Hairy root cultures of Silybum marianum (L.) Gaertn in bioreactor and evaluation of stress enzyme activity and silymarin production in response to elicitation

Silymarin, chemical bioactive constituent of extract from the seeds of milk thistle (Silybum marianum (L.) Gaertn) is mostly used for liver disease treatment. Hairy root culture of S. marianum was established by genetic transformation through Agrobacterium rhizogenes for higher secondary metabolite production. The first part of this study focused on making optimal physiological condition, pH (5, 5.7, 6 and 7) and L-phenylalanine concentration as the precursor for silymarin production in shake flasks. Furthermore, the stimulatory effect of different concentrations of phenylalanine (0, 1, 10 and 100 μM) on phenylalanine ammonia-lyase activity, naringenin content, root biomass was investigated in S. marianum hairy roots. Phenylalanine 100 μM after 72h was found optimal feeding condition for production of silymarin (1.84 fold higher than that of the control). The maximum biomass and silymarin accumulation occurred in root cultures grown in pH, 5 (0.45 g and 0.26 mg g_1 DW) and higher lipoxigenase activity at this pH level indicated oxidative stress. As the second part, we evaluated the mass cultivation of S. marianum roots and the effects of 100 μM methyl jasmonate (MJ) elicitation on the accumulation of silymarin in 2 l bioreactor. The extent of MJ-induced oxidative damage was assayed by measuring guaiacol peroxidase (G-POD) and ascorbate peroxidase (APX) activities in MJ-induced production of silymarin in bioreactor hairy root cultures of S. marianum. The flavonolignans were analyzed by high performance liquid chromatography method and the antioxidant enzymes activities were measured using a UV visible spectrophotometer. Silymarin accumulation was increased from 0.13 in non-treated hairy roots to 0.22 mg g_1 DW in MJ- treated hairy roots 72h after treatment. G-POD and APX was activated by MJ 72h after treatment, being 3.2 and 1.3 higher than that of the control. An increase in G-POD and APX activity suggests increased scavenging of reactive oxygen species (ROS), indicating the tolerance to MJ stress. At the final stage, we established the bioreactor hairy root culture with optimal condition which was resulted from previous experiments.