Atropa belladonna is the most important commercial source of pharmaceutical tropane alkaloids such as Atropine, Scopolamine, and Hyoscyamine.The current study aims to investigate the effects of 6 different helium-neon laser doses (0.0, 10, 15, 20, 25 and 30 J cm^-2) and exposure conditions on callus fresh weight and apply biotechnological tools as alternative approach for increasing the accumulation of active agents (atropine) of A. belladonna plant using abiotic elicitors salicylic acid precursor feeding. After treatment they were analyzed by a photograph illustrating the banding profile produced by the twelve ISSR primers DNA under the six investigated laser radiation doses and HPLC technique was applied to determine Atropine, Scopolamine and Hyoscyamine contents. The results showed that the highest callus fresh weight, callus dry weight was achieved by callus derived from (leaves and stem) explants, which was cultured on (MS) medium supplemented with different combinations among different concentrations of growth regulators as NAA, 2,4-D, Kin and BAP (at 0.5 and 1.0 mg/l). On the other hand all treatments of callus tissue had affected the content of medically active constituents of Atropa belladonna. This study suggests a more efficient method for commercially large-scale production of Atropine, Scopolamine, and Hyoscyamine by A. belladonna plants.