CRISPR/Cas9-based gene-editing platform development by targeting genes encoding magnesium chelatase and phytoene...

Sugarbeet (Beta vulgaris ssp. vulgaris, L.), is a vital temperate crop, supplying nearly 40% of the world's sugar. However, its high susceptibility to bacterial, fungal, and viral diseases creates an urgent need for improved, disease-resistant cultivars. The CRISPR/Cas9 system has rapidly advanced plant genetic engineering by enabling precise and targeted genome modifications. Our goal is to establish a gene-editing platform in sugarbeet to support future development of disease-resistant lines by targeting the candidate genes. In this study, we applied CRISPR/Cas9 to generate targeted mutations in two genes involved in chlorophyll biosynthesis and carotenoid-mediated leaf pigmentation: magnesium chelatase (Mg-chelatase) and phytoene desaturase (PDS). Two CRISPR/Cas9 constructs, each carrying an sgRNA targeting either Mg-chelatase or PDS, were developed and mobilized into Agrobacterium tumefaciens. A total of 233 and 200 hypocotyl explants were transformed with constructs targeting Mg-chelatase and PDS, resulting in regeneration efficiencies of 8% and 14% on kanamycin selection medium, respectively. Light green, yellow, variegated yellow-green, and albino phenotypes were observed among the putative transformants, whereas non-edited transformed lines resembled untransformed control plants. Targeted mutations, including insertions, deletions, and substitutions of nucleotides, were identified at both genomic loci, with editing efficiencies of 60.0% for Mg-chelatase and 68.75% for PDS underscoring the effectiveness of this approach in sugarbeet, a recalcitrant crop. Deletions ranged from 5 to 28 bp in Mg-chelatase and 2 to 21 bp in PDS, while insertion events consisted of single-base additions in Mg-chelatase edited lines and larger insertions of 7-16 bp in PDS mutants. The results demonstrate the successful deployment of CRISPR/Cas9 for targeted genome engineering in sugarbeet and establish a reliable platform for future gene-editing efforts aimed at enhancing resistance to a wide range of pathogens and diseases affecting the crop.