Abstract
To determine the possibility of generating the Podophyllotoxin accumulation using Podophyllum hexandrum adventitious roots derived from root segments, several nutrient constituents (carbon sources, media strength, initial medium pH, ammonium and nitrate proportion and phosphate ratio) were evaluated in culture. The maximum biomass accumulation was recorded in 0.50 MS medium containing 3 mg/l indole-3-butyric acid and 2 % sucrose, and the maximum accumulation of Podophyllotoxin was documented in the same strength of MS medium with 6 % of sucrose. When the initial medium pH was on 6 in the optimized MS medium, the biomass and Podophyllotoxin accumulations were highest. The lower concentration of ammonium (10 mM) in combination with a moderate concentration of nitrate (20 mM) was found ideal for maximum accumulations of biomass and Podophyllotoxin. Maximum Podophyllotoxin accumulation (6.4 mg/g dry weight) was recorded at the higher concentration of phosphate (2.25 mM), and lower concentration of phosphate (1.25 mM) showed highest growth accumulation. The outcome of the present work will be helpful for the large-scale cultivation of adventitious root for the production of Podophyllotoxin.
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Abbreviations
- 2,4-D:
-
2,4-Dichlorophenoxy acetic acid
- DW:
-
Dry weight
- FW:
-
Fresh weight
- HPLC:
-
High performance liquid chromatography
- IAA:
-
Indole-3-acetic acid
- IBA:
-
Indole-3-butyric acid
- MS:
-
Murashige and Skoog medium
- NAA:
-
1-Naphthaleneacetic acid
- NH4 +/NO3 − :
-
Ammonium/nitrate ratio
- PGRs:
-
Plant growth regulators
- PTOX:
-
Podophyllotoxin
- RT:
-
Retention time
References
Anbazhagan VR, Ahn CH, Harada E, Kim YS, Choi YE (2008) Podophyllotoxin production via cell and adventitious root cultures of Podophyllum peltatum. In Vitro Cell Dev Biol Plant 44:494–501
Baque MA, Lee EJ, Paek KY (2010a) Medium salt strength induced changes in growth, physiology and secondary metabolite content in adventitious roots of Morinda citrifolia: the role of antioxidant enzymes and phenylalanine ammonia lyase. Plant Cell Rep 29:685–694
Baque MA, Hahn EJ, Paek KY (2010b) Growth, secondary metabolite production and antioxidant enzyme response of Morinda citrifolia adventitious root as affected by auxin and cytokinin. Plant Biotechnol Rep 4:109–116
Baque MA, Elgirban A, Lee EJ, Paek KY (2012a) Sucrose regulated enhanced induction of anthraquinone, phenolics, flavonoids biosynthesis and activities of antioxidant enzymes in adventitious root suspension cultures of Morinda citrifolia (L.). Acta Physiol Plant 34:405–415
Baque MA, Moh SH, Lee EJ, Zhong JJ, Paek KY (2012b) Production of biomass and useful compounds from adventitious roots of high-value added medicinal plants using bioreactor. Biotechnol Adv 30:1255–1267
Bensaddek L, Gillet F, Saucedo JEN, Fliniaux MA (2001) The effect of nitrate and ammonium concentrations on growth and alkaloid accumulation of Atropa belladonna hairy roots. J Biotechnol 85:35–40
Chattopadhyay S, Srivastava AK, Bhojwani SS, Bisaria VS (2002) Production of podophyllotoxin by plant cell cultures of Podophyllum hexandrum in bioreactor. J Biosci Bioeng 93:215–220
Chaurasia OP, Ballabh B, Tayade A, Kumar R, Phani Kumar G, Singh SB (2012) Podophyllum L.: an endangered and anticancerous medicinal plant-an overview. Indian J Tradit Knowl 11:234–241
Chen YQ, Fei Y, Cai M, Lou JX (2003) Effects of amino acids, nitrate, and ammonium on the growth and taxol production in cell cultures of Taxus yunnanensis. Plant Growth Reg 41:265–268
Choi SM, Son SH, Yun SR, Kwon OW, Seon JH, Paek KY (2000) Pilot-scale culture of adventitious roots of ginseng in a bioreactor system. Plant Cell Tissue Organ Cult 62:187–193
Cui XH, Murthy HN, Wu CH, Paek KY (2010) Sucrose-induced osmotic stress affects biomass, metabolite, and antioxidant levels in root suspension cultures of Hypericum perforatum L. Plant Cell Tissue Organ Cult 103:7–14
Cui HY, Abdullahil Baque Md, Lee EJ, Paek KY (2013) Scale-up of adventitious root cultures of Echinacea angustifolia in a pilot-scale bioreactor for the production of biomass and caffeic acid derivatives. Plant Biotechnol Rep 7:297–308
Deus B, Zenk MH (1982) Exploitation of plant cells for the production of natural compounds. Biotechnol Bioeng 24:1965–1974
Farkya S, Bisaria VS, Srivastava AK (2004) Biotechnological aspects of the production of the anticancer drug podophyllotoxin. Appl Microbiol Biotechnol 65:504–519
Folkes LK, Wardman P (2001) Oxidative activation of indole-3-acetic acids to cytotoxic species–a potential new role for plant auxins in cancer therapy. Biochem Parmacol 6:129–136
George EF (2008) Plant tissue culture procedure-background. In: George EF, Hall MA, Klerk G-JD (eds) Plant propagation by tissue culture. Springer, Dordrecht, pp 1–28
Huang T, Gao WY, Wang J (2010) Selection and optimization of a high-producing tissue culture of Panax ginseng C.A. Meyer. Acta Physiol Plant 32:765–772
Kim YS, Lim S, Choi YE, Anbazhagan VR (2007) High frequency plant regeneration via somatic embryogenesis in Podophyllum peltatum L., an important medicinal plant for source of anticancer drug. Curr Sci 92:662–666
McDonald KA, Jackman AP (1989) Bioreactor studies of growth and nutrient utilization in alfalfa suspension cultures. Plant Cell Rep 8:455–458
Murashige T, Skoog F (1962) A revised medium for rapid growth and bioassays with tobacco tissue culture. Physiol Plant 15:473–497
Praveen N, Manohar SH, Naik PM, Nayeem A, Jeong JH, Murthy HN (2009) Production of andrographolide from adventitious root cultures of Andrographis paniculata. Curr Sci 96(5):694–697
Qazi P, Rashid A, Shawl SA (2011) “Podophyllum hexandrum”—a versatile medicinal plant. Int J Pharm Pharm Sci 3:261–268
Sagar PS, Zafar R (2005) In Vitro enhanced production of Podophyllotoxin in phytohormonal induced and regenerated roots of Podophyllum hexandrum. Pharm Biol 43:404–410
Sivakumar G (2006) Bioreactor technology: a novel industrial tool for high-tech production of bioactive molecules and biopharmaceuticals from plant roots. Biotechnol J 1:1419–1427
Sivanandhan G, Arun M, Mayavan S, Rajesh M, Mariashibu TS, Manickavasagam M, Selvaraj N, Ganapathi A (2012a) Chitosan enhances withanolides production in adventitious root cultures of Withania somnifera (L.) Dunal. Ind Crop Prod 37:124–129
Sivanandhan G, Arun M, Mayavan S, Rajesh M, Jeyaraj M, Kapil Dev G, Manickavasagam M, Selvaraj N, Ganapathi A (2012b) Optimization of elicitation conditions with methyl jasmonate and salicylic acid to improve the productivity of withanolides in the adventitious root culture of Withania somnifera (L.) Dunal. Appl Biochem Biotechnol 168:681–696
Sivanandhan G, Rajesh M, Arun M, Jeyaraj M, Kapil Dev G, Manickavasagam M, Selvaraj N, Ganapathi A (2012c) Optimization of carbon source for hairy root growth and withaferin A and withanone production in Withania somnifera. Nat Prod Commun 7:1271–1272
Sivanandhan G, Rajesh M, Arun M, Jeyaraj M, Kapil Dev G, Arjunan A, Manickavasagam M, Muthuselvam M, Selvaraj N, Ganapathi A (2013a) Effect of culture conditions, cytokinins, methyl jasmonate and salicylic acid on the biomass accumulation and production of withanolides in multiple shoot culture of Withania somnifera (L.) Dunal using liquid culture. Acta Physiol Plant 35:715–728
Sivanandhan G, Kapil Dev G, Jeyaraj M, Rajesh M, Arjunan A, Muthuselvam M, Manickavasagam M, Selvaraj N, Ganapathi A (2013b) Increased production of withanolide A, withanone and withaferin A in hairy root cultures of Withania somnifera (L.) Dunal elicited with methyl jasmonate and salicylic acid. Plant Cell Tissue Organ Cult 114:121–129
Sultan P, Shawl AS, Ramteke PW, Jan A, Chisti N, Jabeen N, Shabir S (2006) In vitro propagation for mass multiplication of Podophyllum hexandrum: a high value medicinal herb. Asian J Plant Sci 5:179–184
Wu CH, Dewir YH, Hahn EJ, Paek KY (2006) Optimization of culturing conditions for the production of biomass and phenolics from adventitious roots of Echinacea angustifolia. J Plant Biol 49:193–199
Yin S, Gao W, Liang Y, Wang J, Liu H, Wei C, Zuo B (2013) Influence of sucrose concentration and phosphate source on biomass and metabolite accumulation in adventitious roots of Pseudostellaria heterophylla. Acta Physiol Plant 35:1579–1585
Yu KW, Gao WY, Hahn EJ, Peak KY (2001) Effect of macro elements and nitrogen source on adventitious root growth and ginsenoside production in ginseng (Panax ginseng C. A. Meyer). J Plant Biol 44(4):179–184
Zenk MH, El-Shagi H, Arens H, Stöckigt J, Weiler EW, Deus B (1977) Formation of the indole alkaloids serpentine and ajmalicine in cell suspension cultures of Catharanthus roseus. In: Barz W, Reinhard E, Zenk MH (eds) Plant tissue culture and its bio-technological application. Springer, Berlin, pp 27–43
Zhang Y, Zhong JJ, Yu JT (1995) Effect of osmotic pressure on cell growth and production of ginseng saponin and polysaccharide in suspension cultures of Panax notoginseng. Biotechnol Lett 17:101–106
Zhang J, Gao WY, Wang J, Li XL (2011) Effects of explant types and media salt strength on growth and secondary metabolite accumulation in adventitious roots of Periploca sepium Bunge. Acta Physiol Plant 33:2447–2452
Zhang J, Gao WY, Wang J, Li XL (2012) Effects of sucrose concentration and exogenous hormones on growth and periplocin accumulation in adventitious roots of Periploca sepium Bunge. Acta Physiol Plant 34:1345–1351
Acknowledgments
Prof. A. Ganapathi is thankful to University Grants Commissions (UGC), Government of India, for the award of UGC-BSR (Basic Scientific Research Fellowship). Dr. M. Manickavasagam is grateful to University Grants Commissions (UGC), Government of India, for providing UGC-START UP Grant. First author is thankful to Prof. S. K. Nandi, G. B. Pant Institute, Almora, Uttaranchal and Dr. Lokho Puni IFS, Forest Research Institute, Dehradun, Uttaranchal for helping in collection of seeds. The corresponding author is thankful to DRDO, Government of India, for providing financial support (DLS/81/48222/LSRB–171 BTB/2008) to carry out this work.
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Communicated by K.-Y. Paek.
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Rajesh, M., Sivanandhan, G., Arun, M. et al. Factors influencing podophyllotoxin production in adventitious root culture of Podophyllum hexandrum Royle. Acta Physiol Plant 36, 1009–1021 (2014). https://doi.org/10.1007/s11738-013-1479-3
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DOI: https://doi.org/10.1007/s11738-013-1479-3