## 4 Discussion
However, in a study of the distribution of strictoisidine synthase (STR), a crucial enzyme for CPT biosynthesis in different tissues of _O. pumila_, STR activity was detected in stem, root, and hairy root extracts, but no activity was detected in leaf and callus extracts [1]. It has been suggested that roots and stems are the predominant tissues in which CPT biosynthesis occurs [1]. The distribution of STR activity is correlated with the accumulation of mRNA and CPT production in _O. pumila_ tissues. Presence of MeJA as a signaling molecule on the plasma membrane could initiate a signal transduction network inside the cells, leading to the expression of biosynthetic genes involved in the production of plant secondary metabolites of CPT in _Ophiorrhizza_[21; 32]. In the present study, following elicitation with MeJA (50 uM) for 12 h, the total CPT accumulation of whole plants of 5-month-old _O. ridleyana_ cultured _in vitro_ in solid medium was increased by approximately 1.8-fold as compared with that of the control (Table 3). In comparison with plant tissue culturing in liquid medium, plant culture on solid medium is simpler, requires less expensive equipment, consumes less energy, and is easier for large-scale production. Furthermore, the use of electrical stimulation for CPT accumulation in tissue culture plants of _O. ridleyana_ was investigated. The DC current at 10-40 mA with the total treatment time of 15 min was applied to 5-month-old _O. ridleyana_ plants _in vitro_. Effects of electrical stimulation on some secondary plant metabolisms have been previously reported. Enhanced lycopene, \(\beta\)-carotene, total phenols contents and antioxidant activity were attained with a DC treatment of 500 mA with an application time of 15-30 min on _Solanum Lycopersicon_ L. after an adaptation time of 2-24 h [27]. Electrical stimulation was also found to increase anthocyanin and resveratrol contents in berries of grapevines relative to those of control plants and electrode-treated plants [33]. Previously, the treatment by DC current demonstrated effects on the accumulation of some bioactive compounds in leaves. Applied DC induced alteration of cell membrane permeability due to DC could have improved physiological processes, leading to enhanced accumulation of bioactive compounds such as chlorophylls carotenoids in African nightshade leaves [28]. Biosynthesis of secondary plant compounds is usually associated with plant defense response. Plant defense response can be induced by an elicitor and/or physiological stimulation such as changes in membrane potential and ion fluxes to trigger oxidative burst, resulting in the activation of defense-related genes for synthesis of secondary compounds [26; 34]. It was suggested that DC influences the voltage-gated ion channels and increases the membrane permeability for calcium cations (Ca\({}^{2+}\)) at the cellular level, followed by a rapid influx of Ca\({}^{2+}\) through cation channels. Voltage-gated calcium (Ca\({}^{2+}\)) channels are key transducers of membrane potential changes into intracellular Ca\({}^{2+}\) transients that initiate many physiological events. Ca\({}^{2+}\) plays an important role in signal transduction and in the absorption of nutrients across the cell membranes [35; 36] and helps in the upregulation of respective genes for biosynthesis of secondary metabolites in plants such as polyphenols, flavonoid [37]. Therefore, electrical stimulation could be considered as physiological stimulation to induce plant secondary metabolism. The additional potential induced from external electric field in plants was reported to raise pressure on the membrane due to attraction between opposite charges on both sides of the membrane, whereby a faster transport of ions was induced simultaneously and this could cause an accumulation of minerals and bioactive compounds [28]. In the present study, the treatment by applied DC current at 20 mA could enhance the CPT content in roots, stems and leaves at 1.5-, 1.7- and 1.4-fold higher than that of the controls, respectively.