A developmentally regulated Cre-lox system to generate marker-free transgenic Brassica napus plants

In this chapter, a strategy for engineering marker-free Brassica napus plants is described. It is based on the Cre-lox site-specific recombination system and includes three essential steps. At first, the binary vector pLH-nap-lx-cre-35S-bar-lx-vst has been designed. In this vector, the cre gene and the bar expression cassette are flanked by two lox sites in direct orientation. The lox-flanked sequence is placed between a seed-specific napin promoter and a coding region for the vstI gene. At the second step, the cre-bar vector was transferred into B. napus hypocotyl explants by Agrobacterium tumefaciens-mediated transformation. Finally, T1 progeny was tested for excision of the marker gene at phenotypic and molecular levels. PCR, sequencing, and Southern blot analysis confirmed complete and precise deletion of the lox-flanked DNA region. This developmentally regulated Cre-lox system can be applied to remove undesirable DNA in transgenic plants propagated by seeds.