Callus from Nicotiana tabacum is used as a model in plant developmental research. We tested several phytohormone (Indoleacetic acid – IAA; 2,4-Dichlorophenoxyacetic acid – 2,4-D; kinetin – KIN; 6-Benzylaminopurine – BAP) combinations to compare different approaches to callus induction directly from the seeds of Nicotiana tabacum. Callus formation was observed up to 4 weeks after sowing and the most effective were 0.5 mg/L of 2,4-D with 0.25 mg/L of BAP and 2 mg/L 2,4-D with 1 mg/L of BAP. The calli were green, photosynthetically active and after 6 weeks of growth, no stress symptoms (estimated on the basis of fluorescence of chlorophyll a in photosystem II) were noticed.