الفهرس 
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Abstract
This study was funded by the EU Commission, joint master’s degree, tempus project no.543865. It is a scientific cooperation between Genetics and Genetic engineering dept., Faculty of Agriculture, Benha University, Egypt, and Plant Physiology dept., Faculty of Pharmacy, Barcelona University, Spain.In this study, the enhancing effect of gamma irradiation (200, 600 GY) and colchicine (0.05%) on silymarin (secondary metabolites) production using cell suspension culture of Silybum marianum L. was investigated. Also, chalcone synthase (CHS) genes expression was evaluated in all treatments as well as control using qRT-PCR, quantitative real time PCR To achieve these goals, the M3 stable mutated seeds of Silybum marianum L. which were treated with different doses of gamma irradiation (200GY, 600GY) in previous study for El-Garhy et al., 2016 were used. In addition, we used the M1 seeds soaked in colchicine (0.5 mg/L) for 30 min comparing with control seeds. Hence, calli were obtained from irradiated and colchicine treated seedlings using MS medium. Moreover, the cell viability of the obtained callus was determined where viable cells appeared as fluorescein green while dead cells appeared as fluorescein red. After confirming the callus cells viability, cell suspensions were established from 3-month-old undifferentiated callus. In addition, the fresh weight, dry weight and growth rate were evaluated after 12 days of incubation periods under the effect of the studied different treatments. Also, the flavonoid in both dry seeds and cell suspension culture were determined by HPLC. The total RNA was extracted from all the studied treatments as well as the control of S. marianum callus samples for evaluating the differential expression of CHS1, 2, 3 genes in response to gamma radiation and colchicine. On the other hand, conventional PCR using cDNA as a template was performed to confirm the amplicon lengths (101, 184, 105 and 134bp) and primer specificity of CHS1, CHS2, CHS3 genes and NADH, internal reference gene for qRT-PCR.