BROADCASTS.com

b'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.'