Optimal media compositions were worked out for efficient callus induction and consequent plantlet regeneration using different explants (leaf, node and internode) of potato (Solanum tuberosum L.). Among seventeen media compositions used, MS medium supplemented with 3.0 mg/L 2,4-D + 0.25 mg/L BAP exhibited maximum callus induction and proliferation response. Well-proliferated calli were exposed to different treatments of physical (gamma rays 10 to 50 Gy) and chemical (EMS and NaN₂; 1 to 5 mg/L) mutagens and were then screened against partially purified toxin of Alternaria solani. Plants regenerated from callus, insensitive to toxin, were supposed to be resistant and were then screened against two different isolates of A. solani under field conditions. After two year of field trials four out of 63 in vitro screened lines of gamma irradiation; four out of 52 in vitro screened lines of sodium azide treated cultures and five out of 59 screened lines of EMS treated cultures were selected according to disease rating scales. Variation was further confirmed using various biochemical and molecular markers.