Functional Analysis of Naturally Integrated Rol Genes in Sweet Potato via CRISPR/Cas9 Genome Editing

Sweet potato (<i>Ipomoea batatas</i>) is a globally important crop and one of a growing number of plants recognized as naturally transgenic, harboring <i>Agrobacterium</i>-derived T-DNA genes whose functions remain largely uncharacterized. In this proof-of-concept study, we applied CRISPR/Cas9 technology to generate targeted knockouts of the <i>Ib-rolB/C</i> and <i>Ib-rolD-like</i> genes located within the sweet potato cellular T-DNA2 (<i>Ib</i>T-DNA2) region. Mutations were introduced into sweet potato callus cultures using an optimized genome editing protocol, with most edits consisting of single-nucleotide insertions. Knockout of <i>Ib-rolB/C</i> did not affect callus growth but significantly reduced levels of chlorogenic acid derivatives. Validation <i>in planta</i> using transient expression in <i>I. batatas</i> leaves confirmed the suppressive effect of <i>Ib-rolB/C</i> disruption on polyphenol content. In contrast, <i>Ib-rolD-like</i> knockout lines showed reduced biomass accumulation and downregulation of cell cycle-related genes, but did not display significant changes in metabolite content in either callus cultures or leaf tissues. These findings suggest that <i>Ib-rolB/C</i> and <i>Ib-rolD-like</i> may differentially contribute to growth and secondary metabolism in sweet potato.