Generation of marker-free plastid transformants using a transiently cointegrated selection gene.
Genetic engineering of higher plant plastids typically involves stable introduction of antibiotic resistance genes as selection markers. Even though chloroplast genes are maternally inherited in most crops, the possibility of marker transfer to wild relatives or microorganisms cannot be completely excluded. Furthermore, marker expression can be a substantial metabolic drain. Therefore, efficient methods for complete marker removal from plastid transformants are necessary. One method to remove the selection gene from higher plant plastids is based on loop-out recombination, a process difficult to control because selection of homoplastomic transformants is unpredictable. Another method uses the CRE/lox system, but requires additional retransformation and sexual crossing for introduction and subsequent removal of the CRE recombinase. Here we describe the generation of marker-free chloroplast transformants in tobacco using the reconstitution of wild-type pigmentation in combination with plastid transformation vectors, which prevent stable integration of the kanamycin selection marker. One benefit of a procedure using mutants is that marker-free plastid transformants can be produced directly in the first generation (T0) without retransformation or crossing.
Klaus
Sebastian M J
SM
Huang
Fong-Chin
FC
Golds
Timothy J
TJ
Koop
Hans-Ulrich
HU
D001483:Base Sequence; D002736:Chloroplasts / Q000235:genetics*; D018506:Gene Expression Regulation, Plant / Q000235:genetics*; D018014:Gene Transfer Techniques; D005818:Genetic Engineering / Q000379:methods*; D005819:Genetic Markers / Q000235:genetics*; D005822:Genetic Vectors / Q000235:genetics; D008969:Molecular Sequence Data; D030821:Plants, Genetically Modified / Q000235:genetics*; D018087:Plastids / Q000235:genetics; D012641:Selection, Genetic; D014026:Nicotiana / Q000235:genetics*; D014170:Transformation, Genetic / Q000235:genetics
D005819:Genetic Markers