New selection systems are required to extend plastid transformation to a more significant number of plant species. After demonstrating that a bacterial mutant glutamate 1-semialdehyde aminotransferase (GSA) gene is functional in tobacco plastids and confers resistance to the phytotoxin gabaculine, in this work, we transformed the tobacco plastome with a mutated GSA gene derived from alfalfa, along with the?aadA?selectable marker gene conferring spectinomycin resistance. Selection exploiting gabaculine was not effective to directly regenerate transplastomic events, but some events regenerated using conventional spectinomycin selection acquired gabaculine resistance during the second or third regeneration rounds with gabaculine selection. Gabaculine selection with our plant mutant GSA gene can be further investigated for plastid transformation of species that are not transformable with the?aadA-spectinomycin selection system.