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 containing a cre recombinase gene under the control of a germline-specific promoter, transgenic plants become genetically programmed to lose the marker when its presence is no longer required (i.e. after the initial selection of primary transformants). Using promoters with different germline functionality, 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 this work are derived from the APETALA1 and SOLO DANCERS genes from Arabidopsis (Arabidopsis thaliana) Columbia-0 conferring common germline and single germline functionality, respectively. Introduction of the genetic program did not reduce 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.
Verweire
Dimitri
D
Laboratory of Plant Genetics, Institute for Molecular Biology and Biotechnology, Vrije Universiteit Brussel, B-1050 Brussels, Belgium.
Verleyen
Kristof
K
De Buck
Sylvie
S
Claeys
Martine
M
Angenon
Geert
G
D017360:Arabidopsis / Q000235:genetics* / Q000378:metabolism; D029681:Arabidopsis Proteins / Q000235:genetics; D016213:Cyclins / Q000235:genetics; D053837:DNA Cleavage; D004269:DNA, Bacterial; D005818:Genetic Engineering; D005819:Genetic Markers; D005854:Germ Cells / Q000378:metabolism; D026161:MADS Domain Proteins / Q000235:genetics; D030821:Plants, Genetically Modified / Q000378:metabolism; D011401:Promoter Regions, Genetic; D019076:Transgenes
C091535:AP1 protein, Arabidopsis; D029681:Arabidopsis Proteins; D016213:Cyclins; D004269:DNA, Bacterial; D005819:Genetic Markers; D026161:MADS Domain Proteins; C036483:T-DNA; C464574:solo dancers protein, Arabidopsis