Chloroplast Transformation and Reverse Genetics

Abstract

For chloroplast transformation, the most efficient method to introduce DNA is particle bombardment. To then select the cells which harbor a transformed plastid, two classes of markers are available. With one class, selection is based on the rescue of a non-photosynthetic mutant with the wild-type chloroplast gene. With the other class, selection is based on a mutation or a foreign gene conferring resistance to an antibiotic or a herbicide. Transforming DNA is integrated by homologous recombination, and only in exceptional cases is it maintained extrachromosomally. The modified and wild-type copies of the highly polyploid plastid genome usually segregate rapidly, although in some circumstances a heteroplasmic mixture of genomes is retained. The available technology and markers readily allow chloroplast gene inactivation and site-directed mutagenesis. These possibilities are enhanced by strategies such as co-transformation or the repeated use of unstable markers.