Markerfreie transplastome Tabakpflanzen

The genetic engineering of higher plant chloroplasts typically
involves the stable integration of antibiotic resistance genes as
dominant selectable markers. The potential spread of these genes
through pollen release or to soil microbes via horizontal gene
transfer is of considerable environmental concern. The use of
chloroplast transformation alleviates the first of these problems
to some degree since plastid-encoded genes are maternally inherited
in most important crop species. However, the complete removal of
antibiotic resistance genes from transplastomic plants is
preferable for improved safety standards. In this work a new system
was established for the production of marker-free chloroplast
transformants using the approach of pigment mutant reconstitution
in combination with a novel transformation vector. The
complementation of a plastid mutant was shown to be an efficient
and rapid system for the generation of plastid transformants
supported by the phenotype-assisted selection and the restoration
of photosynthesis. Furthermore, new results regarding the
recombination process between standard transformation vectors and
the plastid genome led to the idea to clone the antibiotic
resistance gene outside and not between the homologous flanks used
for stable gene introduction. In this configuration the selection
marker can never become stably integrated into the plastome and
gets simply lost once the antibiotic selection pressure is removed.
Phenotype-assisted selection of plastid transformants in
combination with a transient selection marker is a new efficient
system for the rapid production of marker-free plastid
transformants in the first generation.