Abstract
Roots of Rauwolfia serpentina, also known as “Sarpagandha” possess high pharmaceutical value due to the presence of reserpine and other medicinally important terpene indole alkaloids. Ever increasing commercial demand of R. serpentina roots is the major reason behind the unsystematic harvesting and fast decline of the species from its natural environment. Considering Agrobacterium rhizogenes-mediated hairy root cultures as an alternative source for the production of plant-based secondary metabolites, the present optimized protocol offers a commercially feasible method for the production of reserpine, the most potent alkaloid from R. serpentina roots. This end-to-end protocol presents the establishment of hairy root culture from the leaf explants of R. serpentina through the infection of A. rhizogenes strain A4 in liquid B5 culture medium and its up-scaling in a 5 L bench top, mechanically agitated bioreactor. The transformed nature of roots was confirmed through PCR-based rol A gene amplification in genomic DNA of putative hairy roots. The extraction and quantification of reserpine in bioreactor grown roots has been done using monolithic reverse phase high-performance liquid chromatography (HPLC).
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References
Harisaranraj R, Suresh K, Saravanababu S (2009) Evaluation of the chemical composition Rauwolfia serpentina and Ephedra vulgaris. Adv Biol Res 3(5–6):174–178
Pathania S, Randhawa V, Bagler G (2013) Prospecting for novel plant-derived molecules of Rauvolfia serpentina as inhibitors of aldose reductase, a potent drug target for diabetes and its complications. PLoS One 8, e61327
Campbell-Tofte J (2006) Anti-diabetic extract isolated from Rauvolfia vomitoria and Citrus aurantium, and methods of using same. US Patent 749395 B2
Pistelli L, Giovannini A, Ruffoni B, Bertoli A, Pistelli L (2010) Hairy root cultures for secondary metabolites production. Adv Exp Med Biol 698:167–184
Mehrotra S, Goel MK, Rahman LU, Kukreja AK (2013) Molecular and chemical characterization of plants regenerated from Ri mediated hairy root cultures of Rauwolfia serpentina. Plant Cell Tiss Organ Cult 114:31–38
Goel MK, Goel S, Banerjee S, Shanker K, Kukreja AK (2010) Agrobacterium rhizogenes-mediated transformed roots of Rauwolfia serpentina for reserpine biosynthesis. Med Aromat Plant Sci Biotechnol 4:8–14
Hooykass PJJ, Klapwijk PM, Nuti PM, Shilperoot RA, Rorch A (1977) Transfer of the Agrobacterium tumefaciens Ti plasmid to avirulent Agrobacteria and Rhizobium ex planta. J Gen Microbiol 98:477–484
Goel MK, Mehrotra S, Kukreja AK, Shanker K, Khanuja SP (2009) In vitro propagation of Rauwolfia serpentina using liquid medium, assessment of genetic fidelity of micropropagated plants, and simultaneous quantitation of reserpine, ajmaline, and ajmalicine. In: Jain SM, Saxena PK (eds) Protocols for in vitro cultures and secondary metabolite analysis of aromatic plants, vol 547, Methods in molecular biology. Humana Press, Springer, USA, pp 17–33
Khanuja SPS, Shasany AK, Srivastava A, Kumar S (1998) Assessment of genetic relationship in Mentha species. Euphytica 111:121–125
Mehrotra S, Goel MK, Srivastava V, Rahman LU (2015) Hairy root biotechnology of Rauwolfia serpentina: a potent approach for the production of pharmaceutically important terpenoid indole alkaloids. Biotechnol Lett. doi:10.1007/s10529-014-1695-y
Acknowledgement
The authors are grateful to Director, CIMAP (CSIR) for providing the necessary facilities to carry out the work. Postdoctoral Fellowship provided by Department of Science and Technology (DST), Govt. of India to S.M is acknowledged.
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Mehrotra, S., Srivastava, V., Goel, M.K., Kukreja, A.K. (2016). Scale-Up of Agrobacterium rhizogenes-Mediated Hairy Root Cultures of Rauwolfia serpentina: A Persuasive Approach for Stable Reserpine Production. In: Jain, S. (eds) Protocols for In Vitro Cultures and Secondary Metabolite Analysis of Aromatic and Medicinal Plants, Second Edition. Methods in Molecular Biology, vol 1391. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-3332-7_17
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DOI: https://doi.org/10.1007/978-1-4939-3332-7_17
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