High-efficiency generation of fertile transplastomic Arabidopsis plants

The development of technologies for the stable genetic transformation of plastid (chloroplast) genomes has been a boon to both basic and applied research. However, the extension of the transplastomic technology to major crops and model plants has proven extremely challenging, and the species range of plastid transformation is still very much limited in that most species currently remain recalcitrant to plastid genome engineering. Here we report an efficient plastid transformation technology for the model plant Arabidopsis thaliana that relies on root-derived microcalli as source tissue for biolistic transformation. The method produces fertile transplastomic plants at high frequency when combined with a CRISPR/Cas9-generated knock-out allele of a nuclear locus that enhances sensitivity to the selection agent used for isolation of transplastomic events. Our work makes the model organism of plant biology amenable to routine engineering of the plastid genome, facilitates the combination of plastid engineering with the power of Arabidopsis nuclear genetics, and informs the future development of plastid transformation protocols for other recalcitrant species.