Application of New Genomic Techniques (NGTs) to reduce the pathogen susceptibility in grapevine

Abstract

Preserving the genetic heterogeneity of grapevine provides a valuable reservoir of allelic combinations that can offer genetic traits tocontrast biotic and abiotic stresses, highly advantageous for breeding programs. Next-generation viticulture will need to intensify the contribution of genomics and biotechnologies to solve phytosanitary and qualitative problems. The control of pathogens in viticultureoften 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. Conventional breeding has played a key role in domesticating grapevine varieties, but it is a time-consuming process to develop new cultivars with desirable traits for cultivation, such as pathogen resistances. New plant genomic techniques (NGTs) offer a potential revolution in grapevine cultivation, and genome editing has been shown promising for targeted mutagenesis. The success of these biotechnological approaches relies on efficient in vitro regeneration protocols, particularly through somatic embryogenesis (SE). Although this method has proven to be successful in some Vitis vinifera species, itseffectiveness varies due to the genotype-dependent nature of many cultivars. Considering the heterozygous nature and the widespread use of vegetative propagation of grapevine, protoplasts have proven to be particularly suitable for transgene-free genome editing applications. Despite, plant regeneration from protoplasts remains generally considered challenging in grapevine, DNA-free approaches preserve the genetic identity of the cultivars, which would otherwise be altered through crossing.

The focus of this study is to enhance in vitro plant regeneration process via SE and isolate and regenerate plants from protoplasts derived from embryogenic calli of Corvina and Garganega, two grapevine varieties of significant economic importance for wine production in the Veneto region, Italy. Protoplasts represent a versatile platform for DNA-free genome editing via the CRISPR/Cas9 system, enabling the targeted knockout of MLO and DMR susceptibility (S) genes associated with powdery mildew and downy mildew susceptibility in grapevine. This research aims to accelerate the development of grapevine varieties with improved traits, overcoming the limitation of conventional breeding methods and this advancement aligns with European legislative demand about NGT plants.