<Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated genetic transformation of grapevine (<Emphasis Type="Italic">Vitis vinifera</Emphasis> L.) with a novel stilbene synthase gene from Chinese wild <Emphasis Type="Italic">Vitis pseudoreticulata</Emphasis>

A novel stilbene synthase gene (STS), cloned from Chinese wild Vitis pseudoreticulata (W. T. Wang) and responsible for synthesis of the phytoalexin resveratrol in grapevine, was successfully transferred into V. vinifera L. cv. Thompson Seedless via Agrobacterium tumefaciens-mediated transformation. Using transformation procedures developed in the present study, 72% GFP-positive germinated embryos were produced with about 38% of transformed embryos regenerated into normal plantlets. Integration of the STS gene into the transgenic plants was verified by PCR and Southern blot analysis. Expression of the STS gene was detected by high performance liquid chromatography (HPLC), which showed that the resveratrol concentration in the transgenic plants was 5.5 times higher than that in non-transformed control plants. Chaohong Fan and Ni Pu contributed equally to this work.     

Hairy root culture optimization and resveratrol production from <Emphasis Type="Italic">Vitis vinifera</Emphasis> subsp. <Emphasis Type="Italic">sylvesteris</Emphasis>

Resveratrol is a polyphenolic compound produced in very low levels in grapes. To achieve high yield of resveratrol in wild grape, three Agrobacterium rhizogenes strains, Ar318, ArA4 and LBA9402, were used to induce hairy roots following infection of internodes, nodes or petioles of in vitro grown Vitis vinifera subsp. sylvesteris accessions W2 and W16, and cultivar Rasha. The effects of inoculation time, age of explants, bacterial concentration and co-cultivation times were examined on the efficiency of the production of hairy roots. Strains Ar318, ArA4 and LBA9402 all induced hairy roots in the tested genotypes, but the efficiency of ArA4 strain was higher than the other strains. The highest hairy root production was with using internodes as explants. The transformation of hairy roots lines was confirmed by PCR detection of rolB gene. Half Murashige and Skoog (MS) medium was better for biomass production compared with MS medium. HPLC analysis of resveratrol production in the hairy root cultures showed that all the genotypes produced higher amounts of resveratrol than control roots. The highest amount of resveratrol was produced from W16 internode cultures, which was 31-fold higher than that of control root. Furthermore, TLC analysis showed that treatments of hairy roots with sodium acetate and jasmonate elevated resveratrol levels both in hairy root tissue and excreted into the half MS medium. These results demonstrate that endogenous and exogenous factors can affect resveratrol production in hairy root culture of grape, and this strategy could be used to increase low resveratrol production in grapes.     

Early detection of grapevine leafroll virus in <Emphasis Type="Italic">Vitis vinifera</Emphasis> using <Emphasis Type="Italic">in vitro</Emphasis> micrografting

Micrografting of grapevine was investigated for its use as a tool in virus indexing of grapevine stock. Cabernet franc and Cabernet sauvignon scions infected with grapevine leafroll-associated closterovirus III (GLRaVIII) were grafted on to virus-free indicator rootstocks of LN 33 and Cabernet sauvignon growing in tissue culture. The two rootstocks and two scions were grafted in all four possible combinations along with two control grafts (virus-free scion on virus-free rootstock). A modified MS Murashige and Skoog (1962) tissue culture medium supplemented with 0.5 mg l^–1 6-benzylaminopurine was sufficient to induce multiple shoots. Shoots and micrografts readily produced roots in the basal medium. Micrografting gave an overall success rate of 77.8%, with no significant difference between LN 33 rootstock and Cabernet sauvignon. When leafroll infected scion material was micrografted on to virus-free rootstock, the rootstock leaf turned red (23.5% in LN 33 and 63.9% in Cabernet sauvignon) or it showed leafrolling (28.5%, no significant difference between LN 33 and Cabernet sauvignon) within 2–3 weeks. After 12 weeks in culture, the extent of viral symptoms in the micrografted material was high (81.3%), with no significant difference between LN 33 and Cabernet sauvignon; however, the expression of symptoms was more severe on Cabernet sauvignon than on LN 33 rootstock. Double antibody sandwich-enzyme linked immunosorbent assay (DAS-ELISA) was used to validate the visual symptoms and the presence of virus was confirmed in 80% of the rootstock with visual symptoms of infection. Results indicate that micrografting is an effective method for viral indexing of grapevines. The method can be used in conjunction with wood indexing for post-entry quarantine to identify infected material and reject it much earlier than is currently possible.     

Photorespiration and photoprotection of grapevine (<Emphasis Type="Italic">Vitis vinifera</Emphasis> L. cv. Cabernet Sauvignon) under water stress

In order to investigate the photoprotective function of photorespiration in grapevine under water stress, potted grapevines (Vitis vinifera L. cv. Cabernet Sauvignon) were randomly divided into three uniform groups for well-watered [watered every morning to keep the relative water content (RWC) of soil over 70 %], water-stress adapted (drought-adapted at 30 % relative soil water content for 30 days), and water stress without adaptation treatment (water-stressed to 30 % relative soil water content for 3 days). Net assimilation rate (A _N), stomatal conductance (g _s), substomatal CO₂ concentration (C _i), transpiration rate (E), actual photochemical efficiency of PSII (Φ_PSII), and maximum photochemical efficiency of PSII (F_v/F_m) were recorded by combining measurements of gas exchange and chlorophyll fluorescence. Gross photorespiration (P_r), photosynthetic electron partitioning (J_C/J_T), photochemical quenching coefficient (qP), and non-photochemical quenching (NPQ) were also calculated. The ratio of net assimilation rate to transpiration rate (A _N/E) was used as an indicator of water use efficiency (WUE). A _N, apparent P_r, Φ_PSII, F_v/F_m, qp, and g _s decreased, NPQ increased, and gross P_r sustained at a high level under water stress. This suggests that both photorespiration and energy dissipation play important roles in protecting photosynthetic apparatus against photoinhibition. C _i in water-stressed plants without adaptation treatment increased, which indicates the leaves suffered a non-stomatal limitation, while the water-stress adaped plants only suffered a stomatal limitation indicated by low C _i.     

Development of SCAR markers linked to powdery mildew (<Emphasis Type="Italic">Uncinula necator</Emphasis>) resistance in grapevine (<Emphasis Type="Italic">Vitis vinifera</Emphasis> L. and <Emphasis Type="Italic">Vitis</Emphasis> sp.)

Sequence-characterized amplified regions markers (SCARs) were developed from six randomly amplified polymorphic DNA (RAPD) markers linked to the major QTL region for powdery mildew (Uncinula necator) resistance in a test population derived from the cross of grapevine cultivars “Regent” (resistant) × “Lemberger”(susceptible). RAPD products were cloned and sequenced. Primer pairs with at least 21 nucleotides primer length were designed. All pairs were tested in the F1 progeny of “Regent” × “Lemberger”. The SCAR primers resulted in the amplification of specific bands of expected sizes and were tested in additional genetic resources of resistant and susceptible germplasm. All SCAR primer pairs resulted in the amplification of specific fragments. Two of the SCAR markers named ScORA7-760 and ScORN3-R produced amplification products predominantly in resistant individuals and were found to correlate to disease resistance. ScORA7-760, in particular, is suitable for marker-assisted selection for powdery mildew resistance and to facilitate pyramiding powdery mildew resistance genes from various sources.     

A <Emphasis Type="Italic">Vitis vinifera</Emphasis> xanthine dehydrogenase gene, <Emphasis Type="Italic">VvXDH</Emphasis>, enhances salinity tolerance in transgenic <Emphasis Type="Italic">Arabidopsis</Emphasis>

Xanthine dehydrogenase (EC1.1.1.204; XDH) plays an important role in purine catabolism that catalyzes the oxidative hydroxylation of hypoxanthine to xanthine and of xanthine to uric acid. Long attributed to its role in recycling and remobilization of nitrogen, recently, XDH is implicated in plant stress responses and acclimation, such research efforts, however, have thus far been restricted to Arabidopsis XDH-knockdown/knockout studies. This study, using an ectopic overexpression approach, is expected to provide novel findings. In this study, a XDH gene from Vitis vinifera, named VvXDH, was synthesized and overexpressed in Arabidopsis, the transgenic Arabidopsis showed enhanced salt tolerance. The VvXDH gene was investigated and the results demonstrated the explicit role of VvXDH in conferring salt stress by increasing allantoin accumulation and activating ABA signaling pathway, enhancing ROS scavenging in transgenic Arabidopsis. In addition, the water loss and chlorophyll content loss were reduced in transgenic plants; the transgenic plants showed higher proline level and lower MDA content than that of wild-type Arabidopsis, respectively. In conclusion, the VvXDH gene has the potential to be applied in increasing allantoin accumulation and enhancing the tolerance to abiotic stresses in Arabidopsis and other plants.     

Genetic diversity of <Emphasis Type="Italic">Vitis vinifera</Emphasis> L. in Azerbaijan

To examine the genetic diversity of Vitis vinifera L., growing in the region near the Caspian Sea of Azerbaijan Republic, nuclear genomes of 31 cultivated and 34 wild grapevine accessions were studied at population and individual levels using five ISSR primers. In total, 51 fragments were amplified, of which 45 were found to be polymorphic. A high level of polymorphism was revealed (the mean PPF and PIC values constituted 87.69% and 0.94, respectively). High values of the EMR, MI, and RP indices showed the effectiveness of the application of ISSR primers and the possibility of their use in further investigations in this direction. Cluster analysis based on Nei’s genetic distance values showed that all genotypes could be grouped into seven main clusters. Furthermore, no differences between the wild and cultivated grape wine accessions were revealed. For instance, there was no distinct distribution of the accessions according to their geographical localization. On the basis of the PIC values, the group of cultivars from Absheron Peninsula was distinguished by the highest polymorphism level (PIC = 0.36). Natural populations from the Guba and Shabran regions were characterized by a relatively low polymorphism level (PIC = 0.31 and PIC = 0.28, respectively), and a wild population from Nabran demonstrated the lowest polymorphism level (PIC = 0.25). The data obtained confirmed paleontological and historical data of different periods and provided the supposition that Azerbaijan was the center of diversity of V. vinifera L. In addition, our data indicate that Azerbaijan grape landraces originated from local wild forms.     

Stress effects of flumioxazin herbicide on grapevine (<Emphasis Type="Italic">Vitis vinifera</Emphasis> L.) grown in vitro

Among the herbicides used in vineyards, the pre-emergence soil-applied flumioxazin (FMX) is a recently synthesized molecule that inhibits chlorophyll biosynthesis in weed species. The aim of this work was to characterize the effects of FMX on non-target grapevine (Vitis vinifera L. cv. Chardonnay) plantlets grown in vitro. FMX treatment (from 1 to 100 M) represented a stress, as revealed by measurement of several parameters. Stem and leaves underwent dehydration and a decrease in both water- and osmotic-potential. Treated plantlets exhibited concomitant accumulation of soluble carbohydrates in all tissues and of free proline in stems and leaves. Moreover, FMX caused lipid peroxidation and electrolyte leakage in leaf tissues. These results indicate that the herbicide FMX is toxic for grapevine grown in vitro. In addition to inhibiting protoporphyrinogen IX oxidase, it causes water stress and membrane alteration in tissues and, as a consequence, generates the accumulation of carbohydrates and free proline.Abbreviations FMX Flumioxazin - MDA Malondialdehyde - PROTOX Protoporphyrinogen IX oxidase - TBARS Thiobarbituric acid reactive substances - TFAA Total free amino acids - TSS Total soluble sugarsCommunicated by S. Gleddie     

Proteomic analysis of somatic embryogenesis in <Emphasis Type="Italic">Vitis vinifera</Emphasis>

Two dimensional gel electrophoresis coupled to mass spectrometry has been used to study the somatic embryogenesis in Vitis vinifera, by comparing embryogenic and non embryogenic calluses of the Thompson seedless cv. More than 1,000 spots were reproducibly resolved in colloidal Coomassie brilliant blue stained gels over a pI nonlinear range of 3–10 in the first dimension and using homogeneous 12.5% polyacrylamide gels in the second dimension. The expression pattern of 35 spots differed significantly between the two samples. These spots were processed by mass spectrometry analysis and the protein identity was assigned by using both the non-redundant protein and EST databases. Several responsive proteins, some already known to be involved in the somatic embryogenesis process while others, for the first time put into relation with this process, have been described. Moreover, they have been subdivided in functional categories, and their putative role is discussed in terms of their relevance in the somatic embryogenesis process.     

Secreted proteins produced by fungi associated with <Emphasis Type="Italic">Botryosphaeria</Emphasis> dieback trigger distinct defense responses in <Emphasis Type="Italic">Vitis vinifera</Emphasis> and <Emphasis Type="Italic">Vitis rupestris</Emphasis> cells

Grapevine trunk diseases (Eutypa dieback, esca and Botryosphaeria dieback) are caused by a complex of xylem-inhabiting fungi, which severely reduce yields in vineyards. Botryosphaeria dieback is associated with Botryosphaeriaceae. In order to develop effective strategies against Botryosphaeria dieback, we investigated the molecular basis of grapevine interactions with a virulent species, Neofusicoccum parvum, and a weak pathogen, Diplodia seriata. We investigated defenses induced by purified secreted fungal proteins within suspension cells of Vitis (Vitis rupestris and Vitis vinifera cv. Gewurztraminer) with putative different susceptibility to Botryosphaeria dieback. Our results show that Vitis cells are able to detect secreted proteins produced by Botryosphaeriaceae, resulting in a rapid alkalinization of the extracellular medium and the production of reactive oxygen species. Concerning early defense responses, N. parvum proteins induced a more intense response compared to D. seriata. Early and late defense responses, i.e., extracellular medium alkalinization, cell death, and expression of PR defense genes were stronger in V. rupestris compared to V. vinifera, except for stilbene production. Secreted Botryosphaeriaceae proteins triggered a high accumulation of δ-viniferin in V. vinifera suspension cells. Artificial inoculation assays on detached canes with N. parvum and D. seriata showed that the development of necrosis is reduced in V. rupestris compared to V. vinifera cv. Gewurztraminer. This may be related to a more efficient induction of defense responses in V. rupestris, although not sufficient to completely inhibit fungal colonization. Overall, our work shows a specific signature of defense responses depending on the grapevine genotype and the fungal species.     

In planta transformation of <Emphasis Type="Italic">Notocactus scopa</Emphasis> cv. <Emphasis Type="Italic">Soonjung</Emphasis> by <Emphasis Type="Italic">Agrobacterium </Emphasis><Emphasis Type="Italic">tumefaciens</Emphasis>

Notocactus scopa cv. Soonjung was subjected to in planta Agrobacterium tumefaciens-mediated transformation with vacuum infiltration, pin-pricking, and a combination of the two methods. The pin-pricking combined with vacuum infiltration (20-30 cmHg for 15 min) resulted in a transformation efficiency of 67-100%, and the expression of the uidA and nptII genes was detected in transformed cactus. The established in planta transformation technique generated a transgenic cactus with higher transformation efficiency, shortened selection process, and stable gene expression via asexual reproduction. All of the results showed that the in planta transformation method utilized in the current study provided an efficient and time-saving procedure for the delivery of genes into the cactus genome, and that this technique can be applied to other asexually reproducing succulent plant species.     

Photoinhibition of photosynthesis in leaves of grapevine (<Emphasis Type="Italic">Vitis vinifera</Emphasis> L. cv. Riesling). Effect of chilling nights

Photoinhibition of photosynthesis was investigated in control (C) and chilling night (CN) leaves of grapevine under natural photoperiod at different sampling time in a day. The degree of photoinhibition was determined by means of the ratio of variable to maximum chlorophyll fluorescence (F_v/F_m) and photosynthetic electron transport measurements. When the potential efficiency of photosystem (PS) 2, F_v/F_m was measured at midday, it markedly declined with significant increase of F₀ in CN leaves. In isolated thylakoids, the rate of whole chain and PS2 activity were markedly decreased in CN leaves than control leaves at midday. A smaller inhibition of PS1 activity was also observed in both leaf types. Later, the leaves reached maximum PS2 efficiencies similar to those observed in the morning during sampling at evening. The artificial exogenous electron donors diphenyl carbazide, NH₂OH, and Mn²⁺ failed to restore the PS2 activity in both leaf types at midday. Thus CN enhanced inactivation on the acceptor side of PS2 in grapevine leaves. Quantification of the PS2 reaction centre protein D1 following midday exposure of leaves showed pronounced differences between C and CN leaves. The marked loss of PS2 activity in CN leaves noticed in midday samples was mainly due to the marked loss of D1 protein of the PS2 reaction centre.     

<Emphasis Type="Italic">VpUR9</Emphasis>, a novel RING-type ubiquitin ligase gene from <Emphasis Type="Italic">Vitis pseudoreticulata</Emphasis>, is involved in powdery mildew response in transgenic <Emphasis Type="Italic">V. vinifera</Emphasis> plants

Ubiquitination plays important roles in disease resistance in plants. We report the identification and functional characterization of the RING-type ubiquitin ligase gene VpUR9 from Chinese wild Vitis pseudoreticulata accession Baihe-35-1. VpUR9, encodes 164 amino acids and possesses a RING conserved motif. It is homologously cloned from the cDNA library of the high powdery mildew (Erysiphe necator [Schw.] Burr) resistant V. pseudoreticulata accession Baihe-35-1 inoculated with E. necator. The gene is induced in response to powdery mildew and salicylic acid. VpUR9 fused with FLAG-tag controlled by 35S promoter was transformed into 15 regenerated V. vinifera L. cv. Red Globe lines via Agrobacterium tumefaciens-mediated transformation. Twelve of these lines were confirmed by Western blot of FLAG-tag. As a result, the powdery mildew-resistance of Red Globe transformed with VpUR9 was repressed. Furthermore, the expression of some disease-resistant related genes (NPR1, PR1, PR10 and PAL) of the transgenic Red Globe declined compared with wild type grapes when inoculated with powdery mildew or salicylic acid. When treated with jasmonic acid methyl ester, its PR1 gene expression decreased, while the expressions of NPR1, PR10 and PAL all increased, contrasting with the wild type grape.     

Differences between sympatric populations of <Emphasis Type="Italic">Eotetranychus carpini</Emphasis> collected from <Emphasis Type="Italic">Vitis vinifera</Emphasis> and <Emphasis Type="Italic">Carpinus betulus</Emphasis>: insights from host-switch experiments and molecular data

Eotetranychus carpini (Oudemans) is an important pest of grapevine (Vitis vinifera L.) in southern Europe. This mite is also found on a number of different plants, including Carpinus betulus L., which commonly occurs in stands and hedgerows bordering vineyards, where it may serve as a potential mite reservoir. The economic importance of this pest has motivated a number of studies aimed at investigating whether the mites found on V. vinifera and C. betulus are conspecific. The results obtained to date have been inconclusive. In this study, we used biological and molecular approaches to investigate this issue. First, we conducted host-switch experiments to test the ability of E. carpini to develop on an alternative host plant, using mite populations originally collected on either C. betulus or V. vinifera plants from the same area. Second, we investigated DNA-based differentiation using nucleotide sequences of the ITS1-5.8S-ITS2 region of the ribosomal DNA of individual E. carpini from the populations examined in our host-plant experiments. We also analyzed sequences of individuals collected in other regions (Italy and Slovenia) to estimate species variation. The results from our host-switch experiments suggest the differentiation of mites collected on the two hosts. Mites collected from C. betulus did not survive and reproduce on V. vinifera and vice versa. Our molecular work revealed significant genetic differentiation between the mites collected from the two hosts, but no evidence of genetic variation among specimens collected from the same host species. Our results indicate the existence of host races of E. carpini.     

Characterisation of the <Emphasis Type="Italic">Vitis vinifera</Emphasis>PR10 multigene family

Background  

Genes belonging to the pathogenesis related 10 (PR10) group have been studied in several plant species, where they form multigene families. Until now, such an analysis has not been performed in Vitis vinifera, although three different PR10 genes were found to be expressed under pathogen attack or abiotic stress, and during somatic embryogenesis induction. We used the complete genome sequence for characterising the whole V. vinifera PR10 gene family. The expression of candidate genes was studied in various non-treated tissues and following somatic embryogenesis induction by the auxin 2,4-D.     

Chitosan improves development,and protects<Emphasis Type="Italic"> Vitis vinifera</Emphasis> L. against<Emphasis Type="Italic"> Botrytis cinerea</Emphasis>

We evaluated the potential of chitosan both to stimulate plant development and to induce protection from Botrytis cinerea in Vitis vinifera L. plantlets. The presence of 1.75% (v/v) chitogel in the culture medium was the optimal concentration for in vitro grapevine plantlet growth, as determined by measurements on enhancement of root and shoot biomass. Photosynthesis and related parameters were also stimulated in chitogel-treated plantlets. Chitogel reduced the development of Botrytis cinerea and induced cytological alterations to the pathogen. When challenged with the fungus, a significant decrease in disease incidence was observed in plants growing on medium supplemented with chitogel. Furthermore, exogenous foliar applications of chitogel to plantlets growing on chitogel-free medium sensitized them so as to be protected against Botrytis cinerea attack. Our results indicate that chitogel can be used in the vineyard as a means to attain protection against Botrytis cinerea and that its application may counteract the wide use of chemical pesticides.Communicated by S. Gleddie     

Selection and regeneration of <Emphasis Type="Italic">Vitis vinifera</Emphasis> Chardonnay hydroxyproline-resistant calli

Proline (Pro) accumulation protects plant cell under abiotic stress. Hydroxyproline (Hyp) as selection agent is a toxic analog of proline and promotes Pro overaccumulation. In this study, Chardonnay calli were firstly irradiated with different dosages of ⁶⁰Co and then cultured on a Hyp-added medium. Finally, some stable hydroxyproline-resistant (HR) calli were obtained. When calli were cultured on 4 mM Hyp medium for 7 days, intracellular Pro content of the HR calli was five times higher than that detected in the normal calli. The regeneration of HR calli into plantlets was much slower than that of normal ones. When cultured on woody plant medium (WPM) containing 10 mM NaCl for 14 days, HR plantlets still grew well with lower Pro than withered normal plantlets. qRT-PCR results of Pro biosynthesis-related genes in HR plantlets showed that three genes VvP5CS, VvOAT, and VvP5CDH were conducive for Pro accumulation. These results confirmed that HR plantlets acquired salt tolerance ability. We prospect that this procedure to obtain salt-tolerant plants may be valuable to breed programs and improve grapevine genotypes with increased tolerance to salt and other abiotic stresses.