Assessment of prime editing in grapevine protoplasts

Grapevine is one of the most culturally and economically significant plants cultivated over a vast area both in our country and around the world. In grapevine cultivation, the powdery mildew disease caused by the fungus Uncinula necator leads to significant yield loss and economic damage. With the increasing world population and climate change, it is predicted that traditional plant breeding methods will be insufficient for growing agricultural products needed for human nutrition. Therefore, the use of genome editing tools like CRISPR, which can make targeted changes in the genome using nucleases without incorporating transgenes, is becoming increasingly important in agricultural production as an alternative to classical breeding techniques. Genome editing studies in plants are progressing rapidly with prime editing, a new adaptation of the CRISPR-Cas9 system, and the dual-epegRNA approach, which allows for more successful results in plants. The longest and most expensive step in genome editing studies in plants is plant tissue culture applications. Therefore, testing and verifying genome editing components in plant protoplasts is of great importance. In this thesis, DNA constructs obtained using prime editing and the dual-epegRNA approach, targeting pLL3 and pLL13, pectate lyase-like genes, and the PDS gene related to resistance to powdery mildew disease, were tested in Chardonnay protoplasts, and their editing efficiencies were compared. Within the scope of the thesis, the highest protoplast yield of 75x106/g leaf was obtained using the optimized protoplast isolation protocol from Chardonnay leaves, and protoplast viability was determined to be 91%. Using the optimized PEG-mediated protoplast transformation method, the average transformation efficiency for prime editing constructs was obtained in the range of 20.25%-27.79%. After transformation, protoplast DNAs were analyzed by amplicon sequencing, and the genome editing efficiencies of the constructs obtained within the scope of the study were found to be in the range of 0.16-0.76%. It was confirmed that the genome editing technique used worked successfully in grapevine protoplasts and could achieve the target mutations. The protoplast isolation and transformation protocols optimized within the scope of the study are considered to be an important resource for researchers working in this field. Keywords: Chardonnay, grapevine (V. vinifera L.), pLL genes, prime editing, dual-epeg, protoplast isolation, transient gene expression.