The cross between Nicotiana tabacum L. (line 329 - oriental tobacco, Djebel type) and N. sanderae Hort. is difficult to carry out because of the existent barriers of incompatibility. When N. sanderae was used as pollinator, a small amount of underdeveloped seeds was obtained. They were cultured on Murashige and Skoog agar medium for embryos, supplemented with 0,05 mgl/L of gibberellic acid, kinetin (KIN) and α-naphthaleneacetic acid (NAA) respectively, where they grew to viable F₁ plants that reached florescence. With their straight, non-branched stems and slightly curled leaves the hybrids obtained resembled N. tabacum , while their flowers in size (flower tube 4,5-5 cm, corolla diameter 4,5-5 cm), fragrance and violet color were close to N. sanderae . Electrophoretic spectra of malate dexydrogenase (E.C.1.1.1.37) and glutamate oxalacetate transaminase (E.C.2.1.1.1) in leaves of F₁ plants combined the isozymes of both parents. All N. tabacum x N. sanderae hybrids were male and female sterile. In order to overcome the male sterility, stem pit parenchyma was cultured in vitro on MS solid medium with KIN (0,5 mg/L) + NAA (2 mg/L) for callusogenesis and KIN (2 mg/L) + NAA (0,5 mg/L) for organogenesis. Some of the regenerants, obtained after longer cultivation (9th passage) were with 62,5% viable pollen and formed seeded capsules after self-pollination. They were mixoploid with chromosome number 2n=44-82. These plants are included in our breeding program.