Plastid engineering in land plants: a conservative genome is open to change.

We have developed efficient transformation protocols to modify each of the 500-10,000 plastid genome copies in a tobacco cell. The transforming DNA is introduced on the surface of microscopic tungsten particles by the biolistic process. Selection for transplastomes is by spectinomycin resistance based on expression of aminoglycoside-3"-adenyltransferase from a chimeric aadA gene in the transforming DNA. Manipulations that are now feasible include replacement of endogenous plastid genes with DNA sequences modified in vitro, targeted gene disruption, and insertion of reporter genes into the plastid genome. Alternative methods for plastid genome manipulations may be developed utilizing an extrachromosomal element which was identified during the transformation studies. Introduction of foreign genes under control of plastid gene expression elements results in duplication of endogenous regulatory sequences. A sensitive somatic assay to detect deletions via such direct repeats confirmed that these sequence duplications do not result in significant genome instability. The ability to transform plastids will facilitate the study of plastid gene regulation, and the application of genetic engineering to crop improvement.