Increasing abiotic stress tolerance in grapevines using CRISPR/Cas9 technology

Introduction. Global climate change-associated drought and low-temperature stresses significantly reduce grapevine (Vitis vinifera L.) productivity and fruit quality. Due to the high heterozygosity and vegetative propagation of grapevine, conventional breeding for stress tolerance remains slow and labor-intensive. This study aimed to identify candidate genes associated with abiotic stress tolerance and develop a CRISPR/Cas9-based genome editing strategy for local grapevine cultivars.

Methods. Candidate genes were selected through literature analysis and RNA-seq data evaluation. Members of the DREB transcription factor family, LEA proteins, and the VvESK1 gene were analysed using the Vitis vinifera PN40024.v4 reference genome. Gene structure, conserved domains, and promoter cis-regulatory elements were investigated using BLAST, Clustal Omega, PFAM, InterProScan, and PlantCARE platforms. Guide RNAs targeting VvESK1 were designed using CRISPR-associated computational tools. A binary CRISPR/Cas9 vector was assembled and introduced into Agrobacterium tumefaciens strain LBA4404. Embryogenic calli from local grapevine cultivars were used for transformation and regeneration through somatic embryogenesis.

Results. More than 30 DREB genes and multiple LEA genes associated with abiotic stress responses were identified and characterised in the grapevine genome. Promoter analysis revealed stress-responsive cis-regulatory elements related to drought and cold tolerance. The VvESK1 gene was selected as a promising target associated with cold stress resistance. Designed guide RNAs exhibited high predicted editing efficiency and low off-target potential. The CRISPR/Cas9 construct was successfully validated and transferred into Agrobacterium. In addition, embryogenic callus induction and regeneration conditions were optimised, resulting in putative transformant embryos.

Conclusions. This study established a CRISPR/Cas9-based genome editing platform for grapevine improvement. The DREB, LEA, and VvESK1 genes were identified as promising targets for enhancing abiotic stress tolerance. Furthermore, an efficient regeneration and Agrobacterium-mediated transformation system was optimised, providing a valuable molecular platform for developing climate-resilient grapevine cultivars.