Background
Ursolic (UA), oleanolic (OA) and rosmarinic (RA) acids are bioactive metabolites found in Lepechinia caulescens that have generated interest for their health benefits, which include antimicrobial, antioxidant, antimutagenic, gastroprotective, antidiabetic, antihypertensive and anti-inflammatory properties, among others. To date, very few attempts have been made to evaluate the potential for simultaneous production of these bioactive compounds, using a biotechnological approach. Hairy root cultures offer a biotechnology approach that can be used to study the factors affecting the biosynthesis and the production of UA, OA and RA. In the current study, we established hairy root cultures of L. caulescens and evaluated the effect of sucrose on biomass accumulation, and the effect of different concentrations and times of exposure of methyl jasmonate (MeJA), on the accumulation of UA, OA and RA.
Methods
Leaves from plants of L. caulescens were inoculated with Agrobacterium rhizogenes strain ATCC 15834. PCR of rolB gene confirmed the transgenic nature of hairy roots. Hairy roots were subcultured in semisolid MSB5 medium, supplemented with 15, 30, 45 or 60 g/L sucrose and after 4 weeks, dry weight was determined. The accumulation of UA, OA and RA of wild plants and hairy roots were determined by HPLC. Finally, the hairy roots were treated with 0, 100, 200 and 300 µM of MeJA and the content of bioactive compounds was analyzed, after 24, 48 and 72 h.
Results
High frequency transformation (75%) was achieved, using leaf explants from axenic seedlings, infected with A. rhizogenes. The hairy roots showed an enhanced linear biomass accumulation, in response to the increase in sucrose concentration. The hairy root cultures in MSB5 medium, supplemented with 45 g/L sucrose, were capable to synthesizing UA (0.29 ± 0.00 mg/g DW), OA (0.57 ± 0.00 mg/g DW) and RA (41.66 ± 0.31 mg/g DW), about two, seven and three times more, respectively, than in roots from wild plants. Elicitation time and concentration of MeJA resulted in significant enhancement in the production of UA, OA and RA, with treatments elicited for 24 h, with a concentration of 300 µM of MeJA, exhibiting greatest accumulation.
Conclusion
This is the first report on development of hairy root cultures of L. caulescens. Future studies should aim towards further improving triterpenes and polyphenolic compound production in hairy roots of L. caulescens, for use in the pharmaceutical and biotechnological industry.
Methyl jasmonate enhances ursolic, oleanolic and rosmarinic acid production and sucrose induced biomass accumulation, in hairy roots of Lepechinia caulescens