The growth and secondary metabolite accumulation by the embryos of E. senticosus, cultivated in bioreactor cultures are presented in Table 1 and Table 2. The embryos in the untreated cultures reached 102.65 g L-1 fresh weight and 11.32 g L-1 dry weight. Growth of embryos was significantly affected by the application of MJ. There was slight increment in fresh weight of embryos (104.66 g L-1) when compared to the control (Table 1). However, the fresh weight, dry weight and growth ratio were decreased with increasing MJ concentration. On the other hand, eleutheroside content was significantly enhanced by the addition of MJ. Amount of total eleutherosides and chlorogenic acid increased with increasing MJ concentration, and reached a maximum at 200 µM MJ representing 7.3 fold (649.95 µg g-1 DW) and 3.9 fold (4.48 mg g-1 DW) increases over controls respectively. There were 1.4, 3.4 and 14.9 fold increments in eleutheroside B, E and E1 respectively compared to the control.
The accumulation of secondary metabolites in plants is part of the defense response against pathogenic attack, which is triggered and activated by elicitors, the signal compounds of plant defense responses (Zhao et al. 2005). Therefore, the treatment of plant cells with biotic and/or abiotic elicitors has been a useful strategy to enhance secondary metabolite production in cell cultures. The most frequently used elicitors in previous studies were fungal carbohydrates, yeast extract, MJ and chitosan. MJ, a proven signal compound, is the most effective elicitor of taxol production in Taxus chinensis Roxb. (Wu and Lin, 2003) and ginsenoside production in Panax ginseng C.A. Meyer(Yu et al. 2000; Yu et al. 2002; Kim et al. 2004; Thanh et al. 2005) cell/organ culture. In the present study, the effect of different concentrations of MJ on embryogenic cell growth and eleutheroside accumulation was tested and results reveled that addition of 200 µM MJ was suitable for optimum accumulation of eleutheroside B, E, E1 and chlorogenic acid. However, addition of MJ at higher concentration (above 100 µM) was detrimental for biomass accumulation. Similar to the present results, MJ inhibited the cell growth and promoted the secondary metabolite production with cell/adventitious root cultures of Bupleurum falcatum L. (Aoyagi et al. 2001), Taxus spp. (Yukimune et al. 1996; Ketchum et al. 1999) and Panax ginseng C.A. Meyer (Kim et al. 2004; Thanh et al. 2005). Differential accumulation of eleutherosides was observed during elicitation experiments (Table 2). Eleutheroside E1 content was highest among the different eleutherosides produced by the suspended somatic embryos. Similar to the present observation differential accumulation of secondary compounds have been reported during cell/organ cultures of Panax ginseng (Kim et al. 2004; Thanh et al. 2005).
The results from this study demonstrate that MJ elicitation strategy was quite useful to improve the yield of eleutherosides and chlorogenic acid in embryogenic cell cultures of E. senticosus. The biomass produced in the bioreactor cultures may be used as source of medicinal raw material for the extraction of eleutherosides and chlorogenic acid.
One of the authors (H.N. Murthy) is thankful to Korean Federation of Science and Technology Societies (KOFST) for the award of Brain Pool Fellowship (016S-4-3-0030). We thank Dr. A.M. Reddy for critical review of this manuscript.