Plastid Transformation in Nicotiana tabacum and Nicotiana sylvestris by Biolistic DNA Delivery to Leaves

The protocol we report here is based on biolistic delivery of the transforming DNA to tobacco leaves, selection of transplastomic clones by spectinomycin resistance and regeneration of plants with uniformly transformed plastid genomes. Because the plastid genome of Nicotiana tabacum derives from Nicotiana sylvestris, and the two genomes are highly conserved, vectors developed for N. tabacum can be used in N. sylvestris. Also, the tissue culture responses of N. tabacum cv. Petit Havana and N. sylvestris accession TW137 are similar, allowing plastid engineering protocols developed for N. tabacum to be directly applied to N. sylvestris. However, the tissue culture protocol is applicable only in a subset of N. tabacum cultivars. Here we highlight differences between the protocols for the two species. We describe updated vectors targeting insertions in the unique and repeated regions of the plastid genome as well as systems for marker excision. The simpler genetics of the diploid N. sylvestris, as opposed to the allotetraploid N. tabacum, make it an attractive model for plastid transformation.