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’ 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.