Trasformazione genetica di Vitis vinifera L. con costrutti che inducono il silenziamento genico post-trascrizionale

GENETIC TRANSFORMATION OF Vitis vinifera L. WITH CONTRUCTS THAT ELICIT THE POST-TRANSCTRIPTIONAL GENE SILENCING Grapevine (Vitis vinifera L.) is one of the most important crop species in the world. Main purposes of grapevine genetic breeding are resistance to biotic stresses and improvement of fruit production and quality. The aim of this doctoral thesis is the genetic transformation of grapevine plants with constructs that elicit post transcriptional gene silencing (PTGS) in order to induce virus resistance or to study the function of a newly-discovered gene family (Aucsia) involved in auxin-mediated regulation of fruit development. To attain the final targets of the PhD project, we applied to wine (Pinot noir and Corvina) and table grape (Vitroblack) varieties and to a grape rootstock (1103 Paulsen) a genetic transformation protocol based on organogenesis (Mezzetti et al., 2002). This method consists in the formation of a meristematic bulk with a high regenerative capacity, starting from adventitious shoots proliferating in vitro. The meristematic bulk is subsequently used for both grape regeneration and genetic transformation. The first objective has been the development of a genetic engineering method for conferring resistance to Grapevine Fan Leaf Virus (GFLV) and Grapevine Leaf Roll associated Viruses (GLRaV) through the induction of PTGS. A chimeric hairpin construct (hpViruses GFLV-GLRaV) aimed to induce resistance against both GFLV and GLRaV-3 was built. The hpViruses GFLV-GLRaV construct was used for genetic transformation of the model plant Nicotiana benthamiana and grapevine. For these experiments we used two different grapevine cultivars (Corvina and Pinot noir) and one rootstock (1103 Paulsen). Two rooted hpVirusesGFLV-GLRaV lines of Corvina resulted positive to PCR analysis. Regenerated shoots of Pinot noir and 1103 Paulsen transformed with the hpViruses GFLV-GLRaV construct, have been obtained by selection at increasing concentrations of kanamycin starting from 25 mg/l and are now in the rooting phase. Experiments on hpViruses GFLV-GLRaV N. benthamiana and control plants challenged with either ArMV or GFLV were performed. The second objective concerns the identification of the grapevine orthologous of the AUCSIA tomato genes, the analysis of their expression during fruit development and the development of tools for investigating their function through PTGS-mediated suppression. The expression of VvAUCSIA genes was studied in Silcora table grape variety comparing wild type plants and plants transformed with the auxin-synthesising DefH9-iaaM gene at different phases of berry growth from dormant buds to mature berries (dormancy-harvest). The genetic transformation of Vitroblack variety with a construct (hpVvAUCSIA1) designed to elicit PTGS against AUCSIA genes has been performed. Selection of the putative transformants has been done using substrates enriched with the selective agent kanamycin at concentrations 25 mg/l. Seven rooted lines analyzed by PCR resulted positive.