Materials and Methods

Plant Material.Seeds of male sterile N.tabacum cultivars were kindly supplied by Drs.S.J.Sheen.University of Kentucky：J.Berbec'.Department of Tobacco Breeding and Cultivation.Pulway.Poland：prof.L.Burk，USDA，Oxford.N.C.，and Mr.G.W.Pittarelli，USDA，Beltsville，Md.Tobacco seedlings of approximately 1-inch in height were transplanted into 5-inch soil pots in the greenhouse.Plants were allowed to grow for 4 to 8 weeks depending on the season after transplanting and then covered with black cloth 48 h before the leaves were harvested.

Purification of Chloroplasts.Chloroplasts were purified by the procedure previously described by Rhodes et al.（1981）.Freshly harvested leaves were homogenized in a metal Waring blender with liquid nitrogen（Rhodes and Kung，1981）.When the liquid nitrogen had evaporated and the temperature had warmed to-5℃at room temperatureure，three weight volumes of isolation buffer（Walbot，1977）were added to the powder with rapid mixing.The suspension was filtered through miracloth and centrifuged at 1，500×g for 15 min.The crude chloroplast pellets were gently resuspended in the same buffer and purified through a discontinuous silica sol gradient（Walbot，1977）containing 0.24 mol/L sucrose throughout.

Preparation of Chloroplast DNA.Chloroplast DNA was prepared from the purified chloroplasts according to the method of Kolodner and Tewari（1975）.The chloroplasts were lysed in 2%sarkosyl and DNA was fractionated by CsCl density gradient centrifugation.The purity of ct-DNA was occassionally checked by the denaturation and renaturation procedure described by Kung and Williams（1969）.(https://www.daowen.com)

Restriction Enzyme Analysis of Chloroplast DNA.Chloroplast DNA was digested with EcoRl，BamHI，SmaI，or SalI as directed by the supplier，New England Biolabs.The restricton fragments were separated by electrophoresis in a 0.8%or 1.5%agarose slab gel according to the procedure of Helling et al.（1974）.Ethidium bromide-stained bands were visualized with a Model C-62 Chromatovue transilluminator（Ultra-Violet Product，Inc.）.The EcoRI and HindⅢ single or double digest fragments of lambda phage DNA were used as molecular weight markers.