Summary
Rauwolfia serpentina holds an important position in the pharmaceutical world because of its immense anti-hypertensive properties resulting from the presence of reserpine in the oleoresin fraction of the roots. Poor seed viability, low seed germination rate, and enormous genetic variability are the major constraints for the commercial cultivation of R. serpentina through conventional mode. The present optimized protocol offers an impeccable end to end method from the establishment of aseptic cultures to in-vitro plantlet production employing semisolid as well liquid nutrient culture medium and assessment of their genetic fidelity using polymerase chain reaction based rapid amplification of polymorphic DNA analysis. In vitro shoots multiplied on Murashige and Skoog basal liquid nutrients supplemented with benzo[a]pyrene (1.0 mg/L) and NAA (0.1 mg/L) and in-vitro rhizogenesis was observed in modified MS basal nutrient containing NAA (1.0 mg/L) and 2% sucrose. In-vitro raised plants exhibited 90-95% survival under glass house/field condition and 85% similarity in the plants regenerated through this protocol. Field established plants were harvested and extraction of indole alkaloid particularly reserpine, ajmaline and ajmalicine and their simultaneous quantitation was performed using monolithic reverse phase high-performance liquid chromatography (HPLC)
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CSIR, (1969). Wealth of India: raw materials. Council of Scientific and Industrial Research. N. Delhi, pp. 376–390
Mehrota, S., Goel, M.K., Kukreja, A.K., and Mishra, B.N. (2007). Efficiency of liquid culture systems over conventional micropropagation: a progress towards commercialization. Afr. J. Biotechnol. 6(13): 1484–1492
Goel, M.K., Kukreja, A.K., and Khanuja, S.P.S. (2007). Cost-effective approaches for in vitro mass propagation of Rauwolfia serpentina Benth. Ex. Kurz. Asian J. Plant Sci. 6: 957–961
Goel, M.K. (2007). Applications of bioreactors for micropropagation and secondary metabolite production in Rauwolfia serpentina L. Benth. Ex. Kurz. Ph.D Thesis submitted to the H.N.B. Garhwal University, Srinagar, Garhwal (India)
Khanuja, S.P.S., Shasany, A.K., Srivastava, A., and Kumar, S. (1998). Assessment of genetic relationship in Mentha species. Euphytica 111: 121–125
Murashige, T., and Skoog, F. (1962). A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol. Plant. 15: 473–497
Khanuja, S.P.S., Shasany, A.K., Darokar, M.P., and Kumar, S. (1999). Rapid isolation of DNA from dry and fresh samples of plants producing large amounts of secondary metabolites and essential oils. Plant Mol. Biol. Rep. 17: 74–80
Goel, M.K., Kukreja, A.K., and Khanuja, S.P.S. (2006). PCR based RAPD analysis of in vitro raised plants of Rauwolfia serpentina Benth. Ex. Kurz. In: National Symposium on Plant Biotechnology. Dehradun, India
Nei, M., and Li W. (1979). Mathematical model for studying genetic variation in terms of restriction endonucleases. Proc. Natl. Acad. Sci. U.S.A. 76: 5269–5273
Srivastava, A., Tripathi, A.K., Pandey, R., Verma, R.K., and Gupta, M.M. (2006). Quantitative determination of reserpine, ajmaline, and ajmalicine in Rauwolfia serpentina by reversed-phase high-performance liquid chromatography. J. Chromatogr. Sci. 44: 557–560
Acknowledgments
Financial assistance from Council of Scientific and Industrial Research (CSIR) Government of India to MKG is gratefully acknowledged. Authors are also thankful to Director, CIMAP, for providing the facilities.
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Goel, M.K., Mehrotra, S., Kukreja, A.K., Shanker, K., Khanuja, S.P.S. (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, S.M., Saxena, P.K. (eds) Protocols for In Vitro Cultures and Secondary Metabolite Analysis of Aromatic and Medicinal Plants. Methods in Molecular Biology, vol 547. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-60327-287-2_2
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DOI: https://doi.org/10.1007/978-1-60327-287-2_2
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