Visible marker excision via heat-inducible Cre/LoxP system and Ipt selection in tobacco

Numerous studies have reported on the heat-inducible deletion of a marker gene in plant transformation. However, these deletions seldom display manifestations that are visible to the naked eye to easily identify whether excision has occurred. In this study, an isopentenyl-transferase (ipt) gene from Agrobacterium tumefaciens was adopted to render abnormal bunched and underdeveloped leaf phenotypes to transgenic buds and/or shoots, and a synthesized heat shock (HS) promoter HSP70m was used to control the Cre/LoxP recombination system. Results showed that the HSP70m promoter could be highly induced by HS, even reaching the strength of the CaMV 35S promoter. The HSP70m was also sufficiently strong to drive Cre/LoxP system to obtain marker-free tobacco plants, meanwhile ipt-induced abnormal buds and shoots served as a visible selectable marker for transgenics, and normal shoot development after heat shock functioned as a positive selectable marker for removal of marker gene including ipt. This screening method is more convenient than the traditional heat-inducible marker excision system. Therefore, the combination of heat-inducible Cre/LoxP system and ipt selection has the potential to be a reliable tool for generating marker-free transgenic plants in genetic manipulation.