PLASTID ENGINEERING IN LAND PLANTS - A CONSERVATIVE GENOME IS OPEN TOCHANGE

We have developed efficient transformation protocols to modify each of the 500-10 000 plastid genome copies in a tobacco cell. The transform ing DNA is introduced on the surface of microscopic tungsten particles by the biolistic process. Selection for transplastomes is by spectino mycin resistance based on expression of aminoglycoside-3''-adenyltrans ferase from a chimeric aadA gene in the transforming DNA. Manipulation s that are now feasible include replacement of endogenous plastid gene s with DNA sequences modified in vitro, targeted gene disruption, and insertion of reporter genes into the plastid genome. Alternative metho ds for plastid genome manipulations may be developed utilizing an extr achromosomal element which was identified during the transformation st udies. Introduction of foreign genes under control of plastid gene exp ression elements results in duplication of endogenous regulatory seque nces. A sensitive somatic assay to detect deletions via such direct re peats confirmed that these sequence duplications do not result in sign ificant genome instability. The ability to transform plastids will fac ilitate the study of plastid gene regulation, and the application of g enetic engineering to crop improvement.