Agrobacterium-mediated capsicum annuum gene editing in two cultivars, hot pepper CM334 and bell pepper dempsey

Abstract

Peppers (Capsicum annuum L.) are the most widespread and cultivated species of Sola-naceae in subtropical and temperate countries. These vegetables are economically attractive world-wide. Although whole-genome sequences of peppers and genome-editing tools are currently avail-able, the precision editing of peppers is still in its infancy because of the lack of a stable pepper transformation method. Here, we employed three Agrobacterium tumefaciens strains-AGL1, EHA101, and GV3101-to investigate which Agrobacterium strain could be used for pepper trans-formation. Hot pepper CM334 and bell pepper Dempsey were chosen in this study. Agrobacterium tumefaciens GV3101 induced the highest number of calli in cv. Dempsey. All three strains generated similar numbers of calli for cv. CM334. We optimized a suitable concentration of phosphinothricin (PPT) to select a CRISPR/Cas9 binary vector (pBAtC) for both pepper types. Finally, we screened transformed calli for PPT resistance (1 and 5 mg/L PPT for cv. CM334 and Dempsey, respectively). These selected calli showed different indel frequencies from the non-transformed calli. However, the primary indel pattern was consistent with a 1-bp deletion at the target locus of the C. annuum MLO gene (CaMLO2). These results demonstrate the different sensitivity between cv. CM334 and Dempsey to A. tumefaciens-mediated callus induction, and a differential selection pressure of PPT via pBAtC binary vector.