TECHNOLOGY DEVELOPMENT FOR ANDROGRAPHOLIDE PRODUCTION FROM HAIRY ROOT CULTURES OF Andrographis paniculata
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Date
2018
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Indira Gandhi Krishi Vishwavidhyalaya, Raipur
Abstract
Andrographolide is a pharmaceutically important compound synthesized in Andrographis paniculata herbal plant which is used as an anti diabetic, anti cancer and hepatoprotective agent. It is present in very low quantity in roots (0.15%). Its demand is met by collecting the whole plant which is resulting in approximately 39 % of population reduction. So, there is a need of alternatives to meet the industrial demands. In this research, development of hairy root cultures of A. paniculata was done. Hairy root cultures are becoming a promising technique for production of secondary metabolites. Recent progress in scaling up of hairy root cultures enables us for industrial exploitation of this system. In the current work, apical meristem explant was infected with Agrobacterium rhizogenes (MTCC 532). Different media for co-cultivation was used for hairy root induction. For hairy root mass multiplication, liquid and semi-solid media were tested to find out the higher root growth and elicitation of andrographolide was done. The findings of the work indicated that co-cultivation media having half strength Murashige and Skoog medium supplemented with acetosyringone 400μM responded with highest (62.83 +1.69 %) hairy root induction percentage in 10.2 + 1.25 days. The integration of T-DNA in explant was confirmed by PCR analysis with rolC gene primer which showed 1000bp band confirming gene from Ri plasmid of MTCC 532 strain. The best media for mass multiplication was found to be semi solid media (Half strength MS with 3 % sucrose, cefotaxime 250mg/L and agar 0.8% with gradual decrease in cefotaxime 125 and 0 mg/L and agar agar with 0.7 and 0.55 % respectively) with 40.8 fold increase in total biomass yield (fresh weight) after six weeks of culturing. It was found that optimum period of harvesting was at 6th week after hairy root inoculation.
After hairy root mass multiplication, the roots were subjected to various abiotic elicitor treatments to increase andrographolide content. Four different elicitors were used; heat and cold based elicitor, jasmonic acid elicitor and sodium chloride. Among different treatments, highest andrographolide content (4695.9 µg/gm DW) with 3.0 fold increase was obtained as compared to control when hairy root cultures were exposed to 500C temperature for one hour. This was followed by hairy roots exposed at 0 0C for 6 hours 4503.0 µg/gm DW obtained with 3.2 folds increase compared to control and jasmonic acid treated hairy root cultures 100µM for 14 hour (4107.86 µg/g DW) with 2.7 folds increase. The lowest andrographolide (3119.5 μg/g DW) was obtained in hairy root cultures without salt stress. The results of elicitation revealed that high temperature treatment of 500C temperature for 1 hour should be used to elicit andrographolide in hairy root cultures. For obtaining maximum hairy root biomass of A.paniculata under in vitro condition, we recommend to use apical meristem as explant in co-cultivation media of half strength MS supplemented with acetosyringone 400μM for cocultivation with MTCC 532 strain of A. rhizogenes for high hairy root induction. For higher biomass production, semi soild media(Half strength MS with 3 % sucrose, cefotaxime 250mg/L and agar 0.8% with gradual decrease in cefotaxime 125 and 0 mg/L and agar agar with 0.7 and 0.55 % respectively) should be used followed by exposing roots to 500C temperature for one hour. This will give biomass yield of 6.81 gm/L (dry weight) which will produce 32000 μg/6.81 gm DW andrographolide. So the complete technology developed under this research work can be utilized for hairy root production with high andrographolide content .
Description
TECHNOLOGY DEVELOPMENT FOR ANDROGRAPHOLIDE PRODUCTION FROM HAIRY ROOT CULTURES OF Andrographis paniculata
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