Breaking the glass ceiling of stable genetic transformation and gene editing in the popular pepper cv Cayenne

Abstract Chili pepper (Capsicum spp.) is one of the oldest domesticated crops and the world's most widely cultivated spice. However, functional genetics research and gene editing in pepper are limited by the low efficiency of stable genetic transformation methods. To address this issue, we employed an anthocyanin-based visual marker system and an RNAi construct targeting the SUPPRESSOR OF GENE SILENCING 3 (SGS3) gene to enable efficient isolation of transgenic lines with robust transgene expression, achieving effective transformation of the popular pepper cultivar C. annuum cv Cayenne. The efficacy of this approach is further demonstrated through five independent transformation lines, resulting in transgenic plants with heritable transgenes and gene edits. These include the successful disruption of the developmental regulator ARGONAUTE7 (CaAGO7), which produced wiry-leaf phenotypes, and the enzyme-coding gene POLYPHENOL OXIDASE (CaPPO), confirmed by reduced enzymatic activity in the edited plants. The reported optimized method provides a reliable tool for precise genetic engineering in pepper, enabling functional genomics research and targeted breeding to improve its agricultural traits.