Seedling tests for resistance to kanamycin and spectinomycin

Methods pPRV111A::neoSTLS2 construction and transplastomic plants pPRV111A::neoSTLS2 was produced by ligating the HindIII fragment containing neoSTLS2 from plasmid pCMneoSTLS2 (ref. 13) into pPRV111A. This transformation vector was introduced into the plastids of Nicotiana tabacum L. Petit Havana (N,N) by biolistic bombardment, and homoplasmic plants were regenerated as described previously. The homoplasmic plants were transferred to soil and grown in a controlled environment chamber with a 14 h light/10 h dark and 25 8C day/20 8C night growth regime. The photon flux density was approximately 300 mmol m s at the plant surface. The nuclear neoSTLS2 control line was generated by biolistic bombardment with plasmid pCMneoSTLS2 (ref. 13).