Optimized protocol for efficient generation, confirmation, transformation, and CRISPR editing of grapevine hairy roots

Hairy root cultures (HRCs) are powerful tools in plant biotechnology but show variable establishment efficiencies, limiting broader applications. Here, we present a standardized and optimized reference methodology for the routine generation, multiplication, and maintenance of HRCs across diverse grapevine genotypes. Our workflow evaluated three Rhizobium strains, seven grapevine cultivars (three Vitis vinifera cultivars; four Vitis rootstock hybrids), multiple explant types, infection protocols, co-cultivation times, growth media types, and anti-browning agents. The resulting protocol was effective for all grapevine genotypes and, with minor adjustments, also yielded HRCs from two other important South African plant species, namely Sutherlandia frutescens and Aspalathus linearis. Useful molecular tools were developed for transformation and selection of HRCs, including universal multiplex primers for confirmation of transformation, tested antibiotic resistance markers (kanamycin and hygromycin), and fluorescent reporters (DsRed and eyGFPuv), with DsRed found to be particularly versatile. To test the system, we overexpressed the VviMYBA1 transcription factor gene, leading to increased anthocyanin accumulation and red pigmentation in HRCs. Additionally, we achieved CRISPR/Cas9 editing of the VviPUB19 gene, the first report of CRISPR-edited grapevine HRCs. Gene editing combined with HRCs can facilitate rapid gene function studies, offering an efficient alternative or pre-screening system to whole-plant transformations, that could support advanced functional genomics and biotechnological applications in grapevine.