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
vst
I 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.