Current study is to establish a basic plant tissue culture of Nicotiana tabacum TAPM 26 and test the plant tissue on resistancies against 2,2 DCP an active ingredient in herbicide-Dalapon. During micropropagation, the surface sterilization method was ascertained on seeds of tobacco. HgCl2 was used to disinfect tobacco seeds at different concentrations (0.05 gL -1 , 0.2 gL -1 , 0.5 gL -1 and 1.0 gL -1 ) within three minutes. About 70% seeds were survived when exposed to 0.05 gL -1 of HgCl2, whereas, no seeds were germinated when sterilized at concentrations above 0.05 gL -1 of HgCl2. To optimize an efficient protocol of shoots and callus formation during in vitro regeneration, explant types and plant growth were studied. Growth regulators NAA (0.1 mgL -1 , 0.2 mgL -1 , 0.5 mgL -1 , 1.0 mgL -1 and 2.0 mgL -1 ) and BAP (1.0 mgL -1 , 2.0 mgL -1 , 3.0 mgL -1 and 4.0 mgL -1 ) were used. The explants types were one month old leaves and two weeks old cotyledons. The maximum numbers of shoots per explants were obtained from cotyledon with combination 0.1 mgL -1 NAA and 1.0 mgL -1 BAP. The highest callus fresh weight was achieved when NAA 0.5 mgL -1 with BAP 1.0 mgL -1 after four weeks. Thus, the highest number of shoots produced per explants from leaves culture on the MS media containing 0.2 mgL -1 NAA and 4.0 mgL -1 BAP. The best callus fresh weight was obtained with combination of 1.0 mgL -1 NAA and 1.0 mgL -1 BAP by using leaves explant. Finally, Dalapon (5 gL -1 , 10 gL -1 , 15 gL -1 and 20 gL -1 ) were applied onto leaves and cotyledon cultures of N. tabacum to check on the minimum concentration of inhibition. The minimum concentration of inhibition of leaves and cotyledon cultures of N. tabacum was at 5 gL -1 of 2,2DCP but not at 10 gL -1 , 15 gL -1 and 20 gL -1 . This investigation will shed alight for future studies on transgenic tobacco resistant against Dalapon Research article