Leaf mesophyll protoplasts of kanamicine resistant, nitrate reductase deficient mutant Nicotiana tabacum (NR−, Km+) were fused with leaf mesophyll protoplasts of wild type Nicotiana sanderae (wt). Somatic hybrid cell colonies were selected for kanamicine resistance and nitrate reductase proficiency. Nineteen independent cell lines, capable of growth in selection medium were obtained. From 14 of them shoots were regenerated and adapted in soil. All regenerants have intermediate plant and leaf morphology. The chromosome number for regenerants from six lines was determinated, as well as analyses of four isoenzymes: peroxidase, esterase, shikimate dehydrogenase and glutamate-oxaloacetate transminase were performed. Results show that all studied plants represent nuclear somatic hybrids, possessing entire genomes of both parents. Local lesion (hypersensitivity)—type resistance to TSWV has been observed in leaves of these somatic hybrid plants after virus inoculation.