Agrobacterium-mediated gene transformation in 'Black Khismish': targeting MLO and ALS genes with CRISPR/Cas9 system

Economic losses in grape cultivation globally are predominantly linked to the powdery mildew disease caused by Erysiphe necator. It is widely acknowledged that nearly all cultivars of Vitis vinifera L., which represent approximately 90% of the total grape production worldwide, are susceptible to this disease. The widespread prevalence of the pathogen necessitates frequent pesticide applications in viticulture. However, these chemicals not only incur additional production costs for growers but also have detrimental effects on the health of vineyard workers and beneficial organisms in the vineyard environment. Recent studies have explored the potential use of S (susceptible) genes, which facilitate a harmonious interaction between the plant and pathogen, in breeding for resistance. Researchers have investigated the feasibility of conferring powdery mildew resistance to plants by targeting S-genes located in loci known as MLO. In this research, CRISPR technology is utilized to induce mutations in MLO and ALS genes by employing a combination of gene knockout and base editing techniques in ‘Black Khismish’. The CRISPR/Cas9 system, along with Agrobacterium tumefaciens, is utilized to facilitate the selection of both transient and stable transfers of guide RNAs designed for grapevines. Additionally, besides the vector design tailored for knocking out MLO genes, an additional design aimed at targeting ALS genes has been incorporated into the plant using the Agrobacterium tumefaciens EHA105 strain. In the study, embryogenic callus induction was performed with three different media compositions in the ‘Black Khismish’ genotype, and transformation experiments were conducted on the developed calli. The study concluded with the identification of 28 out of 132 regenerated plants in the ‘Black Khismish’ cultivar exhibiting stable transformation. These findings are anticipated to be beneficial for subsequent gene transfer investigations in ‘Black Khismish’.