Rapid and proven production of transplastomic tobacco plants by restoration of pigmentation and photosynthesis

Tobacco chloroplast transformation is typically achieved using dominant, selectable antibiotic resistance genes such as aadA, nptII and aphA-6. An improvement would be the combination of such a marker with a visual screening system for the early and conclusive detection of plastid transformants. As such, we investigated the use of three photosynthesis-deficient plastid mutants, DeltapetA, Deltaycf3 and DeltarpoA, for the development of a phenotypic selection system. Mutant plants were used as an alternative to the wild-type as source tissue for transformation, re-introducing deleted plastid sequences and using the aphA-6 gene as a selection marker. The reconstitution of the deleted genes in transformed regenerants resulted in shoots with a visually distinct phenotype comparable to the wild-type. This transformation/selection system overcomes the common problems associated with plastid transformation, e.g. the recovery of spontaneous mutants or nuclear insertions. In addition to the benefits offered by phenotypic selection, phenotype reconstitution leads to restoration of photosynthesis, which we assume drives reconstituted plants rapidly towards homoplasmy. As such, repeated cycles of regeneration in the presence of an antibiotic selection agent are no longer required.