Enhancement of anthraquinone production and release by combination of culture medium selection and methyl jasmonate elicitation in hairy root cultures of Rubia tinctorum

Anthraquinones (AQs) are secondary metabolites widely distributed in nature. Interesting applications of plant extracts containing AQs include the treatment of Hepatitis C and cancer. Plant in vitro culture is an alternative for producing plant-derived pharmaceuticals in controlled conditions and with low environmental impact. Moreover, it allows the application of different strategies for enhancing secondary metabolite production. Hairy root cultures, obtained after Agrobacterium rhizogenes infection, are able to produce high amounts of secondary metabolites at high growth rates. In this work, growth kinetics of hairy root cultures of Rubia tinctorum and AQ production were evaluated in two different culture media, Gamborg B5 with half of the saline strength (B5(1/2)) and Lloyd & Mc Cown's Woody Plant Medium (WPM). Although WPM allowed higher biomass production (58.6% higher) than B5(1/2), specific AQ production was higher in B5(1/2) (between 1.2 and 2.1 fold increases from day 21 to the end of the experiment). Moreover, AQ release to the culture medium was observed in B5(5/2) (similar to 10% of total AQs). The different performance of hairy roots in these culture media may be due to a limiting nutrient (other than carbon source) in B5(1/2). Elicitation in B5(1/2) with methyl jasmonate (100 mu M) resulted in a massive accumulation of intracellular (between 1.5 and 2.4-fold increases) and also extracellular AQs (up to 8.1 fold-increase compared with control at 4 days post-elicitation), which could ease AQ purification. These results prove the usefulness of combining different approaches to enhance secondary metabolite accumulation in plant in vitro cultures, in order to develop an optiMized productive process.