Silybum marianum L. (Milk thistle) extracts are the main source of silymarin that is a mixture of various flavonolignan (silybin (silibinin), silydianin and silychristin). Silymarin of milk thistle has a hepatoprotective activity for liver cirrhosis and chronic inflammatory. Silybum marianum regeneration from hypocotyl explants and evaluation of their callogenesis, growth and total flavolignan (silymarin) upon copper sulphate (as abiotic elicitor) elicitation was targeted. Copper sulphate (CuSO4) was applied in concentrations of 0, 3, 5, 7 and 9 µM to elicit the silymarin production in cultures. The elicitation periods used in this study were 2, 4, 7, 14 and 28 days. Half-strength MS medium recorded better results relative to full-strength MS one and seed incubation in the darkness at room temperature resulted in rapid germination and reached to the gar lid after 10 days. Callus fresh and dry weights as well as growth index were gradually increased with increasing the copper sulphate concentration till 5 µM while decreased thereafter at any elicitation period. With the increase of the elicitation period, the increase of the previous parameters was observed. Flavonolignan (silymarin) was positively correlated with CuSO4 levels since all levels of copper sulphate significantly enhanced its content in relative to the control. Additionally, more silymarin was accumulated after 4 or 7 days and the accumulation significantly decreased when the elicitation period reached 14 days more. The highest silymarin (flavolignan) content (11.79 and 11.67 mg g–1 DW) was obtained when 5 or 7 µM copper sulphate levels were combined with 4 days elicitation period, being about five-fold of the control.
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