New Genomic Techniques applied to grapevine: production of oenologically and resilience-improved NGT1 prototypes and bioelectric stimulation to enhance regeneration efficiency

In 2025, viticulture must tackle with several challenges related primarily to climate change, insurgence of resistance in pathogens, change in market trend and consuming and the rising demand for a more sustainable management of the vineyards. The indiscriminate use of phytosanitary products has led to loss of effectiveness and the insurgence or resistant strains. In 2019, the European Commission launched the Green Deal project with the goal of achieving net-zero climate impact by 2050. The Farm to Fork and Biodiversity 2030 strategies, in line with the Green Deal, promote the commitment to reduce pesticide use by 50% by 2030. The implementation of integrated pest management approaches is pivotal to contain pest diseases in vineyards and agricultural practise plays a fundamental role in the restriction of climate change effect. However, although effective, these approaches are often insufficient. The combination of agricultural practices and biotechnological approaches is fundamental to provide a more sustainable management of the vineyards and meet the strategic objective prefixed. Nowadays, New Genetic Techniques (NGTs) have revolutionized the frontiers of the genetic improvement in plants worldwide. In particular, CRISPR/Cas9 system has become one of the smartest tools to introduce precise target mutations in specific genes while preserving the genetic background and thus the distinctive varietal characteristics at the basis of the wine social, cultural, and economic tradition. This system is widely studied in grapevine thus demonstrating to be an efficient tool to manipulate precisely target genes leading to improved traits while at the same time preserving the genetic background of the varieties. Nevertheless, the success of application of genome editing in grapevine relies on the availability of efficient in vitro regeneration protocols, especially through somatic embryogenesis (SE). Indeed, to produce edited NGT1 grapevine plants it is of paramount importance to dispose of an efficient delivery system to introduce the preassembled ribonucleoproteins (RNP) and to ensure the subsequent step of regeneration of the edited plant. Protoplasts are the best platform for this purpose: a highly regenerative system accessible to most transformation techniques. The regeneration is possible through the somatic embryogenesis process, in which a single cell goes through morphological and physiological changes developing a somatic embryo that will become a functional plant. This study revolves around the establishment of an efficient grapevine multiple gene editing protocol through a DNA-free approach in two wine cultivars of national and international interest respectively: Glera and Cabernet Sauvignon. Regeneration studies were initially conducted to find the best parameters to manipulate each variety. The genes of interest for genome editing in this study are susceptibility genes to downy mildew and powdery mildew. Mutating these genes would lead to the genesis of prototypes potentially resistant against major biotic agents, thereby achieving the coveted goal of increasing the intrinsic resistance of the plant leading to the reduction of the use of phytosanitary products. In this scenario, this work reports the first achievement of a quadruple gene editing event in grapevine using a DNA-free approach in Glera variety, demonstrating the feasibility of simultaneous multiple gene editing. Moreover, gene editing to improve both oenological and resilience traits was also achieved in Cabernet Sauvignon, a well known recalcitrant red-berry variety. Although the editing process was successfully carried out, the overall regeneration efficiency was low for both varieties. To overcome this limiting step, in this study, approaches with the potential of enhancing the regeneration process, such as the exposure to bioelectric stimulation was investigated with promising preliminary results in Arabidopsis thaliana. Further studies are needed to understand whether this process is applicable also in grapevine to improve its regeneration attitude thereby improving the outcome of regeneration processes. The success of this application for the improvement of multiple target traits establishes a foundation for a resilient and environmentally conscious winegrowing system.