Marker-free transgenic plants through genetically programmed auto-excision

We present here a vector-system to obtain homozygous marker-free transgenic plants without the need of extra handling and within the same time frame as compared to transformation methods in which the marker is not removed. By introducing a germline-specific auto-excision vector (GSA vector) containing a cre recombinase gene under control of a germline-specific promoter transgenic plants become genetically programmed to lose the marker when its presence is not longer required, i.e. after the initial selection of primary transformants. By using promoters with different germline functionalities, two modules of this genetic program were developed. In the first module the promoter, placed upstream of the cre gene, confers CRE functionality in both the male and the female germline or in the common germline (e.g. floral meristem cells). In the second module a promoter conferring single-germline specific CRE functionality was introduced upstream of the cre gene. Promoter sequences used in the work presented here are derived from the APETALA1 and SOLO DANCERS genes from Arabidopsis thaliana Col-0 conferring common-germline and single-germline functionality, respectively. The introduction of the genetic program did not reduce the transformation efficiency. Marker-free homozygous progeny plants were efficiently obtained regardless of which promoter was used. In addition, simplification of complex transgene loci was observed.