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Culturing conditions and the dose of a chemical mutagen N-nitroso-N-methylurea were worked out for selecting auxin-independent (auxin-habituated) cell lines of tobacco (Nicotiana tabacum L.). A clone (strain B-4) that steadily grew on an auxin-free nutrient medium and did not express any traits of morphogenesis emerged at the frequency of 1 per 7 × 107 plated cells. Mutation was the cause of habituation in this cell line as corroborated by a low frequency of its emergence, the appearance of the habituation trait only after the mutagenic treatment, and the stability of habituation trait for many years when the cells were subcultured in both selective and nonselective media. The auxin-habituated strain B-4 grew more rapidly on an auxin-free medium, was cytokinin-dependent, resistant to high exogenous IAA concentrations, and incapable of morphogenesis.