Regeneration of plants from embryogenic callus-derived protoplasts of Garganega and Sangiovese grapevine (Vitis vinifera L.) cultivars

Protoplasts are useful research tools for basic and applied plant science, but the regeneration of whole plants from protoplasts is challenging in most of agronomically important crops, including grapevine (Vitis vinifera L.). Here we describe an efficient protocol for the induction of embryogenic callus, the isolation of protoplasts, and the regeneration of whole grapevine plants in two Italian grapevine cultivars. Embryogenic callus was induced successfully from stamens collected from immature flowers. Isolated protoplasts were tested to confirm their viability and then cultivated using the disc-culture method, at a density of 1\u2009 7\u2009105 protoplasts/mL in solid Nitsch\u2019s medium supplemented with 2 mg/L 1-naphthaleneacetic acid and 0.5 mg/L 6-benzylaminopurine. After 3\u20134 months, the protoplasts of both cultivars regenerated with similar efficiency into cotyledonal-stage somatic embryos. The somatic embryos were transferred to solid Nitsch\u2019s medium supplemented with 30 g/L sucrose and 2 g/L gellan gum, and were maintained in the dark for 4 weeks. This step was necessary for the embryo to complete germination, allowing subsequent shoot elongation in response to light on a medium with 4 \ub5M 6-benzylaminopurine. Then root elongation occurred after transferring on a medium with 0.5 \ub5M 1-naphthaleneacetic. After\u2009~\u20096 months from the isolation of protoplasts, normal plants were regenerated, which were moved to the greenhouse. The protoplasts could also be transfected using the polyethylene glycol method, as confirmed using a plasmid carrying the yellow florescent protein marker gene. The new method is therefore compatible with biotechnological applications such as gene transfer and genome editing

Esthesioneuroblastoma is not a member of the primitive peripheral neuroectodermal tumour-Ewing’s group

Esthesioneuroblastoma (ENB) is a rare, site-specific, locally aggressive neuronal malignancy so far thought to belong to primitive peripheral neuroectodermal tumour-Ewing's tumour (pPNETs-ETs). Its anatomical location, in addition to morphologic, immunophenotypic and ultrastructural features, suggests its origin in the neuronal or neuroendocrine cells of the olfactory epithelium. However, the cytogenetic and molecular data currently available appear controversial on the presence of the typical translocation t(11;22)(q24;q12) and of trisomy 8, chromosomal changes that characterize the tumours belonging to the pPNETs-ETs. Herein we have analysed five ENB tumour specimens for trisomy 8 by fluorescence in situ hybridization (FISH), for the presence of EWS gene rearrangements by FISH, reverse transcription polymerase chain reaction and Southern blot analyses, as well as for the expression of the Ewing sarcoma-associated MIC2 antigen by immunohistochemistry. Neither EWS/FLI-I, EWS/ERG and EWS/FEV fusion genes nor MIC2 expression were found in any tumour, whereas trisomy 8 was found in one case only. Moreover, DNA from three cases analysed by Southern blot did not show EWS gene rearrangements. Our results support the evidence that ENB is not a member of the pPNETs-ETs. © 1999 Cancer Research Campaig

A major QTL is associated with berry grape texture characteristics

Berry texture and berry skin mechanical properties are traits with high agronomic relevance because they are related to quality parameters and marketing requirements of wine, table, and raisin grapes. Searching for QTLs linked to berry texture, an F1 population of 152 individuals and their parents were used in this study. These F1 plants were obtained crossing Raboso Veronese, a seeded black wine grape cultivar, and Sultanina, a seedless white grape variety, especially used for raisins. Density flotation was applied for berry sorting improving the management of many and highly variable genotypes, irrespective of the quantification of specific molecule classes. Berries were evaluated for technological ripeness parameters and mechanical properties. Texture parameters were taken as raw data and as data normalised on berry dimensions, i.e., berry diameter or surface or volume. SSR molecular markers were used to produce a genetic map and a major QTL for berry texture was found on chromosome 18 with traits related to berry firmness showing a phenotypical explained variance higher than 60 %, and traits related to berry resilience, springiness and cohesiveness showing a variance higher than 50 %. Surprisingly, this QTL showed to be associated with SSR markers linked to VviAGL11, the main gene linked to seedlessness. VviAGL11 expression and co-expression profiling during grape ripening was evaluated using available information; this data suggested a role for this gene on the texture of a ripe berry

Lo sviluppo delle conoscenze genomiche in vite e il loro potenziale utilizzo nella viticoltura attuale e futura

Nel 2007 sono stati pubblicati i risultati del sequenziamento e dell\u2019analisi dettagliata del genoma di Vitis vinifera frutto di due iniziative indipendenti, una italo-francese e l\u2019altra italo-americana. Questi risultati costituiscono la base per studi futuri e gettano le fondamenta per l\u2019adozione di metodologie innovative per sviluppare e rafforzare la viticoltura italiana e mondiale del XXI secolo. In questa review verranno illustrate le potenzialit\ue0 delle tecnologie derivate dalla conoscenza del genoma come ad esempio lo sviluppo di strumenti per l\u2019analisi di espressione genica su larga scala, e riportati alcuni esempi inerenti al loro impiego attuale e futuro in viticoltura

The complex microbial community of withered berries of cv. Corvina as revealed by metagenomic analysis

Corvina is the most important of Verona\u2019s red grape varieties and forms the basis for the production of unique wines, such as Amarone and Recioto of the Valpolicella area (Italy). The distinctive features of such wines are linked to the peculiar winemaking techniques and especially to the post-harvest grape withering process, when the grapes are partially dried in attics for three/four months to concentrate sugars up to about 27% (w/v). Dehydratation increases the concentration of constituent substances in berries, determining also specific expression of grape genes. During this process, grapes are colonized with a complex microbiota to an extent which depends on environmental temperature and humidity. In this study, a whole metagenome sequencing (WMS) approach was used to analyse the microbial consortia present on Corvina berries at the end of the withering process performed in two different conditions (\u201ctraditional\u201d or \u201cfast\u201d). Representative berry samples were collected and washed to avoid grapevine DNA contamination. Bioinformatic analyses, performed on reads and scaffolds, revealed that traditionally withered berries were dominated by Clostridiales and Pseudomonadales, while the fast procedure determined the presence of Enterobacteriales and Lactobacillales. On the contrary, few consistent differences characterized the eukaryotic fraction, dominated by Aspergillus and Penicillium for both samples. Interestingly, the \u201cbinning\u201d procedure revealed 15 most abundant genomes characterizing the two conditions. These results provide insights into the microbial community of Corvina withered berries and reveal relevant variations attributable to environmental withering conditions. Further studies will be performed to determine whether the different microbial compositions could lead to significant chemical variations of the musts, with an impact on the organoleptic properties of wine. This study underlines how novel technologies, like WMS, could open novel perspectives in the knowledge and management of traditional processes as the withering process of Corvina, with an impact on the winemaking of important Italian wines

Grapevine field experiments reveal the contribution of genotype, the influence of environment and the effect of their interaction (GXE) on the berry transcriptome

Changes in the performance of genotypes in different environments are defined as genotype x environment (G x E) interactions. In grapevine (Vitis vinifera), complex interactions between different genotypes and cli- mate, soil and farming practices yield unique berry qualities. However, the molecular basis of this phe- nomenon remains unclear. To dissect the basis of grapevine G x E interactions we characterized berry transcriptome plasticity, the genome methylation landscape and within-genotype allelic diversity in two genotypes cultivated in three different environments over two vintages. We identified, through a novel data-mining pipeline, genes with expression profiles that were: unaffected by genotype or environment, genotype-dependent but unaffected by the environment, environmentally-dependent regardless of geno- type, and GxE-related. The GxE-related genes showed different degrees of within-cultivar allelic diversity in the two genotypes and were enriched for stress responses, signal transduction and secondary metabo- lism categories. Our study unraveled the mutual relationships between genotypic and environmental vari- ables during GxE interaction in a woody perennial species, providing a reference model to explore how cultivated fruit crops respond to diverse environments. Also, the pivotal role of vineyard location in deter- mining the performance of different varieties, by enhancing berry quality traits, was unraveled

Increasing the source/sink ratio in Vitis vinifera (cv Sangiovese) induces extensive transcriptome reprogramming and modifies berry ripening

<p>Abstract</p> <p>Background</p> <p>Cluster thinning is an agronomic practice in which a proportion of berry clusters are removed from the vine to increase the source/sink ratio and improve the quality of the remaining berries. Until now no transcriptomic data have been reported describing the mechanisms that underlie the agronomic and biochemical effects of thinning.</p> <p>Results</p> <p>We profiled the transcriptome of <it>Vitis vinifera </it>cv. Sangiovese berries before and after thinning at veraison using a genome-wide microarray representing all grapevine genes listed in the latest V1 gene prediction. Thinning increased the source/sink ratio from 0.6 to 1.2 m²leaf area per kg of berries and boosted the sugar and anthocyanin content at harvest. Extensive transcriptome remodeling was observed in thinned vines 2 weeks after thinning and at ripening. This included the enhanced modulation of genes that are normally regulated during berry development and the induction of a large set of genes that are not usually expressed.</p> <p>Conclusion</p> <p>Cluster thinning has a profound effect on several important cellular processes and metabolic pathways including carbohydrate metabolism and the synthesis and transport of secondary products. The integrated agronomic, biochemical and transcriptomic data revealed that the positive impact of cluster thinning on final berry composition reflects a much more complex outcome than simply enhancing the normal ripening process.</p

Towards a scientific interpretation of the terroir concept: plasticity of the grape berry metabolome

BACKGROUND: The definition of the terroir concept is one of the most debated issues in oenology and viticulture. The dynamic interaction among diverse factors including the environment, the grapevine plant and the imposed viticultural techniques means that the wine produced in a given terroir is unique. However, there is an increasing interest to define and quantify the contribution of individual factors to a specific terroir objectively. Here, we characterized the metabolome and transcriptome of berries from a single clone of the Corvina variety cultivated in seven different vineyards, located in three macrozones, over a 3-year trial period. RESULTS: To overcome the anticipated strong vintage effect, we developed statistical tools that allowed us to identify distinct terroir signatures in the metabolic composition of berries from each macrozone, and from different vineyards within each macrozone. We also identified non-volatile and volatile components of the metabolome which are more plastic and therefore respond differently to terroir diversity. We observed some relationships between the plasticity of the metabolome and transcriptome, allowing a multifaceted scientific interpretation of the terroir concept. CONCLUSIONS: Our experiments with a single Corvina clone in different vineyards have revealed the existence of a clear terroir-specific effect on the transcriptome and metabolome which persists over several vintages and allows each vineyard to be characterized by the unique profile of specific metabolites.Andrea Anesi, Matteo Stocchero, Silvia Dal Santo, Mauro Commisso, Sara Zenoni, Stefania Ceoldo, Giovanni Battista Tornielli, Tracey E. Siebert, Markus Herderich, Mario Pezzotti and Flavia Guzz