Enhancing fungal resistance in local Vitis vinifera cultivars: from optimized somatic embryogenesis process to NGTs application and phenotyping characterization

Preserving the genetic heterogeneity of grapevine provides a valuable reservoir of allelic combinations that can offer genetic traits to contrast biotic and abiotic stresses, highly advantageous for breeding programs. The control of pathogens in viticulture often relies on the application of massive amounts of pesticides, especially fungicides, which comes at great costs for viticulture and poses a considerable risk for human health and the environment. Next-generation viticulture will need to intensify the contribution of genomics and biotechnologies to solve phytosanitary and qualitative problems. New plant Genomic Techniques (NGTs) offer a potential revolution in grapevine cultivation, and genome editing has shown promise for targeted mutagenesis. The success of these biotechnological approaches relies on efficient in vitro regeneration protocols, particularly through somatic embryogenesis (SE). This method has proven successful in Vitis vinifera, but its effectiveness is very genotype-dependent and varies for the different cultivars. Additionally, protoplasts, cells without a cell wall, have proven to be particularly suitable for genome editing applications. These approaches preserve the genetic identity of the cultivars, which would otherwise be altered through crossing. In this PhD thesis, tissue culture approaches were explored to improve in vitro plant regeneration in local grapevine cultivars, with particular focus on Corvina and Garganega, two varieties representative of viticulture in north-eastern Italy. The work focused on: (i) the optimization of in vitro plant regeneration through somatic embryogenesis and the isolation and regeneration of plants from protoplasts derived from embryogenic calli of Corvina and Garganega; (ii) the application of DNA-free genome editing via RNP-Cas9 complex in grapevine protoplasts to knock out Susceptibility genes, MLO and DMR6, associated with powdery mildew and downy mildew disease ; and (iii) the use of advanced high-throughput phenotyping and spectroscopic sensing technologies to characterize grapevine responses to biotic stress and sunburn-related conditions, supporting future genetic improvement and adaptive viticultural strategies.