Agrobacterium-free genome editing in grapevine: biolistic transformation of somatic embryos

Abstract

Grapevine cultivation is heavily dependent on chemical pesticides to manage biotic or abiotic stressors. However, increasingenvironmental and health concerns, alongside climatic and economic pressures, necessitate the development of more sustainable agricultural practices. Traditional grapevine breeding is hindered by long generation times and high heterozygosity, making thedevelopment of improved cultivars both time-consuming and costly. To enhance biotic and abiotic stress tolerance while reducingpesticide dependence, next-generation genetic engineering, specifically genome editing, offers a rapid and precise alternative. In thisstudy, we demonstrate a proof-of-concept for targeted mutagenesis in grapevine somatic embryos (SE) using a single-plasmid CRISPR-Cas9 system. The use of an SE-based in vitro system ensures the maintenance of the desired genotype and facilitates efficient whole-plant regeneration. We targeted the VvPDS locus, an established marker gene in carotenoid biosynthesis whose disruption should result in a clearly observable phenotype. Transformation was achieved via biolistics (particle bombardment) to deliver DNA-coated microparticles into the embryonic tissues. Following bombardment, we successfully regenerated grapevine plants and analysed sequence alterations at the VvPDS target site. The induced mutations were found not to be biallelic, which was a necessary for theappearance of visible bleached sectors. The reason for that was a hidden heterozygocity at the target locus which was not immediatelyobvious form the genome sequence. Our findings underscore a critical bottleneck in grapevine editing: despite the availability of qualityreference genomes, there is a fundamental necessity for initial, cultivar-specific biallelic genotyping of any targeted genomic region. Nevertheless, we demonstrated that somatic embryogenesis provides a valuable platform upon which more precise and efficient genome-editing strategies can be developed.