidant capacity tended to increase in berries grown under shading. The sugars content was, mostly, not negatively influenced by the shading. Conversely, the organic acids content, in some instances, increased along with the shading percentage. CONCLUSIONS: It can be concluded that it is possible to combine the greenhouse production of high-quality berries (with a particular focus on polyphenols, bioactive compounds valuable for human health) with the production of renewable energy, in the context of sustainable agriculture.

This study investigates the effects of tomato puree fortification with several anthocyanin-rich food colorants on bioactive compound content (phenolics, isoprenoids), antioxidant capacity, in vitro biological activities and consumer acceptance. Tomato puree (tp) was added with different anthocyanin extracts from black carrot (Anthocarrot), grape fruit skins (Enocolor), elderberry fruits (Elderberry) or mahaleb cherry fruits (Mahaleb), thus obtaining a 'functional tomato puree' (ftp). The consumer acceptance (colour, flavor, taste, visual appearance) was at high level, except for Mahaleb-added ftp. Compared to the control (tp), the addition of colouring extracts increased significantly the total phenolic content, before pasteurization, in addition to the expected anthocyanin content. However, after pasteurization, mostly Anthocarrot-ftp preserved an increased phenolic (+53%) content, as well as a higher antioxidant capacity (50%), more than the other added-extracts. Consistently, against tp, Anthocarrot-ftp exhibited an increased anti-inflammatory capacity as showed by the reduced expression of vascular cell adhesion molecule (VCAM)-1 in human cultured endothelial cells, under inflammatory conditions.

Artemisia annua L. is a herbaceous plant belonging to the Asteraceae family, known for producing, although at low levels, the sesquiterpene lactone artemisinin (AN), which is highly effective against malaria. In this study, an in vitro regeneration process of A. annua L. using ‘Artemis’ progeny was established and the potential of tissue culture for inducing new variability in terms of AN metabolism of in vitro regenerated plants was investigated. Among the plant growth regulators tested, the cytokinin 6-benzyladenine (BA) at 4.4 μM in combination with the auxin indole-butyric acid (IBA) at 0.35 μM yielded the greatest frequency of shoot induction. The optimal multiplication medium contained BA at 0.9 μM and naphthaleneacetic acid (NAA) at 0.05 μM. Regenerated plants (RPs), after transferring to the greenhouse and subsequently to the field, were analyzed during the growth cycle at different sampling times, showing a peak of AN content 20 days before blossom. Variability among different RPs and sampling times, in terms of AN and its precursors dihydroartemisinic acid (DHAA) and artemisinic acid (AA) was observed. This suggests that adventitious shoot induction could provide a useful strategy to induce variability influencing artemisinin metabolism as a consequence of in vitro manipulation.

Fruits are an important part of the human diet and a major source of bioactive compounds (BCs) with human health benefits. Among different BCs present in fruits are polyphenols, carotenoids and some vitamins (C), which are all correlated with antioxidant potential of fruits. In the last thirty years, the genetic improvement of fruit trees has mainly been driven by productivity issues and consumers preference; the quality traits have only recently been recognized as important as the commercial ones. The nutraceutical characterization has become one issue to address when dealing with genetic improvement of fruits. The content of BCs in fruits can vary depending on different factors, primarily due to the genetic base, and the environmental conditions where fruit trees are cultivated. Effects of light, temperature, cultural practices, nutrient management, elicitors and biostimulants are presented here as outcomes on how research can contribute to enhance and improving sustainable crop production.

Plant extracts are a rich source of natural compounds with antimicrobial properties, which are able to prevent, at some extent, the growth of foodborne pathogens. The aim of this study was to investigate the potential of polyphenolic extracts from cladodes of Opuntia ficus-indica (L.) Mill. to inhibit the growth of some enterobacteria and the biofilm formation by Staphylococcus aureus. Opuntia ficus-indica cladodes at two stages of development were analysed for total phenolic content and antioxidant activity by Oxygen Radical Absorbance Capacity (ORAC) and Trolox equivalent antioxidant capacity (TEAC) (in vitro assays) and by cellular antioxidant activity in red blood cells (CAA-RBC) (ex vivo assay). The Liquid Chromatography Time-of-Flight Mass Spectrometry (LC/MS–TOF) analysis of the polyphenolic extracts revealed high levels of piscidic acid, eucomic acid, isorhamnetin derivatives and rutin, particularly in the immature cladode extracts. Opuntia cladodes extracts showed a remarkable antioxidant activity (in vitro and ex vivo), a selective inhibition of the growth of Gram-positive bacteria, and an inhibition of Staphylococcus aureus biofilm formation. Our results suggest and confirm that Opuntia ficus-indica cladode extracts could be employed as functional food, due to the high polyphenolic content and antioxidant capacity, and used as natural additive for food process control and food safety.

Prunus mahaleb L. is a marginal fruit crop producing cherry-like dark purple drupes with a very bitter taste, rich in polyphenolic compounds. A mahaleb fruit concentrated extract ( mfce ) has been assayed for its biological activities. In this work, we report results on the in vitro effects of mfce including: i) anti-proliferative, gap junction intercellular communication (GJIC) modulation and pro-apoptotic properties in a breast cancer cell line (MCF-7), ii) anti-inflammatory properties in human vascular endothelial cells (HUVEC), iii) anti-mutagenic effect on yeast Saccharomyces cerevisiae strain D7. Mfce exerted a dose-dependent anti-proliferative effect on MCF-7 cells together with a significant time-dependent increase in GJIC and a pro-apoptotic effect. Furthermore, mfce significantly reduced levels of endothelial inflammatory antigens in a concentration-dependent manner. Finally, mfce protected S. cerevisiae cells against H 2 O 2 cytotoxicity by direct radical scavenging activity. This study demonstrated multiple healthful biological effects of mfce , highlighting it as a potential nutraceutical product.

Background The production and consumption of cherries has increased recently due to consumer awareness of their health benefits, since they are rich in polyphenolics (namely anthocyanins and hydroxycinnamic acids). Global sweet cherry production increased over the last 16 years from 1.9 to 2.32 million tons, with Turkey, USA, Iran, as the main producers. Sour cherry production has been static during the same period at around 1.1–1.3 million tons concentrated in Europe, with the Russian Federation as the predominant producer. Scope and approach This review presents a comprehensive survey of the current literature on sweet and sour cherries, from the origin and distribution of the fruit crop to their antioxidant and health properties. Moreover, possible further investigations are highlighted after summarizing the existing body of knowledge. Key findings and conclusions Sweet cherries are mostly consumed fresh, and are also highly perishable, whereas sour cherries are most frequently processed. Sweet cherries are appreciated for their organoleptic aspects, such as taste, texture and color, whereas processed sour cherries are recognized for their higher bioactive content. Both sweet and sour cherries exert beneficial health effects to counteract oxidative stress, reduce inflammation, modulate blood glucose, and enhance cognitive function. Sour cherries supplementation in particular, has been reported to benefit in recovery from exercise-induced muscle damage. The health benefits derived from consuming cherries should increase this fruit tree crop cultivation and improve the agricultural practices and food processing technology to provide consistent and reliable supply of these fruits.

Background The identification of novel plant-based functional foods or nutraceutical ingredients that possess bioactive properties with antioxidant function has recently become important to the food, nutraceutical and cosmetic industries. This study evaluates the polyphenolic composition, identifies bioactive compounds and assays the total antioxidant capacity of Prunus mahaleb L. fruits collected from different populations and sampling years in the countryside around Bari (Apulia Region, Italy). Results We identified nine polyphenolic compounds including major anthocyanins, coumaric acid derivatives and flavonols from P. mahaleb fruits. The anthocyanin content (in some populations > 5 g kg(-1) fresh weight; FW) in the fruit was comparable to that reported for so-called superfruits such as bilberries, chokeberries and blackcurrants. Coumaric acid derivatives comprised a large portion of the total polyphenolic content in the P. mahaleb fruits. Antioxidant activities, assessed using ORAC and TEAC assays, measured up to 150 and 45 mmol Trolox equivalents kg(-1) FW, respectively. Therefore antioxidant capacity of P. mahaleb fruits is relatively high and comparable to that of superfruit varieties that are often used in commercial nutraceutical products. Conclusion Our findings suggest that mahaleb fruit (currently not consumed fresh or used in other ways) could serve as a source of bioactive compounds and therefore find interest from the functional food and nutraceutical industries, as a natural food colorant and antioxidant ingredient in the formulation of functional foods. © 2015 Society of Chemical Industry.

There is an increasing consumer and retailer demand for new natural colorants. Anthocyanins are a class of molecules already used as water-soluble natural color- ant and for their health effects. Prunus mahaleb L. is a tree native to the Mediterranean area, producing highly pig- mented small-stone fruits currently not used for fresh con- sumption due to their astringent and sour taste. In the pre- sent study, we set up a protocol to obtain a "mahaleb fruit concentrated extract" (mfce) and characterized it chemi- cally by high-performance liquid chromatography/diode array detection. Anthocyanins, flavonols, flavanols, cou- marin, sugars, ascorbic acid and organic acids were identi- fied and quantified. After characterization, the antioxidant capacity of the mfce was assayed in vitro by three different methods: Trolox equivalent antioxidant capacity, Oxygen Radical Absorbance Capacity and Folin-Ciocalteu assays. The concentrated extract produced and characterized in this study showed a high content of anthocyanins, flavonols and coumarin and a strong antioxidant capacity. Therefore, mfce may be of interest as a natural food colorant and as an ingredient in the formulation of functional foods.

Anthocyanins, the naturally occurring pigments responsible for most red to blue colours of flowers, fruits and vegetables, have also attracted interest because of their potential health effects. With the aim of contributing to major insights into their structure–activity relationship (SAR), we have evaluated the radical scavenging and biological activities of selected purified anthocyanin samples (PASs) from various anthocyanin-rich plant materials: two fruits (mahaleb cherry and blackcurrant) and two vegetables (black carrot and “Sun Black” tomato), differing in anthocyanin content (ranging from 4.9 to 38.5 mg/g DW) and molecular structure of the predominant anthocyanins. PASs from the abovementioned plant materials have been evaluated for their antioxidant capacity using Trolox Equivalent Antioxidant Capacity (TEAC) and Oxygen Radical Absorbance Capacity (ORAC) assays. In human endothelial cells, we analysed the anti-inflammatory activity of different PASs by measuring their effects on the expression of endothelial adhesion molecules VCAM-1 and ICAM-1. We demonstrated that all the different PASs showed biological activity. They exhibited antioxidant capacity of different magnitude, higher for samples containing non-acylated anthocyanins (typical for fruits) compared to samples containing more complex anthocyanins acylated with cinnamic acid derivatives (typical for vegetables), even though this order was slightly reversed when ORAC assay values were expressed on a molar basis. Concordantly, PASs containing non-acylated anthocyanins reduced the expression of endothelial inflammatory antigens more than samples with aromatic acylated anthocyanins, suggesting the potential beneficial effect of structurally diverse anthocyanins in cardiovascular protection.

Anthocyanins, the naturally occurring pigments responsible for most red to blue colours of flowers, fruits and vegetables, have also attracted interests because of their potential health effects. With the aim of contributing to major insights into their structure-activity relationship (SAR), we have evaluated the radical scavenging and biological activities of selected purified anthocyanin samples (PASs) from various anthocyanin-rich plant materials: two fruits (mahaleb cherry and blackcurrant) and two vegetables (black carrot and ‘Sun Black’ tomato). PASs from the above-mentioned plant material have been evaluated for their antioxidant capacity, using TEAC and ORAC assays. In human endothelial cells, we analysed the biological activity of different PASs by measuring their effects on the expression of endothelial inflammatory markers, including endothelial adhesion molecules VCAM-1 and ICAM-1. We demonstrated that all the different PASs showed biological activity. They exhibited antioxidant capacity of different magnitude, higher for samples containing non-acylated anthocyanins (typical for fruits) compared to samples containing more complex anthocyanins acylated with cinnamic acid derivatives (typical for vegetables), even though this order was slightly reversed when ORAC assay values were expressed on molar basis. Concordantly, PASs containing non-acylated anthocyanins reduced the expression of endothelial inflammatory antigens more than samples with aromatic acylated anthocyanins, suggesting the potential beneficial effect of structurally diverse anthocyanins in cardiovascular protection.

Plant cell cultures as valuable tools for the production of specific metabolites can be greatly improved by the application of elicitors including cyclodextrins (CDs) for enhancing the yields of the desired plant compounds. Here the effects of 2,6-dimethyl-β-cyclodextrins (DIMEB) on the production of carotenoids and quinones from Artemisia annua L. cell suspension cultures were investigated. The addition of 50 mM DIMEB induced an early increase of intracellular carotenoid and quinone contents, which could be observed to a higher extent for lutein (10-fold), Q9 (3-fold) and Q10 (2.5-fold). Real Time PCR analysis revealed that the expression of 1-deoxy-d-xylulose-5-phosphate reductoisomerase (DXR) gene in DIMEB treated cell cultures after three days was 2.5-fold higher than in untreated samples, thus suggesting that the DIMEB induced increase of carotenoids and quinones could be due to the induction of the plastidial isoprenoid biosynthetic route. In addition, the DIMEB treatment induced an enhanced release of carotenoids and quinones into the culture medium of A. annua cell suspension cultures possibly due to the ability of CDs to form inclusion complexes with hydrophobic molecules.

Plant extracts are a rich source of natural compounds with antimicrobial properties, which are able to prevent, at some extent, the growth of foodborne pathogens. The aim of this study was to investigate the potential of polyphenolic extracts from cladodes of Opuntia ficus-indica (L.) Mill. to inhibit the growth of some enterobacteria and the biofilm formation by Staphylococcus aureus. Opuntia ficus-indica cladodes at two stages of development were analysed for total phenolic content and antioxidant activity by Oxygen Radical Absorbance Capacity (ORAC) and Trolox equivalent antioxidant capacity (TEAC) (in vitro assays) and by cellular antioxidant activity in red blood cells (CAA-RBC) (ex vivo assay). The Liquid Chromatography Time-of-Flight Mass Spectrometry (LC/MS&ndash, TOF) analysis of the polyphenolic extracts revealed high levels of piscidic acid, eucomic acid, isorhamnetin derivatives and rutin, particularly in the immature cladode extracts. Opuntia cladodes extracts showed a remarkable antioxidant activity (in vitro and ex vivo), a selective inhibition of the growth of Gram-positive bacteria, and an inhibition of Staphylococcus aureus biofilm formation. Our results suggest and confirm that Opuntia ficus-indica cladode extracts could be employed as functional food, due to the high polyphenolic content and antioxidant capacity, and used as natural additive for food process control and food safety.

Eugenia myrtifolia Sims fruits have been characterized for their anthocyanin content and quality. Following the common procedures and validated methods for separation, identification and quantification of anthocyanins, we found the presence of malvidin 3,5- O -diglucoside as a unique anthocyanin (32,59 mg/100 g fresh weight of the fruit). An in vitro shoot culture of E. myrtifolia was established in order to explore the possibility to produce pigments in vitro . The presence of the only malvidin 3,5- O -diglucoside could be a useful feature in order to manipulate biotechnologically the anthocyanin biosynthetic pathway in the in vitro material (callus and suspension cultures). Industrial relevance The research interest on the phenolic composition of non-traditional food plants is a leading trend in the looking for biofunctional compounds to be included in “designer foods”. Eugenia myrtifolia , a Myrtaceae species from the South hemisphere, produces red edible fruits, that have been characterized for their anthocyanin composition, revealing the presence of the only malvidin 3,5- O -diglucoside, at relatively low amounts. Since attempts for in vitro callus production and for anthocyanin induction from the callus of E. myrtifolia have been carried out, the presence of the specific anthocyanin compound could be a useful feature in order to manipulate biotechnologically the anthocyanin biosynthetic pathway in the in vitro material, pushing the biosynthetic pathway towards the unique final product. Since malvidin is a final product (methoxylated anthocyanin) of the anthocyanin pathway, this in vitro system could help to study the regulation of the pathway, altering the genetic (regulator genes) or physiological control of the pathway. This could allow to obtain better product yields even in another in vitro systems, possibly overcoming the key bottleneck of the secondary metabolite production by plant cell and tissue cultures.

Plant cell cultures are valuable tools for the production of bioactive compounds. Recently, many efforts have been made in order to develop and optimize strategies for increasing the yields of the desired plant metabolites by eliciting their biosynthesis or improving the efficiency of product recovery. We established Artemisia annua L. (Asteraceae) cell suspension cultures to investigate the biosynthesis of the antimalarial artemisinin (AN) [1] and other healthful phytochemicals such as carotenoids and quinones. The use of culture supplementation with cyclic oligomers ?-cyclodextrins was successful to significantly increase intracellular and extracellular levels of AN, lutein, Q9 and Q10 [2, 3]. In order to investigate the mechanism underlying these effects, other oligosaccharides having a linear structure were explored as putative elicitors in A. annua cell suspension cultures. For this purpose, oligogalacturonides (OGA) were obtained by hydrolysis of polygalacturonic acid with pectolyase from Aspergillus japonicus. Various OGA fractions were used for culture medium supplementation at different concentrations. The results showed that the 4-5 OGA fraction induced significant increases of AN and the intermediate dihydroartemisinic acid. These results suggest that the ability of ?-CDs to elicit the production of bioactive compounds in A. annua cell cultures could be due to their chemical similarity to pectic oligosaccharides often occurring in plants after fungal infection.

In the recent years many studies on anthocyanins have revealed their strong antioxidant activity and their possible use as chemotherapeutics. The finding that sour cherries (Prunus cerasus L) (also called tart cherries) contain high levels of anthocyanins that possess strong antioxidant and anti-inflammatory properties has attracted much attention to this species. Here we report the preliminary results of the induction of anthocyanin biosynthesis in sour cherry callus cell cultures. The evaluation and characterization of the in vitro produced pigments are compared to those of the anthocyanins found in vivo in fruits of several sour cherry cultivars. Interestingly, the anthocyanin profiles found in whole fruit extracts were similar in all tested genotypes but were different with respect to the callus extract. The evaluation of antioxidant activity, performed by ORAC and TEAC assays, revealed a relatively high antioxidant capacity for the fruit extracts (from 1145 to 2592 $\mu $ mol TE/100 g FW) and a lower one for the callus extract (688 $\mu $ mol TE/100 g FW).

In the last years many studies on anthocyanins, the flavonoids responsible for the orange, red and blue colors in different plant organs, have revealed their antioxidant activity and a possible use as chemotherapeutics. The finding that tart cherries (Prunus cerasus L.) contain a great amount of anthocyanins, constituents that possess strong antioxidant and anti-inflammatory activities, have attracted much attention. Here, we report the preliminary results of the induction of anthocyanin biosynthesis in sour cherry callus cell cultures and the characterization of those callus cultures for cell growth and anthocyanin production. Callus cultures have been obtained from the leaf of in vitro grown shoots of two sour cherry cultivars, Amarena Mattarello (A.M.) and Stevnsbaer (S.). Several in vitro culture conditions have been tested to induce the anthocyanin biosynthesis using different media with reduced nitrogen and elevated sucrose contents in order to stimulate anthocyanin accumulation. No stimulatory affect from elevated sucrose concentration was observed, while reduced nitrogen and light seems to be the triggering factors. The pigment production is absent during the first days of growth at the light; afterwards, the pigment accumulation is stimulated by elicitors as light and nutritional factors, and reach a maximum nearly the 20 day of culture, during the exponential phase of growth. The in vitro pigment production system described in cherry callus cultures has given these preliminary but encouraging results, making the plant material very suitable and plastic to the in vitro manipulations. INTRODUCTION Anthocyanins, one of the major group of pigments, belonging to the secondary metabolite group of flavonoids, are responsible for the orange, red and blue colors in fruits, vegetables, flowers and other storage tissues in plants. The best known property of flavonoids in general is their strong antioxidant activity in metabolic reactions due to their ability to scavenge oxygen radicals and other reactive species, implicated in ageing process, cancer incidence and oxidative stress. Since it has been reported that anthocyanins inhibit the growth of cancer cells (Kamei et al., 1993) and act as chemotherapeutics for numerous diseases (Bomser et al., 1996), anthocyanins have become an important substance not only as food additive but also for medicine. The finding that tart cherries (Prunus cerasus L.) contain a great amount of anthocyanin (Wang et al., 1997) has attracted much attention to this species. Although cherry biomass is used as meat additive, the use of purified anthocyanins extracted either from fruit biomasses or from cherry cells cultured in vitro is a world novelty. Anthocyanins from cherry have been demonstrated to possess strong antioxidant and anti-inflammatory activities (Wang et al., 1999). Moreover, cyanidin, the anthocyanin aglycone, showed more efficient anti-inflammatory activity than aspirin. Thus, the production of a “natural aspirin” will represent an alternative pharmaceutical source for people with digestive tract ulcer or allergic to aspirin and to non-steroidal anti-inflammatory compounds. The production of anthocyanins from cherry fruits is restricted to the seasonal production. Proc. XXVI IHC – Future for Medicinal and Aromatic Plants Eds. L.E. Craker et al. Acta Hort. 629, ISHS 2004 Publication supported by Can. Int. Dev. Agency (CIDA) 46 Plant cells/tissue cultures represent a continuous source of plant metabolites. Moreover, plant cell culture is an attractive production source, since it lends itself to scaling up or down according to the needs. Here we report the preliminary results of the induction of anthocyanin biosynthesis in sour cherry (Prunus cerasus L.) callus cell cultures and the characterization of those callus cultures in term of cell growth and anthocyanin production. MATERIALS AND METHODS Plant Material and Callus Induction Callus cultures were inducted from the leaf segments of Prunus cerasus L. ‘Amarena Mattarello’ and ‘Stevnsbaer’ on a Callus Induction Medium (CIM) (Table 1). Leaf explants were taken from plants grown in vitro on a Plant Multiplication Medium (PMM) (Table 1). The explants were then incubated in a growth chamber at 25 ± 2 °C at the dark. Callus cultures were maintained on the same “Callus Induction Medium” (CIM) at the dark and transferred to fresh CIM medium every 3 weeks. Biosynthesis Induction of Anthocyanins In Vitro Callus cultures of both cultivar, at the final growth cycle, were transferred on different “Anthocyanin Induction Media” (AIM) (Table 1) and then incubated under the light (Philips TLD/83, 125 μmolmsec), with a 16 h-light photoperiod. Callus growth on different media, at the light or at the dark, has been reported for 40 days as fresh and dry weight. At the same time, anthocyanins from pigmented calli have been extracted, following the methodology reported by Francis (1982). RESULTS AND DISCUSSION Callus production from explants of both cultivar was different. The cv. Amarena Mattarello (A.M.) produced in a short time (20 days) an abundant and friable callus from nearly 100 % of the explants put on CIM, instead the cv. Stevnsbaer (St.) produced less abundant callus from nearly 40 % of the explants. The time course of A.M. callus growth, as fresh and dry weight, follows the typical curves of cell cultures and calli in vitro (Fig. 1). On AIM3, at the light, the increasing of FW was 2300 % and 2000 % for DW, instead on CIM, at the dark, was 2000 % for FW and 1500% for DW. We tested different media with reduced nitrogen and elevated sucrose contents to stimulate anthocyanin accumulation, according to the experiment of Decendit and Merillon (1996) on Vitis cell cultures, and Mori and Sakurai on strawberry suspended cells (1996). In media with elevated sucrose concentration (AIM1 and AIM2) we did not observe an increased anthocyanin accumulation in respect to the control (AIM0), but rather a slightly reduction in growth (data not shown). In media with a changed ammonium:nitrate ratio (AIM3-AIM8) we observed a slight increase in the anthocyanin accumulation. The time course of anthocyanin accumulation in A.M. callus cultures grown on AIM3 is reported in Fig 2. The pigment production is absent during the first days of growth at the light; afterwards, the pigment accumulation is stimulated by elicitors as light and nutritional factors, and reach a maximum nearly the 20 day of culture, during the exponential phase of growth. Even if these experimental data are preliminary and need to be confirmed by additional experiments, studies to deepen our understanding the aspects of anthocyanin biosynthesis in sour cherry cell cultures is worth investigating. On the other hand, the in vitro pigment production system from cherry callus has given these preliminary but encouraging results, making the plant material very suitable and plastic to in vitro manipulations. Previous studies on in vitro sour cherry propagation (Leva et al., 1981, Blando, 2001), protoplasts production and adventitious regeneration (Kondakova and Druart, 1997; Blando, in prep.) emphasized this aspect. In conclusion, studies on triggering factors for anthocyanin biosynthesis, pigment identification and characterisation of key enzymes in the flavonoid pathway are the future research we intend to approach.

A beta-glucosidase (beta-D-glucoside glucohydrolase, EC 3.2.1.21) was purified to homogeneity from ripe fruits of sweet cherry (Prunus avium L.) by ammonium sulphate precipitation, ion exchange and size exclusion chromatography. The enzyme is a monomer with a molecular mass of approximately 68 kDa and an acidic isoelectric point. N-terminal sequence analysis indicated that sweet cherry beta-glucosidase is related to other plant cyanogenic beta-glucosidases. Substrate specificity studies revealed that the enzyme is able to attack and hydrolyse several synthetic substrates and total cell walls purified from ripe fruit. Biochemical and immunolocalisation studies showed that sweet cherry beta-glucosidases are mainly localised in the cytosol and in the apoplast, at the unripe stage of ripening; in ripe fruit it is also associated with cell wall.

Characterization of polyphenols and antioxidant activity (ORAC) in cherry cultivars of Apulia - Extracts of sweet cherries (Prunus avium L.) grown in a suitable area of cherry cultivation of Apulia (the south-east area around Bari) were characterized. High-performance liquid chromatographic methods were used to identify and quantify phenolics. A total of five phenolic compounds were characterised as hydroxycinnamic acid derivatives (neochlorogenic acid, p-coumaroylquinic acid and chlorogenic acid) and anthocyanins (cyanidin 3-glucoside, cyanidin 3-rutinoside). The total anthocyanin content ranged from 0.17 to 8.43 mg keracyanin equivalents/g dry weight. The oxygen radical absorbance capacity (ORAC) assay indicated that fruits of indigenous and rare genotypes possessed considerable antioxidant activity. The results of these studies can allow to identify genotypes with good antioxidant capacity, in order to enhance the cherry production in Apulia through breeding for improved nutraceutical characteristics.

Eugenia myrtifolia Sims. is an evergreen shrub, native to temperate and tropical rainforests of Australia, which is becoming an important containerized ornamental plant in the US and Mediterranean nursery industries. To satisfy the growing market demands for this new ornamental plant, development of an accelerated propagation method is required. The goal of this study was to investigate the in vitro regeneration potential of E. myrtifolia Sims. seeds at different stages of development, towards establishment of an in vitro multiplication system. Maximum regeneration of adventitious shoots was achieved from immature seeds cultured in the dark on half-strength Murashige and Skoog (MS) macronutrients and full-strength MS micronutrients and vitamins (MS/2) medium supplemented with 2.5 ?M thidiazuron (TDZ). Induction of regeneration occurred after at least two successive subcultures on TDZ-enriched medium, followed by subcultures on expression medium (hormone free MS/2) or multiplication medium [MM; MS medium enriched with 4.4 M 6-benzyladenine and 0.05 M ?-naphthaleneacetic acid], where complete development of shoots occurred. The regenerated shoots were excised and transferred again onto MM for micropropagation, where a proliferation rate of 1:4 was achieved, and finally the shoots were transferred to a hormone-free MS medium for rooting. Following ex vitro transplanting, acclimatization over a period of 15 d was sufficient to establish greenhouse plants. The regenerated plants grown in the field for more than 2 yr showed the same phenotype as that of mother plants. The adventitious regeneration and micropropagation carried out in this study can be used for a large-scale propagation and genetic engineering of E. myrtifolia Sims. © 2013 The Society for In Vitro Biology. Acknowledgements The authors would like to thank Dr. M. Beruto for useful advice. We also thank Mr. Gervasio and Mr. Protasio Tarantino, from ‘Vivai Tarantino’ (Lecce), for providing the plant material. This work was supported by Apulia Region, Italy. Project PS070.

Prunus mahaleb L. is a rootstock used for sweet cherry crop in Apulia (Italy), an important cherry producing and exporting Italian Region. The hydro-alcoholic extract of P. mahaleb fruits has been analyzed for anthocyanin content and antimutagenic activity. HPLC/DAD/MS analysis indicated that 'Mahaleb' cherry fruit is extremely rich in anthocyanins (1751 mg/100 g D.W, more than 500 mg /100g F.W.), more than sweet and sour cherries. Moreover the protective effect of P. mahaleb was investigated by using D7 strain of yeast Saccharomyces cerevisiae. Results showed that treatments with P. mahaleb fruit extract decreased significantly the mutagenesis induced by hydrogen peroxide in S.cerevisiae.

A novel strategy to induce parthenocarpy in tomato fruits by the induction of resveratrol biosynthesis in flower tissues was exploited. Two transgenic tomato lines were considered: a higher resveratrol-producing (35SS) line, constitutively expressing a grape stilbene synthase cDNA, and a lower resveratrol-producing (LoxS) line, expressing stilbene synthase under a fruit-specific promoter. The expression of the stilbene synthase gene affected flavonoid metabolism in a different manner in the transgenic lines, and in one of these, the 35SS line, resulted in complete male sterility. Resveratrol was synthesised either in 35SS or LoxS tomato flowers, at an even higher extent (about 8–10 times) in the former line. We further investigated whether stilbene synthase expression may have resulted in impaired naringenin accumulation during flower development. In the 35SS flowers, naringenin was significantly impaired by about 50%, probably due to metabolic competition. Conversely, the amount of glycosylated flavonols increased in transgenic flowers, thereby excluding the diminished production of flavonols as a reason for parthenocarpy in tomato. We further investigated whether resveratrol synthesis may have resulted changes to pollen structure. Microscopic observations revealed the presence of few and abnormal flake-like pollen grains in 35SS flowers with no germination capability. Finally, the analysis of coumaric and ferulic acids, the precursors of lignin and sporopollenin biosynthesis, revealed significant depletion of these compounds, therefore suggesting an impairment in structural compounds as a reason for pollen ablation. These overall outcomes, to the best of our knowledge, reveal for the first time the major role displayed by resveratrol synthesis on parthenocarpy in tomato fruits.

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