Due to the improvement of the mutant germplasm and its use in the creation of new mutant organisms, these mutations have been instrumental in meeting global food and nutritional protection challenges. Mutagenic therapies for plant breeding and for drug alkaloids development projects have contributed to a wide range of genetic variability. The primary commercial source of drug alkaloids such as atropine is Atropa belladonna. This paper focuses on the effect of gamma rays (γ-rays) on callus of Atropa belladonnaat the molecular level and changes of biochemical metabolisms in callus. Atropa belladonna callus were irradiated with 0.0, 30, 60, 90, 120 and 150 Gy gamma rays after 6 weeks on induction media. Highly significant differences between effect of gamma rays (γ-rays) on callus of Atropa belladonna at 2,4-D 2 mg/L and Kin 0.5 mg/L respectively were recorded on callus fresh weight and callus dry weight. 120 Gy had higher callus fresh weight, dry weight, Dry matter content (%) than the control. The difference in ion levels were measured in control and callus irradiated. The results clearly decrease in ion levels with increase of gamma dose. On the other hand, the deferent of antioxidant activity were cleared between control and gamma ray's callus. The hist antioxidant activity dose 150 Gy which recorded 30.20 comparing with control 20.10. Polyacrylamide gel electrophoresis SDS-protein PAGE profiles were used. In irradiated plants, there were several new protein bands that could be used as markers for each dose. Using DNA-Start codon targeted (SCoT) polymorphism assay. There were two negative molecular markers which were found only in control as compared to irradiated callus, with molecular sizes 940 and 400 bp.