Modern biotechnological approaches toward sustainable viticulture

Conventional breeding does not allow the introgression of single traits without compromising the genetic background that characterize an elite cultivar. The exploitation of the new molecular techniques known as genome editing and cisgenesis make possible to modify or transfer single genes preserving all the characteristics selected with difficulty by breeders over a long-time span. To date, 27 QTLs have been associated with downy mildew disease resistance (Rpv1-Rpv27) and many of these have been employed in breeding programs for the introgression in genotypes of interest. Just in case of Rpv1 and Rpv3, however, the underlying genes have been identified and characterized. In both cases nucleotide-binding leucine rich repeat (NB-LRR) genes are present, codifying for receptors that act as cytoplasmic pathogen sensors, triggering a signal transduction pathway for cell-death mediated defense at the infection site. One of the biggest drawbacks of traditional genetic engineered plants is represented by the presence of transgenes (often selection markers), usually perceived as unsafe by consumers. The cisgenic approach aims at circumventing this problem avoiding the presence of exogenous DNA, introducing only the desired trait by using native genes from Vitis species, interfertile with V. vinifera. Adopting this strategy, the already characterized resistance genes RPV3 and/or RPV1 will be introduced in some elite Vitis vinifera varieties, highly appreciated by the wine industry. The outcomes will reduce the agrochemicals needs and the risks associated with their use, increasing the profitability of the vineyard and consumers\u2019 appreciation. Flower tissues of Glera, Sangiovese and Pinot Nero have been collected from field and fruit cuttings grown plants and from fruiting cuttings and used as explants for the induction of somatic embryos. PCR products of candidate genes, including native promoter and terminator, will be cloned in a suitable vector and transformed into competent E.coli. The gene sequences will be then isolated by PCR and cloned into a binary vector engineered with an inducible excision system. Transient expression assays will be used to evaluate the efficacy of the candidate genes into the different genetic backgrounds of the selected cultivars. The gene construct will be used for the transformation of grapevine embryogenic calli through A. tumefaciens infection. Infected calli will be transferred on selective media for the induction and germination of somatic embryos . Regenerated plantlets will hence be checked for the presence and expression of candidate genes. For the removal of exogenous sequences, chemical or thermal induction of the excision system will be used. Absence of Agrobacterium and backbone sequences will also be checked by PCR on transformants. Resistance and susceptibility to downy mildew will be tested on available material of interest by leaf disc bioassay or whole leaves inoculation of in-vitro and/or acclimatisedacclimatized plantlets

Polyphenol metabolomics of twenty Italian red grape varieties

“Suspect screening analysis”method to study grape metabolomics, was performed. This method is a middle-way “targeted” and “untargeted”approach aiming at identifying the largest number of metabolites in grape samples. A new database of putative grape and wine metabolites (GrapeMetabolomics), which currently contains around 1,100 compounds, was constructed by CREA at Conegliano. By performing high-resolution mass spectrometry analysis of the grape extract in both positive and negative ionization mode, averaging 320- 450 putative compounds are identified. Most of them are grape polyphenols, such as anthocyanins, flavonols and stilbene derivatives. By performing PCA and Cluster Analysis the composition in anthocyanins and flavonols of 20 Italian red grape varieties, was studie

Characterization of some Italian V. vinifera L. grape varieties on the basis of their flavonol profile

"Suspect screening metabolomics" is a mid-way approach between "targeted" and "untargeted" analysis. For this aim, a new database of putative grape and wine metabolites (GrapeMetabolomics ) was expressly constructed. Currently, this database contains around 1,100 compounds. By performing UHPLC/QTOF mass spectrometry analysis in both positive and negative ionization mode, in a grape extract averaging 320–450 putative compounds are identified. Most of these compounds are important grape metabolites, including flavonols, anthocyanins, and stilbene derivatives. In the present study, this approach was focalized on the characterization of flavonols of 18 important Italian red and white grape varieties and the method provided the identification of 15 flavonols. By performing statistical analysis (Principal Component Analysis and Cluster Analysis), the effect of the variety on the flavonol composition of the grapes was studied. Both the red and white samples fell into three different groups, respectively, on the basis of their flavonol profiles. Because the samples were cultivated in the same vineyard, their profile potentially was not affected by cultural or environmental factors. Anyway, these preliminary results will have to be confirmed by the study of grape samples collected in different years and from different vineyards

Parentage Atlas of Italian Grapevine Varieties as Inferred From SNP Genotyping

The Italian grape germplasm is characterized by a high level of richness in terms of varieties number, with nearly 600 wine grape varieties listed in the Italian National Register of Grapevine Varieties and with a plethora of autochthonous grapes. In the present study an extended SNP genotyping has been carried out on Italian germplasm of cultivated Vitis vinifera subsp. sativa and Vitis hybrids. Several hundred Italian varieties maintained in the repositories of scientific Institutions and about one thousand additional varieties derived from previous studies on European, Southern Italy, Magna Graecia and Georgian germplasm were considered. The large genotyping data obtained were used to check the presence of homonyms and synonyms, determine parental relationships, and identify the main ancestors of traditional Italian cultivars and closely-related accessions. The parentage among a set of 1,232 unique varieties has been assessed. A total of 92 new parent-offspring (PO) pairs and 14 new PO trios were identified. The resulted parentage network suggested that the traditional Italian grapevine germplasm originates largely from a few central varieties geographically distributed into several areas of genetic influence: “Strinto porcino” and its offspring “Sangiovese”, “Mantonico bianco” and “Aglianico” mainly as founder varieties of South-Western Italy (IT-SW); Italian Adriatic Coast (IT-AC); and Central Italy with most varieties being offsprings of “Visparola”, “Garganega” and “Bombino bianco”; “Termarina (Sciaccarello)” “Orsolina” and “Uva Tosca” as the main varieties of North-Western Italy (IT-NW) and Central Italy. The pedigree reconstruction by full-sib and second-degree relationships highlighted the key role of some cultivars, and, in particular, the centrality of “Visparola” in the origin of Italian germplasm appeared clear. An hypothetical migration of this variety within the Italian Peninsula from South to North along the eastern side, as well as of “Sangiovese” from South to Central Italy along the Western side might be supposed. Moreover, it was also highlighted that, among the main founders of muscat varieties, “Moscato bianco” and “Zibibbo (Muscat of Alexandria)” have spread over the whole Italy, with a high contribution by the former to germplasm of the North-Western of the peninsula

Mitostatin Is Down-Regulated in Human Prostate Cancer and Suppresses the Invasive Phenotype of Prostate Cancer Cells

MITOSTATIN, a novel putative tumor suppressor gene induced by decorin overexpression, is expressed in most normal human tissues but is markedly down-regulated in advanced stages of mammary and bladder carcinomas. Mitostatin negatively affects cell growth, induces cell death and regulates the expression and activation levels of Hsp27. In this study, we demonstrated that ectopic expression of Mitostatin in PC3, DU145, and LNCaP prostate cancer cells not only induced a significant reduction in cell growth, but also inhibited migration and invasion. Moreover, Mitostatin inhibited colony formation in soft-agar of PC3 and LNCaP cells as well as tumorigenicity of LNCaP cells in nude mice. Conversely, targeting endogenous Mitostatin by siRNA and anti-sense strategies in PC3 and DU145 prostate cancer cells enhanced the malignant phenotype in both cell lines. In agreement of these anti-oncogenic roles, we discovered that Mitostatin was absent in ∼35% (n = 124) of prostate tumor samples and its overall reduction was associated with advanced cancer stages. Collectively, our findings indicate that MITOSTATIN may acts as a tumor suppressor gene in prostate cancer and provide a novel cellular and molecular mechanism to be further exploited and deciphered in our understanding of prostate cancer progression

Sustainability of grape-ethanol energy chain

The aim of this work is to evaluate the sustainability, in terms of greenhouse gases emission saving, of a new potential bio-ethanol production chain in comparison with the most common ones. The innovation consists of producing bio-ethanol from different types of no-food grapes, while usually bio-ethanol is obtained from matrices taken away from crop for food destination: sugar cane, corn, wheat, sugar beet. In the past, breeding programs were conducted with the aim of improving grapevine characteristics, a large number of hybrid vine varieties were produced and are nowadays present in the Viticulture Research Centre (CRA-VIT) Germplasm Collection. Some of them are potentially interesting for bio-energy production because of their high production of sugar, good resistance to diseases, and ability to grow in marginal lands. Life cycle assessment (LCA) of grape ethanol energy chain was performed following two different methods: i) using the spreadsheet BioGrace, developed within the Intelligent Energy Europe program to support and to ease the Renewable Energy Directive 2009/28/EC implementation; ii) using a dedicated LCA software. Emissions were expressed in CO2 equivalent (CO2eq). These two tools gave very similar results. The overall emissions impact of ethanol production from grapes on average is about 33 g CO2eq MJ–1 of ethanol if prunings are used for steam production and 53 g CO2eq MJ–1 of ethanol if methane is used. The comparison with other bio-energy chains points out that the production of ethanol using grapes represents an intermediate situation in terms of general emissions among the different production chains. The results showed that the sustainability limits provided by the normative are respected to this day. On the contrary, from 2017 this production will be sustainable only if the transformation processes will be performed using renewable sources of energy

Integrated Bayesian Approaches Shed Light on the Dissemination Routes of the Eurasian Grapevine Germplasm

The domestication and spreading of grapevine as well as the gene flow history had been described in many studies. We used a high-quality 7k SNP dataset of 1,038 Eurasian grape varieties with unique profiles to assess the population genetic diversity, structure, and relatedness, and to infer the most likely migration events. Comparisons of putative scenarios of gene flow throughout Europe from Caucasus helped to fit the more reliable migration routes around the Mediterranean Basin. Approximate Bayesian computation (ABC) approach made possible to provide a response to several questions so far remaining unsolved. Firstly, the assessment of genetic diversity and population structure within a well-covered dataset of ancient Italian varieties suggested the different histories between the Northern and Southern Italian grapevines. Moreover, Italian genotypes were shown to be distinguishable from all the other Eurasian populations for the first time. The entire Eurasian panel confirmed the east-to-west gene flow, highlighting the Greek role as a “bridge” between the Western and Eastern Eurasia. Portuguese germplasm showed a greater proximity to French varieties than the Spanish ones, thus being the main route for gene flow from Iberian Peninsula to Central Europe. Our findings reconciled genetic and archaeological data for one of the most cultivated and fascinating crops in the world

Ricerche su organogenesi, embriogenesi somatica e trasformazione genetica in alcune specie del genere Prunus

Dottorato di ricerca in produttivita' delle piante coltivate. 7. ciclo. A.a. 1992-95. Tutore G. Ponchia. Coordinatore P. ParriniConsiglio Nazionale delle Ricerche - Biblioteca Centrale - P.le Aldo Moro, 7, Rome; Biblioteca Nazionale Centrale - P.za Cavalleggeri, 1, Florence / CNR - Consiglio Nazionale delle RichercheSIGLEITItal