Agrobacterium rhizogenes mediated transformation of grapevine (Vitis vinifera L.)

Genetically transformed grapevine (Vitis vinifera L.) roots were obtained after inocultation of in vitro grown whole plants (cv. Grenache) with Agrobacterium rhizogenes. The strain used contains two plasmids: the wild-type Ri plasmid pRi 15834 and a Ti-derived plasmid which carries a chimaeric neomycin phosphotrans-ferase gene (NPT II) and the nopaline synthase gene. Expression of the NPT II gene can confer kanamycin resistance to transformed plant cells. Slowly growing axenic root cultures derived from single root tips were obtained. Opine analysis indicated the presence of agropine and/or nopaline in established root cultures. For one culture, the presence of T-DNA was confirmed by dot-blot hybridization with pRi 15834 TL-DNA. Callogenesis was induced by subculturing root fragments on medium supplemented with benzylaminopurine and indoleacetic acid.Transformation of in vitro cultured grapevine cells has recently been reported (baribault T.J. et al., Plant Cell Rep (1989) 8: 137–140). In contrast with the results presented here, expession of the NPT II gene Conferred kanamycin resistance to Vitis vinifera calli that was sufficient for selection of trasformed cells.Abbreviations BAP benzylaminopurine - IAA indoleacetic acid - NAA naphtaleneacetic acid - NPT II neomycin phosphostransferase II - EDTA ethylenediaminetetraacetic acid     

Cryopreservation of grapevine (Vitis vinifera L.) in vitro shoot tips

In this work, we compared the efficiency of encapsulation-dehydration and droplet-vitrification techniques for cryopreserving grapevine (Vitis vinifera L.) cv. Portan shoot tips. Recovery of cryopreserved samples was achieved with both techniques; however, droplet-vitrification, which was used for the first time with grapevine shoot tips, produced higher regrowth. With encapsulationdehydration, encapsulated shoot tips were precultured in liquid medium with progressively increasing sucrose concentrations over a 2-day period (12 h in medium with 0.25, 0.5, 0.75 and 1.0 M sucrose), then dehydrated to 22.28% moisture content (fresh weight). After liquid nitrogen exposure 37.1% regrowth was achieved using 1 mm-long shoot tips and only 16.0% with 2 mm-long shoot tips. With droplet-vitrification, 50% regrowth was obtained following treatment of shoot tips with a loading solution containing 2 M glycerol + 0.4 M sucrose for 20 min, dehydration with half-strength PVS2 vitrification solution (30% (w/v) glycerol, 15% (w/v) ethylene glycol, 15% dimethylsulfoxide and 0.4 M sucrose in basal medium) at room temperature, then with full strength PVS2 solution at 0°C for 50 min before direct immersion in liquid nitrogen. No regrowth was achieved after cryopreservation when shoot tips were dehydrated with PVS3 vitrification solution (50% (w/v) glycerol and 50% (w/v) sucrose in basal medium).     

Berry morphology and composition in irrigated and non-irrigated grapevine (Vitis vinifera L.)

The present study was carried out in a 5-year-old vineyard (Vitis vinifera L., cv. Aglianico) located in Southern Italy. Half of the plants (IRR) were fully irrigated, whereas the other half were not irrigated (NIRR). In both of the treatments, plant water status, gas exchange, photosynthetic efficiency and productive performance were determined. The arid conditions resulted in significant decreases in stem water potential in NIRR (minimum values of -1.34 and -1.52 MPa in IRR and NIRR, respectively). The values of yield per plant, cluster weight and total berry weight were significantly higher in IRR. Grape berries were separated into four weight classes, and morphometric and microscopic analyses were carried out to measure and calculate berry skin characteristics. Irrigation determined a marked shift toward heavier (+23% in the class ≥ 1.25 g) and bigger (336.35 mm3 vs 299.15 mm3) berries, and induced significant changes in other morphometric berry parameters. No differences among berry weight classes and irrigation treatments were observed for berry skin thickness. In all of the berry weight classes, total anthocyanins extracted from berry skins were significantly higher in NIRR than in IRR (12301.53 and 9585.52 mg kg?1 fresh berry skin, respectively), and appeared to be positively related to berry weight, whereas total flavonols were not significantly different between the two treatments. Qualitative changes in the levels of single anthocyanin and flavonol compounds were detected between IRR and NIRR. In addition, iron, copper and zinc, whose high concentration can negatively affect wine quality, were significantly higher in the IRR treatment. The results highlighted that the absence of irrigation did not determine decreases in grape quality. Such data can be of primary importance in environments where water availability is by far the most important limiting factor for plant growth.     

An improved protocol for Agrobacterium-mediated transformation of grapevine (Vitis vinifera L.)

An improved protocol for efficient Agrobacterium-mediated transformation of grapevine (Vitis sp.) was developed through modification of cocultivation and subsequent washing procedures. It was determined that Agrobacterium-infected somatic embryos (SE) cocultivated on filter paper exhibited less browning and significantly higher transient GFP and GUS expression than those cultured on agar-solidified medium. Furthermore, such SE, when subjected to a prolonged washing period in liquid medium containing cefotaxime and carbenicillin, followed by another wash in similar medium with kanamycin added, exhibited significantly higher rates of stable transformation compared to previously-described procedures. Transgenic plant recovery was increased 3.5–6 Xs by careful excision of leafy cotyledons from SE that had been induced to germinate on MS medium containing 1 μM of BA. Southern blot analysis revealed the low copy number integration of transgenes in transgenic plants recovered using the improved protocol. These improved cocultivation and plant recovery procedures have been demonstrated to facilitate production of large populations of transgenic plants from V. vinifera ‘Merlot’, ‘Shiraz’ and ‘Thompson Seedless’ as well as Vitis hybrid ‘Seyval Blanc’.     

Partitioning and mobilization of starch and N reserves in grapevine (Vitis vinifera L.)

We followed C and N reserves of grapevines grown in trenches under semi-controlled conditions over a 3-year period after planting. Temporal mobilization of stored C and N and subsequent distribution of reserve materials within the vines were described in parallel with 15N uptake, particularly during the third growing season. Storage C in the perennial tissues (roots, trunk, canes) was mainly made of starch, which accumulated in the ray parenchyma of the wood. In the permanent tissues, starch and total nitrogen contents were found to decrease early in the development (bleeding sap, budbreak) whereas, on a concentration basis, they decreased only after stage 7 (first leaf fully expanded). Starch started to accumulate again in the perennial tissues during flowering. The same observation was made with total nitrogen, although N levels were much lower than those of starch. The 15N study showed that N uptake by the roots started at budbreak and increased with vine development, becoming predominant over reserve mobilization only after the onset of flowering. Taken together, these results indicate that the spring growth period can be divided into three main phases: In the first (dormancy to budbreak), significant losses of C and N proceed mainly via root necrosis. In the second period (first leaf to the onset of bloom), a strong mobilization of starch (and, to a lower extent, of N) occurred for supporting vegetative and reproductive growth. At that point, most of the C and N reserves used on the spring flush were those of the roots, rather than those of the old wood (trunk, canes). In the third period (bloom and early berry development), the mobilization process became low and was relieved by N uptake (and CO2 assimilation) supplying nutrients to the sink structures.     

QTLs for muscat flavor and monoterpenic odorant content in grapevine (Vitis vinifera L.)

Little is known about the genetic determinism of muscat flavor in grape, although this trait is of major importance for table grape breeding. We therefore performed a search for QTLs (Quantitative Trait Loci) of both muscat score and berry content in the three main free monoterpene alcohols potentially involved, linalool, nerol and geraniol, based on two years of measures. Parental and consensus framework genetic maps of the cross MTP2687-85 (Olivette × Ribol) × Muscat of Hamburg were built after genotyping the 174 offspring for 139 well-scattered SSR markers. The female, male and consensus framework maps spanned 935, 1365 and 1267 cM, respectively. For QTL detection, simple and composite interval mapping were performed, as well as non parametric Kruskal–Wallis tests. QTLs for muscat score were found on linkage groups (LGs) 1, 5 and 7. For the three ln-transformed monoterpene contents, QTLs with major effects (explaining 17–55 % of total phenotypic variance) were found to be colocated on LG 5, on the male and consensus maps in both years. One additional QTL was found for linalool on LG 2, on female and consensus maps, as well as other colocated ones for nerol and geraniol on LG 13, on male and consensus maps. These additional QTLs had lower effects (9–25%). The contribution of these results to the knowledge of muscat aroma genetic determinism is discussed, as well as their potential usefulness for marker assisted breeding of new aromatic grape varieties.     

Epigenetic repressor-like genes are differentially regulated during grapevine (Vitis vinifera L.) development

Grapevine sexual reproduction involves a seasonal separation between inflorescence primordia (flowering induction) and flower development. We hypothesized that a repression mechanism implicating epigenetic changes could play a role in the seasonal separation of these two developmental processes in grapevine. Therefore, the expression of five grapevine genes with homology to the Arabidopsis epigenetic repressor genes FERTILIZATION INDEPENDENT ENDOSPERM (FIE), EMBRYONIC FLOWER 2 (EMF2), CURLY LEAF (CLF), MULTICOPY SUPPRESSOR OF IRA 1 (MSI1) and SWINGER (SWN) was analyzed during the development of buds and vegetative and reproductive organs. During bud development, the putative grapevine epigenetic repressor genes VvCLF, VvEMF2, VvMSI1, VvSWN and VvFIE are mainly expressed in latent buds at the flowering induction period, but also detected during bud burst and inflorescence/flower development. The overlapping expression patterns of grapevine PcG-like genes in buds suggest that chromatin remodeling mechanisms could be operating during grapevine bud development for controlling processes such as seasonal flowering, dormancy and bud burst. Furthermore, the expression of grapevine PcG-like genes was also detected in fruits and vegetative organs, suggesting that epigenetic changes could be at the basis of the regulation of various proliferation–differentiation cell transitions that occur during grapevine development.     

Proteomic analysis of grapevine (Vitis vinifera L.) tissues subjected to herbicide stress

Two-dimensional gel electrophoresis coupled to mass spectrometry analysis was used to examine for the first time the effect of a herbicide (flumioxazin) on a crop species (Vitis vinifera L.) at the proteome level. Examination of 2-D maps derived from chemically stressed tissues revealed the presence of 33 spots displaying a differential expression pattern. The presence of stress responsive proteins in the different plant organs analysed suggests that flumioxazin could act systemically. Among the responsive proteins, some photosynthesis-related proteins, including several fragments of the enzyme Rubisco, were identified. This effect suggests that photosynthesis could be impaired by the herbicide. The induction of several enzymatic antioxidant systems was also observed, probably as a result of an oxidative stress. Moreover, the photorespiration pathway was stimulated, as suggested by the induction of some key enzymes involved in this process. Changes in carbon metabolism-associated proteins presumably reflect altered patterns of carbon flux in response to impaired photosynthesis and an increased need for osmotic adjustment in affected tissues. Finally, plant defences were stimulated as revealed by the induction of a set of proteins belonging to the pathogenesis-related 10 class, suggesting that they could play an essential role in cell defence mechanisms against flumioxazin.     

Large-scale parentage analysis in an extended set of grapevine cultivars (Vitis vinifera L.)

Inheritance of nuclear microsatellite markers (nSSR) has been proved to be a powerful tool to verify or uncover the parentage of grapevine cultivars. The aim of the present study was to undertake an extended parentage analysis using a large sample of Vitis vinifera cultivars held in the INRA “Domaine de Vassal” Grape Germplasm Repository (France). A dataset of 2,344 unique genotypes (i.e. cultivars without synonyms, clones or mutants) identified using 20 nSSR was analysed with FAMOZ software. Parentages showing a logarithm of odds score higher than 18 were validated in relation to the historical data available. The analysis first revealed the full parentage of 828 cultivars resulting in: (1) 315 original full parentages uncovered for traditional cultivars, (2) 100 full parentages confirming results established with molecular markers in prior papers and 32 full parentages that invalidated prior results, (3) 255 full parentages confirming pedigrees as disclosed by the breeders and (4) 126 full parentages that invalidated breeders’ data. Second, incomplete parentages were determined in 1,087 cultivars due to the absence of complementary parents in our cultivar sample. Last, a group of 276 genotypes showed no direct relationship with any other cultivar in the collection. Compiling these results from the largest set of parentage data published so far both enlarges and clarifies our knowledge of the genetic constitution of cultivated V. vinifera germplasm. It also allows the identification of the main genitors involved in varietal assortment evolution and grapevine breeding.     

Monitoring the stability of Rubisco in micropropagated grapevine (Vitis vinifera L.) by two-dimensional electrophoresis

Plants cultured in vitro suffer from several physiological and biochemical impairments due to the artificial conditions of growth, namely the composition of the heterotrophic media. Upon transfer to ex vitro, the higher irradiances, compared to in vitro, can lead to oxidative stress symptoms, which can be counteracted by CO2 concentrations above atmospheric levels. Here we analyse the stability of Rubisco in in vitro grapevine plantlets, and after transfer to ex vitro under four acclimatization treatments: low irradiance (LL, 150 micromol m(-2)s(-1)) and high irradiance (HL, 300 micromol m(-2)s(-1)) in association with CO2 concentrations of 350 (LCO2) and 700 (HCO2) microL L(-1). Proteins were separated with SDS polyacrylamide gel electrophoresis and two-dimensional electrophoresis and Rubisco degradation peptides were analysed by immunoblotting with anti-LSU antibodies. These degradation products were present in the leaves of plantlets under both in vitro and ex vitro treatments. Under LCO2 they were maintained for almost all of the 28 days of the acclimatization period, while becoming scarcely detected after 14 days under HCO2 and after 7 days when HCO2 was associated with HL. These results appear to confirm the counteraction of HCO2 concentrations over the oxidative stress eventually caused by HL. The patterns of soluble sugars in acclimatizing leaves under HLHCO2 also gave an indication of a faster acquisition of autotrophic characteristics.     

From the cradle of grapevine domestication: molecular overview and description of Georgian grapevine (Vitis vinifera L.) germplasm

Historical information and archaeological and palaeobotanical findings point Georgia, in the South Caucasus, as a cradle for grapevine (Vitis vinifera L.) domestication from its wild form (V. vinifera silvestris Beck.) and subsequent selection and development of varieties with characters suitable for human consumption. The hypothesis of Georgia being a center of domestication, combined with its distance from western countries and the importance of its viticulture and wine production, make Georgian grape germplasm particularly interesting to be investigated under the genetic point of view. Twenty nuclear microsatellite loci were used to genotype 112 Georgian grapevine accessions (V. vinifera sativa Beck.) from germplasm collections and 18 from spontaneous growing plants (V. vinifera silvestris Beck.) found in wild conditions and to compare them to a large international cultivar collection in France. Data analysis shows that Georgian grapevine germplasm has maintained distinctive traits despite arrival of international, foreign varieties and still conserve characteristics of local breeding linked to traditional wine production regions of the country. Results have identified alleles, overall loci, well represented in the Georgian germplasm (cultivated and wild) and absent or poorly represented in other countries, highlighting uniqueness and originality of traits of this viticulture. Moreover, the search for relationships between Georgian and foreign viticulture has evidenced few interesting cases linking the Georgian varieties with Western European ones and with neighboring Caucasian countries, helping to identify the real place of origin in some doubtful cases. In addition, populations or sparse individuals of wild grapevine still preserved in the Georgian natural environments present smaller genetic distances with local cultivars than in other European regions. Principal component analysis (PCA) has also identified special overlapping of the wild compartment with some cultivated varieties. This work provides a highly significant new contribution to applied aspects of Georgian grapevine genetic resources management and use. Uniqueness of the Georgian cultivated grapevine gene pool together with its close relatedness with the wild compartment makes this country a good candidate to address questions regarding domestication and grapevine genetic resource conservation.     

Phenology of flowering and starch accumulation in grape (Vitis vinifera L.) cuttings and vines

BACKGROUND AND AIMS: A reliable protocol for flowering and fruiting in cuttings was developed with the aim of (a) studying inflorescence and flower development in grapevine cuttings and field plants, and (b) assisting haploid plant production. METHODS: Inflorescence and flower development was studied in 'Gewurztraminer' (GW) and 'Pinot Noir' (PN) grape vines and cuttings grown in a glasshouse, along with variations in starch in the flowers. As there is a strong relationship between flower development and starch, the starch content of reproductive structures was estimated. KEY RESULTS: Inflorescence and flower development were similar in the vines and cuttings with consistent differences between the two cultivars. Indeed, the ontogenesis of male and female organs is not synchronous in GW and PN, with both female and male meiosis occurring earlier in PN than in GW. Moreover, changes of starch reserves were similar in the two plant types. CONCLUSIONS: Cuttings have a similar reproductive physiology to vines, and can be used to study grape physiology and to develop haploid plants.     

Linkage disequilibrium in cultivated grapevine, Vitis vinifera L

We present here the first study of linkage disequilibrium (LD) in cultivated grapevine, Vitis vinifera L. subsp. vinifera (sativa), an outcrossing highly heterozygous perennial species. Our goal was to characterize the amount and pattern of LD at the scale of a few centiMorgans (cM) between 38 microsatellite loci located on five linkage groups, in order to assess its origin and potential applications. We used a core collection of 141 cultivars representing the diversity of the cultivated compartment. LD was evaluated with both independence tests and multilocus r ² , both on raw genotypic and reconstructed haplotypic data. Significant genotypic LD was found only within linkage groups, extending up to 16.8 cM. It appeared not to be influenced by the weak structure of the sample and seemed to be mainly of haplotypic origin. Significant haplotypic LD was found over 30 cM. Both genotypic and haplotypic r ² values declined to around 0.1 within 5–10 cM, suggesting a rather narrow genetic base of the cultivated compartment and limited recombination since domestication events. These first results open up a few application opportunities for association mapping of QTLs and marker assisted selection. Electronic Supplementary Material Supplementary material is available for this article at and is accessible for authorized users.     

New 2-naphthyloxyacetates for trunk sucker growth control on grapevine (Vitis vinifera L.)

Eighteen new hexyl esters of 2-naphthyloxyacetic acid (-NOA) were synthesized and their efficiency on sucker control of grapevine was investigated. Different solutions (0.5, 1, and 2%, w/w concentrations) of each compound were sprayed on suckers and the wilting effect after 4, 10, and 30 days after treatment was evaluated. Esters derived from primary and secondary alcohols gave a rapid control of suckers, whereas tertiary alcohol derivatives showed milder activity. Moreover, all compounds proved to cause a long-lasting inhibition effect on the growth of new suckers even 1 year after treatment, especially when applied at a 2% concentration.     

Comparison between modified DNA extraction protocols and commercial isolation kits in grapevine (Vitis vinifera L.)

Various protocols have been developed and used for DNA extraction in grapevine. However, owing to the long duration of the isolation steps in previously developed protocols, researchers have preferred to use isolation kits for studies in recent years. In our study, the DNA yield and purity obtained using six methods - namely three DNA isolation protocols and three commercial DNA isolation kits - were compared. Modifications were made and the isolation steps were shortened in the previously developed DNA isolation protocols to achieve more rapid and practical protocols. The samples were taken from plants grown under vineyard and greenhouse conditions in two periods during spring and autumn. The best results among the six DNA isolation methods were discussed. The results were also supported with polymerase chain reaction analyses conducted with isolated DNAs.     

Low-night temperature (LNT) induced changes of photosynthesis in grapevine (Vitis vinifera L.) plants.

Changes of leaf pigments, ribulose-1,5-bisphosphate carboxylase (RuBPC) and photosynthetic efficiency were examined in grapevine (Vitis vinifera L.) plants grown under ambient irradiation (maximum daily PAR = 1500 micromol m(-2) s(-1)) for 7 days to low night temperature (LNT) of 5 degrees C (daily from 18:00 to 06:00). The contents of chlorophyll (Chl) and carotenoids (Car) per fresh mass were lower in LNT leaves than in control leaves. The contents of alpha + beta carotene and lutein-5,6-epoxide remained unaffected, but the de-epoxidation state involving the components of xanthophyll cycle increased. RuBPC activity and soluble proteins were also significantly reduced in LNT leaves. In isolated thylakoids, a marked inhibition of whole chain (PS I + PS II) and PS II activity were observed in LNT leaves. Smaller inhibition of PS I activity was observed in LNT leaves. The artificial exogenous electron donors, MnCl2, DPC and NH2OH did not restored the loss of PS II activity in LNT leaves. The same results were obtained when F(v)/F(m) was evaluated by Chl fluorescence measurements. The marked loss of PS II activity in LNT leaves was due to the marked loss of D1 protein which was determined by immunological studies.     

Identification and characterization of "rd22" dehydration responsive gene in grapevine (Vitis vinifera L.)

To identify and isolate genes related to abiotic stresses (salinity and drought) tolerance in grapevine, a candidate gene approach was developed and allowed isolating a full-length cDNA of rd22 gene from the Cabernet Sauvignon variety. The latter, named Vvrd22, is a dehydration-responsive gene that is usually induced by the application of exogenous ABA. Details of the physicochemical parameters and structural properties (molecular mass, secondary structure, conserved domains and motives, putative post-translational modification sites...) of the encoded protein have also been elucidated. The expression study of Vvrd22 was carried out at the berry growth stages and at the level of plant organs and tissues as well as under both drought and salt stresses. The results showed that Vvrd22 is constitutively expressed at a low level in all analyzed tissues. Moreover, salt stress induced Vvrd22 expression, particularly for the tolerant variety (Razegui), contrary to the sensitive one (Syrah), which did not display any expression variation during the stress, which means that Vvrd22 is involved in salt stress response and that its expression level depends on regulatory mechanisms that are efficient only for the tolerant variety. On the other hand, under drought stress, Vvrd22 is induced in an identical manner for both tolerant and sensitive varieties. In addition, stress signal molecules such as ABA (lonely applied or in combination with sucrose) induced Vvrd22 expression, even at a low level. A minimal knowledge about the role and the functionality of this gene is necessary and constitutes a prerequisite condition before starting and including Vvrd22 in any program of improvement of grapevine's abiotic stress tolerance.