A plant regeneration platform to apply new breeding techniques for improving disease resistance in grapevine rootstocks and cultivars

Worldwide grapevine cultivation is based on the use of elite cultivars, in many cases strictly linked to local important wine brands. Most of Vitis vinifera cultivars have high susceptibility to fungal and viral diseases therefore, new breeding techniques ( e.g. Cisgenesis, RNAi and gene editing) offer the possibility to introduce new clones of the main cultivars with increased diseases resistance, in order to reduce environmental impact and improve quality in the intensive wine grape industry. This study is finalized to develop efficient in vitro regeneration and transformation protocols to extend the application of these technologies in wine grape cultivars and rootstocks. With this aim, in vitro regeneration protocols based on the production of meristematic bulks ( Mezzetti et al., 2002) were optimized for different grapevine cultivars ( Glera, Vermentino, Sangiovese, Thompson Seedless) and rootstocks ( 1103 Paulsen, and 110 Richter). The meristematic bulks were then used as explants for Agrobacterium mediated genetic transformation protocols, by comparing the use of NPTII and e-GFP as marker genes. Results confirmed the efficiency of meristematic bulks as the regenerating tissue to produce new modified plants in almost all the above genotypes. The highest regeneration efficiency in some genotypes allowed the selection of stable modified lines/calli with only the use of e-GFP marker gene. This protocol can be applied in the use of MYB marker gene for the production of cisgenic lines. Genotypes having the highest regeneration and transformation efficiency were also used for transformation experiments using a hairpin gene construct designed to silence the RNA-dependent RNA polymerase ( RpRd) of the GFLV and GLRaV3, which would induce multiple virus resistances, and the Dicer-like protein 1 ( Bc-DCL1) and Bc-DCL2 to control B. cinerea infection