Abiotic stresses such as drought, salinity, and extreme temperatures are major limiting factors in plant growth and development and pose serious threat to global agricultural production. Here we describe a procedure, using a tobacco plastid transformation vector, to generate transplastomic plants with an enhanced ability to tolerate abiotic stresses such as salinity, drought, or cold stress. The procedure involves biolistic delivery of a plastid transformation vector into explants, antibiotic selection procedures, and �identification of transplastomic lines. The plastid transformation vector contains an aadA gene that encodes resistance to spectinomycin as a selectable marker along with the gene of interest for developing transplastomic plants that are tolerant to abiotic stresses. Shoot buds appear over the surface of bombarded explants following spectinomycin selection. Transplastomic shoots are multiplied following several rounds of �spectinomycin selection. Homoplasmic transplastomic lines are confirmed by spectinomycin and streptomycin double selection over a period of 4–5 weeks. The available reports suggest that transplastomic technology is a useful tool for expressing genes in plastids or chloroplasts for enhancing abiotic stress tolerance in plants.