Engineering the Plastid Genome: Problems and Potential

Abstract

The plastid genome of higher plants is a 120-kb to 160-kb double-stranded DNA present in 1,900 to 50,000 copies per leaf cell. To obtain genetically stable transplastomic lines every one of the plastid genome copies (ptDNA) should be uniformly altered in a plant. Transformation is accomplished through the following steps: (i) introduction of the transforming DNA, encoding antibiotic resistance, by the biolistic process or PEG treatment; (ii) integration of the transforming DNA by two homologous recombination events and (iii) elimination of wild-type genome copies during repeated cell divisions on a selective medium. As integration of foreign DNA always occurs by homologous recombination, plastid transformation vectors contain segments of the plastid genome to target insertions to specific locations. The plastid vectors also contain a marker for selection. Useful, non-selectable genes are cloned next to selectable marker genes, with which they are introduced into the plastid genome. Homology-directed genome manipulations have included introduction of point-mutations and deletion of targeted genes.