Development of chloroplast transformation for five species in the genus <i>Nicotiana</i>

Technologies for the stable genetic transformation of the plastid (chloroplast) genome are currently restricted to a small number of species. The development of highly efficient tissue culture, regeneration, and selection procedures represents the major hurdle that needs to be overcome to extend the species range of the transplastomic technology. Here, we report the development of efficient plastid transformation protocols for five species in the genus Nicotiana: the model species N. benthamiana, the tree tobacco N. glauca, the ornamental plants N. langsdorffii and N. longiflora, and the wild species N. otophora. We have optimized medium composition for efficient regeneration from leaf explants in all five species and determined suitable selection conditions for plastid transformation. We successfully isolated multiple transplastomic lines for each species and also generated lines that express the fluorescent reporter protein DsRed. Molecular and genetic analyses confirmed the homoplasmic state of the transplastomic lines and demonstrated maternal inheritance of the transgenes. Our work makes plastid genome engineering available for a set of new species and enables new applications in horticultural research and ecology. It also informs the future development of plastid transformation technology for other species.