Spatio-temporal regulation of recombinase expression enables efficient autoexcision of selectable marker genes in...

Most plant transformation protocols include the use of a selectable marker gene to enable efficient selection of transformed cells for transgenic regeneration. While the marker gene facilitates the transformation process, it is not needed once transgenic plants have been identified and is removed during commercial development. Autoexcision is a marker removal process, in which a Cre-lox system is used to remove both the selectable marker gene and the Cre gene itself in the T0 generation. One challenge for autoexcision is achieving efficient marker removal while preserving the recovery rate for transgene-positive transformation events. Predictable and robust control of Cre gene expression is essential to maintaining this balance.

In this report, we demonstrate the utility of promoters and other expression elements that were selected based on the RNA-seq-derived, tissue-specific expression patterns of their associated endogenous genes. These elements were selected for their potential to drive Cre in a specific spatio-temporal manner. Functional elements expected to drive Cre primarily in floral meristem, gametes, and/or early embryo enabled efficient autoexcision and recovery of transgenic transformants. Additionally, we confirmed the expected expression patterns of successful elements via GUS staining.

Overall, these elements enable an efficient approach for selectable marker gene removal during commercial development.