Plastid transformants of tomato selected using mutations affecting ribosome structure

Abstract

Tomato plastid transformants were obtained using two vectors containing cloned plastid DNA of either Nicotiana tabacum or Solanum nigrum and including point mutations conferring resistance to spectinomycin and streptomycin. Transformants were recovered after PEG-mediated direct DNA uptake into protoplasts, followed by selection on spectinomycin-containing medium. Sixteen lines contained the point mutation, as confirmed by mapping restriction enzyme sites. One line obtained with each vector was analysed in more detail, in comparison with a spontaneous spectinomycin-resistant mutant. Integration of the cloned Solanum or Nicotiana plastid DNA, by multiple recombination events, into the tomato plastome was confirmed by sequence analysis of the targeted region of plastid DNA in the inverted repeat region. Maternal inheritance of spectinomycin and streptomycin resistances or sensitivity in seedlings also confirmed the transplastomic status of the two transformants. The results demonstrate the efficacy in tomato of a selection strategy which avoids the integration of a dominant bacterial antibiotic resistance gene.