Seeds were treated with sulfuric acid (98%, 10 min) and then surface sterilized with 70% ethanol (v/v) for 1 min and sodium hypochlorite solution (10%, at 10 min). After sterilization, seeds were germinated on MS media (Murashige and Skoog, 1962) containing 7 g/L agar (Duchefa, Netherlands). Cultures were maintained under 16/8 h light/dark. Explants were taken from 4-week-old leaves for inoculation with bacteria strain.ATCC15834 strain of A. rhizogenes was supplied by microbial unit of the National Research Center for Genetic Engineering and Biotechnology, Tehran-Iran. Bacterial cells cultivated on LB (Luria-Bertani) culture medium (Bertani, 1952) on rotary shaker (at 26 ℃ and 180 rpm for 48 h) in the darkened state.The leaves were wounded and inoculated with bacterial suspension for 5 min and transferred to MS media containing 7 g/L agar in darkness at 25 ℃ . After 48 h treated Explants were cultured on the 1/2 MS media containing cefotaxime (500 mg/L) and indole-3-butyric acid (IBA) (2 mg/L). Hairy roots emerged at wounded sites, after 4-weeks of incubation, and then each hairy root line was isolated from explants tissue and was subcultured weekly in new media (1/2 MS hormone-free media) with appropriate antibiotic. The concentration of cefotaxime was decreased gradually and eliminated from the culture medium after 8 subcultures and axenic root cultures were obtained. Then hairy root lines were transferred to the 250 mL Erlenmeyer flasks containing 30 mL hormone- free 1/2 MS liquid medium and incubated on a rotary shaker (120 rpm) at 25 ℃ and subcultured every two week. Hairy root line, which showed sufficient growth in 1/2 MS liquid medium, was selected for further investigations.The genomic DNA was extracted from transformed hairy root lines and plant intact roots with CTAB method . Gene-specific primers from rol B were used for amplification of the 780-bp segment in PCR analysis. The primers sequences were, F:5'-ATGGATCCCAAATTGCTATTCCCCCACGA-3'and R:5'-TTAGGCTTCTTTCATTCGGTTTACTGCAGC-3'. Thirty-five PCR cycles were performed with 5 min initial denaturation at 94 ℃ , annealing steps at 60 ℃ for 80 s, extension at 72 ℃ for 90 s, and final extension step of 72 ℃ for 10 min. The amplimer were analyzed by 1% agarose gel electrophoresis.To investigate the effects of SiO₂ NPs, various concentrations (0, 25, 50, 100 and 200 mg/L) of this elicitor were added to the hairy roots culture medium (1/2 MS + 3% sucrose, pH = 5.7) at the end of log phase of growth stages (21-days-old cultures). Hairy roots were incubated with elicitor for 24 and 48 h of exposure time. Hairy roots were harvested 7 days after elicitation and dried on sterile filter paper to remove excess surface moisture and were weighed before freezing by liquid nitrogen and stored at -80℃ until used to measure growth, biochemical and phytochemical analysis.

The effect of silicon dioxide nanoparticles on production of phenolic compounds and expression rate of pal and ras genes involved in rosmarinic acid biosynthesis pathway has been investigated in D. kotschyi. SiO₂ nanoparticles, used as an abiotic elicitor in our study, has appropriate optical, electrical and catalysts properties and has many applications in various industries as well as agriculture. This study clearly suggested that, in the presence of this nanoparticle, induction, production and accumulation of valuable compounds and corresponding antioxidant activity increased in hairy roots.

According to the results, expression levels of the pal and ras genes were influenced by elicitor concentration and exposure time. The elicitation by SiO₂ NP of 100 mg/L after 48 h of exposure time dramatically increased pal expression compared to the control. Briefly, with increasing SiO₂ NP concentrations after 48 h of exposure time, the expression level of pal was also significantly induced . Similarly, ras expression was significantly raised at 48 h after treatment by increasing SiO₂ NP concentration and enhanced to the greatest extent in 50 mg/L concentration. After 24 h of exposure time, the minimum level of ras expression was observed in the 200 mg/L SiO₂ . Amplification products of real-time PCR were assessed with 1.8% agarose gel which was corresponded to the predicted size.