A transposon‐based cargo system mediates gene trafficking and creates ultra‐clean transgenic plants after stable transformation

Summary Genetically modified crops have profound impacts on cost savings and environmental friendliness conferred by new traits, such as resistance to insects and herbicides. Selectable marker genes are essential for screening transformed cells, but they are undesirable in the final product due to the risks of horizontal gene transfer and extensive safety assessment requirements. Generating marker‐ and backbone‐free lines can enhance the public acceptance of transgenic crops. Here, we established a transposon‐mediated ultra‐clean selectable transformant (TRUST) system for generating marker‐ and backbone‐free transformants in a visibly controllable manner, facilitated by the integration of transposon elements, fluorescence proteins, and the anthocyanin biosynthesis gene. This system creates ultra‐clean transgenic events that retain only the expression cassette of the gene of interest with an average probability of 15.5%. Additionally, long‐read whole‐genome sequencing confirmed the integrity of the expression cassette boundaries. The TRUST system is not only a powerful method for producing backbone‐free transgenic plants but also increases the number of transgenic events originating from one starting event, thereby potentially leading to advances in the genetic engineering of recalcitrant crop varieties.