Wild Nicotiana species possess a high potential of useful features. Interspecific hybridization is a very effective method for the introducing of desired characters into tobacco and achieving crop improvement. Its successful realization is limited due to various incompatibilities. Those barriers of incompatibility could be overcome by applying in vitro techniques (embryocultures for embryo rescue and tissue cultures in order to produce viable F₁ hybrid plants and to improve their fertility). We realized six interspecific crosses through routine hybridization technique. Wild species N. sylvestris, N. plumbaginifolia, N. benthamiana, N. cavicola and N. gossei were used as female and N. tabacum as male parents. Tobacco was used as a mother parent only in the combination with N. sanderae . Embryos from the crosses N. plumbaginifolia × N. tabacum and N. tabacum × N. sanderae were grown in vitro in order to produce viable F₁ plants. Morphological, cytological and biochemical examinations confirmed true hybridity of the obtained interspecific combinations. Male and female sterility and meiotic abnormalities were characteristic for all of them. It was overcome through in vitro growing of stem pit parenchyma for callus induction and organ formation. The regenerants (R) obtained after longer callus cultivation (3^th-9^th passages) were with 31.4%- 62.5% viable pollen and formed seeded capsules via self-pollination. They were tested for resistance to some diseases and pests. R₁ from the combinations N. benthamiana × N. tabacum and N. gossei × N. tabacum were shown to be resistant to aphids. These of N. gossei × N. tabacum were tolerant to powdery mildew and tobacco strain of TMV. F₁ regenerants of N. cavicola × N. tabacum were resistant to ToMV and these of N. tabacum × N. sanderae showed resistance toward TSWV. These plants are included in our breeding program.