Sterile hybrids were produced as a result of hybridization between, N. gossei and N. tabacum, cv. Kroumovgrad 90. Fertile regenerants of N. gossei x N. tabacum were developed by application of in vitro methods. Hybrids in F1 and their regenerants were evaluated for resistance to leaf aphids, powdery mildew and tobacco mosaic virus by artificial inoculation in glasshouse conditions. It was found that the inheritance of the resistance to leaf aphids, powdery mildew and TMV in F1 was dominant. In vitro methods provided possibilities for producing fertile regenerants, some of which were with high resistance to the named insects and diseases. The regenerants are of great interest for immunobreeding of tobacco.