Our experimental results have shown that the

Our experimental results have shown that the accumulation of psoralen in the suspension cultures can be stimulated by biotic elicitors. Therefore, elicitation offers an attractive strategy for increasing the metabolite production in vitro culture system significantly (Goel et al., 2011). The stimulation of psoralen accumulation by biotic elicitors such as A. niger, P. notatum, yeast extract and chitosan has also been observed in the cell cultures of plant species viz. Calendula officinalis (Wiktorowska et al., 2010), Sorbus aucuparia (Gaid et al., 2011) and Abrus precatorius (Karwasara et al., 2010, 2011). The cell wall extract preparation of A. niger possessed an oligosaccharide elicitor that induced high level of shikonin (Wen and Ri-qiang, 1996.) Another significant effect of the elicitors observed in the experiments was the rapid increase in psoralen accumulation with elicitor dosage, further increase led to decrease in the psoralen content in cell suspension cultures (Seung-Mi et al., 2009 and Kundu et al., 2012). Thus, the accumulation of psoralen is a dose elicitor dependent response of P. corylifolia L. cell cultures. Similar results were reported in taxol and ajmalicine production (Yari Khosroushahi et al., 2006 and Namdeo et al., 2002). The growth and accumulation of secondary metabolites were influenced by the type and mode of elicitor preparation (Karwasara et al., 2011) and this was observed in the present study also.
The physiological state of the amc 7 at the moment of transfer from growth to production medium is crucial (Schaltmann et al., 1995). The optimum age of the culture for elicitation is different in different plant cell systems (Seung-Mi et al., 2009). In most of the cases, response of the cells was maximum if they were challenged at the end of the growth phase or at the onset of the stationary phase (Yari Khosroushahi et al., 2006). On the contrary, taxol and baccatin were produced during the initial stationary phase of growth in Taxus baccata cell cultures (Barradas-Dermitz et al., 2010). The response to elicitation is dependent on growth phase of the culture that affects the quantitative response as well as the product pattern (Vázquez-Flota et al., 2009). The amount of metabolite production varied with duration time of incubation with elicitors (Karwasara et al., 2010).
The optimum time of elicitor treatment is very essential to produce the secondary metabolites on a large scale. Therefore, the time required for triggering the elicitation in 16days older culture in all the elicitors was studied and was up to 96h. The results show that the amount of metabolite production varied with the duration of incubation time with elicitors. This result indicates that the duration of cell cultured with elicitors is much important for the maximum psoralen accumulation. An important factor for enhancing the psoralen production was the stage of cell growth for elicitor treatment (Catapan et al., 2009). It is believed that the stronger stimulation of secondary metabolites by biotic elicitor usually occurred in the late exponential growth stage of plant cell culture (Namdeo et al., 2002 and Schaltmann et al., 1995). However, the resveratrol and viniferins production in Vitis vinifera cells were treated with elicitor during the early stage of growth (Santamaria et al., 2011). The high availability of the nutrient in the medium or cells can be used for secondary metabolites biosynthesis (Sun et al., 2012). Thus, it is best to produce large amounts of biomass under rapid growth conditions and then transfer the accumulated biomass to the second stage of culture to promote secondary metabolite production (Schaltmann et al., 1995).

The results of this study show that the biotic elicitors influenced the accumulation of psoralen in the cultured cells. The present result exhibited that the cell cultures treated with all the elicitors showed maximum accumulation of psoralen over the control cell. However, the extract of A. niger (1.0% v/v) was found to be the best for maximum metabolite elicitation. Therefore, these results may contribute to the enhancement of psoralen production by using different biotic elicitors in cell suspension cultures.