Removal of antibiotic resistance genes from transgenic tobacco plastids

Removal of antibiotic resistance genes from genetically modified (GM) crops removes the risk of their transfer to the environment or gut microbes. Int egration of foreign genes into plastid DNA enhances containment in crops th at inherit their plastids maternally. Efficient plastid transformation requ ires the aadA marker gene, which confers resistance to the antibiotics spec tinomycin and streptomycin. We have exploited plastid DNA recombination and cytoplasmic sorting to remove aadA from transplastomic tobacco plants. A 4 .9 kbp insert, composed of aadA flanked by bar and uidA genes, was integrat ed into plastid DNA and selected to remove wild-type plastid genomes. The b ar gene confers tolerance to the herbicide glufosinate despite being GC-ric h. Excision of aadA and uidA mediated by two 174 bp direct repeats generate d aadA-free T-0 transplastomic plants containing the bar gene. Removal of a adA and bar by three 418 bp direct repeats allowed the isolation of marker- free T-2 plants containing a plastid-located uidA reporter gene.