Plants are a rich source of various phytochemicals, proteins, enzymes and other secondary products of immense biotechnological applications. The secondary products and the respective enzymes, particularly those of phenylpropanoid pathway are significantly enhanced under the influence of elicitors. Peroxidase (POD) is one such enzyme associated with the plant defense pathway and is elicited when challenged with elicitors (Gómez-Vásquez et al. 2004). Although a large number of commercial applications of POD are possible, the high cost of presently available horseradish POD hinders such applications, which indicates the need for alternative sources of POD. We reported high levels of POD activity in cultured hairy roots of red beet (Thimmaraju et al. 2005) and the present study reports screening of a number of biotic and abiotic elicitors and the interaction of two complex elicitors leading to over-expression of POD activity.

Culture and maintenance of hairy root clone

Induction of hairy roots and their maintenance conditions have been explained in an earlier communication (Thimmaraju et al. 2004). For testing growth performance, approximately 50 mg of root tips from hairy root clones were subcultured in 50 ml Erlenmeyer’s flasks containing 15 ml of MS basal liquid medium (Murashige and Skoog, 1962) with 3% sucrose and grown on a rotary shaker as described previously.  The biomass accumulation and POD activity were monitored at 5 day intervals.  Fresh weight increase was recorded after removal of the spent medium by suction while the biomass was retained in a Buchner funnel.

Preparation of biotic elicitors

Crude elicitors. Based on earlier reports and the availability of cultures, culture filtrate (CF) and dry cell powder (DCP) of various fungi, yeast and bacteria were used. The fungi used were Aspergillus niger, A. parasiticus, Penicillium notatum, and Rhizophus oligosporus. The Yeast species used was Candida versatilis. Among the bacteria Lactobacillus helveticus was used. The cultures maintained on agar slants, were transferred to 100 ml of the respective liquid medium in 250-ml flasks and incubated at room temperature. The cultures were harvested after they reached their stationary phase (i.e., 3 weeks for fungal cultures, 72 hrs for yeast culture and 48 hrs for bacterial culture). The flasks were autoclaved and the mycelial mat of each fungus which floated at the surface of the medium was carefully removed and washed multiple times with sterile distilled water and allowed to dry in a hot air oven at 40ºC, crushed into powder using mortar and pestle and used as DCP. The remaining medium i.e., CF was centrifuged to remove suspended particles, filtered through Whatman No. 1 filter paper and the clear solution obtained was stored at 4ºC for further use. Similarly, for yeast and bacterial cultures, the culture broth was centrifuged at high speed for 1 hr and the cell sediment was air-dried and used as DCP. The respective spent medium was stored at 4ºC for further use as CF.

Purified biotic elicitors

The compounds and their concentrations were selected based on earlier studies (Suresh et al. 2004). Thus Mej was used at 20, 40, 60, 80, 100 µM whereas GSH was used at different levels such as 0.5 mM, 1.0 mM, 1.5, 2, 4, 6, 8, 10 mM.

Abiotic elicitors

These elicitors in the present study were mainly metal ions such as calcium and magnesium, and an hormonal elicitor - thidiazuron (TDZ). Metal ions were used at various levels such as 2, 4 and 8 folds of their respective concentrations in the normal MS medium. TDZ was used at 0.25 ppm, 0.50 ppm and 1.0 ppm in MS liquid medium.

Among the fungal elicitors used, DCPs of both R. oligosporus (added on 15^th day) and P. notatum (added on 20^th day) caused enhancements to the tune of 3-fold higher activity than the control. High levels (5% v/v) of culture filtrates of A. parasiticus and P. notatum (2.5 - 5% v/v) enhanced POD activity by nearly 3-fold. However, a lower concentration (1%) of culture filtrate of R. oligosporus was needed to cause similar levels of elicitation in a short period of 5 days. The dry cell powders of yeast C. versatilis elicited the activity of POD up to 3.5-fold at very low concentration whereas the culture filtrates suppressed the turn over of enzyme activity. L. helveticus elicited nearly 3-fold activity when added on 15^th day only at lower concentration. Higher levels of yeast DCP were however inhibitory in a dose-dependent manner. An important observation made from the present screening study was that the concentration and time of elicitor contact were very critical factors for efficient elicitation of POD. Among the pure compounds, GSH used at 5 concentrations at three different treatment time caused highest elicitation at lowest concentration of 2 mM producing about 3.44-fold higher accumulation of POD activity than the respective control where a contact period of 10 days was required. Mej, a well-known secondary signaling molecule, when used at 5 different concentrations caused high suppression of POD activity by about 50% when compared to control cultures. Among combinations of the three elicitors such as GSH, CF of A. parasiticus, DCPs of R. oligosporus, and C. versatilis, only the combination of 1 mM GSH with 0.25% DCP of R. oligosporus caused the highest elicitation of about 4-fold with a total POD activity of 10.9 x 10⁶ UL^-1 when compared to the individual components. Other combinations either suppressed or caused very little elicitation of POD activity when compared to the effect of individual components

Although many researchers have studied elicitation in various systems and recorded the suppression of biomass in elicitor treatment, no attempt had been made to check the possibility of effectively using an elicitor at the late exponential growth phase. For pigment we observed that the addition of elicitor at late exponential phase could enhance the overall productivity (Savitha et al. 2006). The activity of POD was generally high at the early exponential phase (Thimmaraju et al. 2005) and therefore, maximum elicitation also occurred at treatments on the 15^th and 18^th days rather than on 20^th day. Therefore, by judiciously selecting and timing the addition of an elicitor, there is a possibility of enhancing the production of both POD and Betalaines in the same process, in which case the process of online recovery of pigment developed earlier by us (Thimmaraju et al. 2004) could further be extended for the online recovery of POD as well, which is also proposed by Agostini et al. (1997) for turnip hairy roots.

Encouragement by Dr. V. Prakash, Director, CFTRI for research activities is gratefully acknowledged.