## 4 Discussion  
Tissue-culture plants can be exploited as an ecofriendly approach to produce secondary metabolites _in vitro_ and protect endangered plants from extinction. The present results demonstrated the development of plant science technologies to enhance the productivity of CPT from \(O\). _ridleyana_ plants _in vitro_. Without the elicitation, the accumulation of CPT of 5-month-old tissue-culture plants of \(O\). _ridleyana_ was higher in roots, followed by stems and the leaves, respectively. Previously, Roja [12] reported CPT contents in plants of \(O\). _rugosa_ at 0.002% DW in roots, trace quantities in stems, and 0.0002% DW in leaves. Significant improvement in CPT production was attained by simple pouring of MeJA solution of 50 \(\mu\)M onto the surface of the solid medium and incubation for 12 h before harvesting, in which the CPT contents in roots and stems were at approximately 1.8- and 2.6- fold increase, respectively compared with that of the controls. The higher CPT content in roots and stems can be explained from the role of MeJA as a signal compound responsible for the induction of signal transduction, the expression of many defense genes followed by the  
Figure 9: Effects of DC intensities (10, 20, 30 and 40 mA) on CPT accumulation in \(O\). _ridleyana_ leaves. The values are expressed as mean \(\pm\) SD (\(n\) = 3); a: significant at \(p<0.05\) vs. control. accumulation of secondary metabolites in plants. MeJA could act as a signal transducer in the enhancement of alkaloid biosynthesis. Previously, upon elicitation of hairy roots in a liquid medium, MeJA significantly enhances alkaloid production, particularly CPT in _O. liukuiensis_ hairy roots; however, this effect is not observed in _O. kuroiwai_ and _O. pumila_[4]. On the other hand, elicitation by salicylic acid and yeast extract showed a repressive effect on alkaloid production [4]. The present results are also compatible with previous research by [PERSON] et al. [30], who observed an increase in triterpenoids production in shake flask cultures of _Imonotus obliquus_ in response to addition of MeJA as an exogenous elicitor and fatty acids as a stimulatory agent in liquid medium; the optimal elicitor concentration was 50 uM MeJA. After elicitation for 6 d, the production of total triterpenoids was increased by 66% compared with that of the control. The kinetics of CPT accumulation in suspension cultures of _Camptotheca acuminate_ in response to elicitation with jasmonic acid showed that CPT accumulation peaked at 4 d after treatment and thereafter, the CPT concentration rapidly decreased [11]. Previously, increased CPT production in multiple shoot cultures of _O. munogos_ elicited with 150 uM MeJA in 1/2 MS liquid medium for 72 h was also reported [21]. In the present study, the maximum increase in CPT content in the roots and stems of tissue-cultured plants of _O. ridleyana_ in 1/2 MS solid medium was observed after the elicitation with 50 uM MeJA for 12 h. However, no significant difference was noticed from the elicitation with pure ethanol. Ethanol has been reported as a small-molecule elicitor, which could alter the expression of secondary metabolic genes in tissue cultures of certain plant species. It was previously reported that addition of absolute ethanol (up to 5% _v/v_) to the suspension culture of _Fusarium solani_ results in enhanced CPT production compared with that of the control [7]. However, in the current study, no significant effect of ethanol on CPT production by tissue-cultured plants of _O. ridleyana_ was observed _in vitro_. These different responses may reflect differences in plant species, age of plants, cultivation methods, and elicitation procedures. Under the 20 and 50 uM MeJA treatments, only slight increases in CPT content in the leaves was observed. Accumulation of secondary plant metabolites, triterpenoids in _Centella asatica_ leaves might be triggered by postharvest application of MeJA [31].