Production and Expedient Screening of Transgene-Free Edited Perennial Mutant Plants

Developing genome-edited and transgene-free asexually propagated perennial crop plants is challenging but highly desirable.We have developed a highly useful method using Agrobacterium-mediated transient Cas9 and sgRNA expression to create transgene-free mutant plants without the need for sexual segregation. We have also developed a rapid, cost-effective and high-throughput mutant screening protocol based on Illumina sequencing followed by high-resolution melting (HRM) analysis. Using tetraploid tobacco as a model species and the phytoene desaturase (PDS) gene as a target, we successfully created and expediently identified transgene-free pds mutant plants that are non-transgenic. Our method may provide a tool to produce genome-edited and transgene-free plants that are heterozygous, perennial or difficult to regenerate.