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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.

Using four different chromatographic steps, β-galactosidase was purified from the ripe fruit of sweet cherry to apparent electrophoretic homogeneity with approximately 131-fold purification. The Prunus avium β-galactosidase showed an apparent molecular mass of about 100 kDa and consisted of four different active polypeptides with pIs of about 7.9, 7.4, 6.9 and 6.4 as estimated by native IEF and β-galactosidase-activity staining. The active polypeptides were individually excised from the gel and subjected to SDS-PAGE. Each of the four native enzymes showing β-galactosidase activity was composed of two polypeptides with an estimated mass of 54 and 33 kDa. Both of these polypeptides were subjected to N-terminal amino acid sequence analysis. The 54 kDa polypeptide of sweet cherry β-galactosidase showed a 43% identity with the 44 kDa subunit of persimmon and apple β-galactosidases and the 48 kDa subunit of carambola galactosidase I. The sweet cherry β-galactosidase exhibited a strict specificity towards p-nitrophenyl β- d -galactopyranoside, a pH optimum of 4.0 and Km and Vmax values of 0.42 mM and 4.12 mmol min−1 mg−1 of protein respectively with this substrate. The enzyme was also active towards complex glycans. Taken together the results of this study prompted a role for this class of enzymes on sweet cherry fruit ripening and softening.

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.

Three different Arabidopsis thaliana transgenic lines overexpressing AtHXK1, AtHXK2 and yeast HXK2 genes, respectively, were analyzed with regard to their hexokinase activity. The methodology employed was a non-denaturing electrophoresis followed by activity-staining. Three distinct hexokinase activities were detected and named, respectively, AtHXK1, AtHXK2 and YHXK2. Arabidopsis HXK1 and HXK2 showed high affinity for glucose, minor affinity for mannose, and very low specificity for fructose. In comparison, YHXK2 exhibited a higher affinity for both mannose and fructose. Galactose, 3-O-methyl glucose and 6-deoxyglucose showed no influence on hexokinase activity, while glucosamine and 2- deoxyglucose produced a clear inhibition at high concentrations. Trehalose did not cause inhibition. Instead, trehalose 6- phosphate (T6P) was an effective inhibitor of YHXK2 but not of AtHXK1 and AtHXK2. As a whole, these results indicate important differences between the properties of plant and yeast HXKs and provide useful information for a discussion concerning the sugar response of transgenic plants overexpressing AtHXK or YHXK2 genes.