Abstract
An efficient transformation system for hairy-root induction in Achyranthes aspera L. was established and investigated as a novel method for the production of 20-hydroxyecdysone. Four Agrobacterium rhizogenes strains (MTCC 2364, NCIM 5140, A4, and ATCC 15834) were examined for their frequency of transformation in the presence of various concentrations of acetosyringone. Strain ATCC 15834 showed significantly higher transformation efficiency and was capable of inducing hairy roots from different explants (young leaf, hypocotyls, cotyledon, and stem segments) of A. aspera in the presence of 100 µM acetosyringone. The hairy root transformation of the lines (AS001 and AS002) was confirmed by PCR amplification of rol B locus using primers specific for rol B gene. Hairy roots grown in the Murashige and Skoog (MS) liquid medium augmented with 30 g/L sucrose exhibited the highest biomass accumulation and this medium was found to be superior to Gamborg’s B5 medium and Whites root culture medium. HPLC and LC-Q-TOF analyses of the hairy-root extract of A. aspera revealed the ability of hairy-root lines to synthesise the phytoecdysteroid 20-hydroxyecdysone.
Similar content being viewed by others
References
Ashraf MF, Zain CRCM, Zainal Z, Noor ZM, Anuar N, Markom M, Ismail I (2015) Establishment of Persicaria minor hairy roots and analysis of secreted β-caryophyllene in medium broth. Plant Cell Tissue Organ Cult 121:11–20
Banerji A, Chadha MS (1970) Insect moulting hormone from Achyranthes aspera. Phytochemistry 9:1671
Banerji A, Chintalwar GJ, Joshi NK, Chadha MS (1971) Isolation of ecdysterone from Indian plants. Phytochemistry 10:2225–2226
Bergamasco R, Horn DHS (1983) Distribution and role of insect hormones in plants. In: Downer RGH, Laufer H (eds) Endocrinology of insects. Liss, New York, pp 627–654
Betsui F, Tanaka-Nishikawa N, Shimmomura K (2004) Anthocyanin production in adventitious root cultures of Raphanus sativus L. cv. Peking Koushin. Plant Biotechnol 21:387–391
Bulgakov VP (2008) Functions of rol genes in plant secondary metabolism. Biotech Adv 26:318–324
Danphitsanuparn P, Boonsnongcheep P, Boriboonkaset T, Chintapakorn Y, Prathanturarug S (2012) Effects of Agrobacterium rhizogenes strains and other parameters on production of isoflavonoids in hairy roots of Pueraria candollei Grah. Ex Benth. var. candollei. Plant Cell Tissue Organ Cult 111:315–322
Dinan L (1995) Distribution and levels of phytoecdysteroids within individual plants of the species of the Chenopodiaceae. Eur J Entomol 92:295–300
Dinan L (2001) Phytoecdysteroids: biological aspects. Phytochemistry 57:325–339
Dinan L (2009) The Karlson lecture. Phytoecdysteroids: what use are they? Arch Insect Biochem Physiol 72:126–141
Dinan L, Lafont R (2006) Effects and applications of arthropod steroid hormones (ecdysteroids) in mammals. J Endocrinol 191:1–8
Felipe DF, Brambilla LZS, Porto C, Pilau EJ, Cortez DAG (2014) Phytochemical analysis of Pfaffia glomerata Inflorescences by LC-ESI-MS/MS. Molecules 19:15720–15734
Gao W, Sun H-X, Xiao H, Ciu G, Hillwig ML, Jackson A, Wang X, Shen Y, Zhao N, Zhang L, Wang X-J, Peters RJ, Huang L (2014) Combining metabolomics and transcriptomics to characterize tanshinone biosynthesis in Salvia miltiorrhiza. BMC Genom 15:73
Gelvin SB (2003) Agrobacterium-mediated plant transformation: the biology behind the ‘‘gene-jockeying’’ tool. Microbiol Mol Biol Rev 67:16–37
Georgiev M, Pavlov A, Bley T (2007) Hairy root type plant in vitro systems as sources of bioactive substances. Appl Microbiol Biotechnol 74:1175–1185
Goyal BR, Goyal RK, Mehta AA (2007) Phytopharmacology of Achyranthes aspera: a review. Pharmacogn Rev 1:143–150
Kim OT, Manickavasagm M, Kim YJ, Jin MR, Kim KS, Seong NS, Hwang B (2005) Genetic Transformation of Ajuga multiflora Bunge with Agrobacterium rhizogenes and 20-Hydroxyecdysone production in hairy roots. J Plant Biol 48:258–262
Lafont R, Dinan L (2003) Practical uses for ecdysteroids in mammals including humans: an update. J Insect Sci 3:7
Matsumoto T, Tanaka N (1991) Production of phytoecdysteroids by hairy root cultures of Ajuga reptans var. atropurpurea. Agric Biol Chem 55:1019–1025
Murashige T, Skoog F (1962) A revised medium for rapid growth and bioassays for tobacco tissue cultures. Physiol Plant 15:473–497
Murthy HN, Dijkstra C, Anthony P, White DA, Davey MR, Power JB, Hahn EJ, Paek KY (2008) Establishment of Withania somnifera hairy root cultures for the production of withanolide A. J Integr Plant Biol 50:975–981
Nagella P, Thiruvengadam M, Jung SJ, Murthy HN, Chung IM (2013) Establishment of Gymnema sylvestre hairy root cultures for the production of gymnemic acid. Acta Physiol Plant 35:3067–3073
Ping HS, Yong-Yue L, Tie-Shan S, Eric TPK (2011) Induction of hairy roots and plant regeneration from the medicinal plant Pogostemon cablin. Plant Cell Tissue Organ Cult 107:251–260
Porter J, Flores H (1991) Host range and implications of plant infection by Agrobacterium rhizogenes. Crit Rev Plant Sci 10:387–421
Saleh NM, Thuc LV (2009) Assessment of hairy roots induction in Solenostemon scutellarioides leaves by different strains of Agrobacterium rhizogenes. Afr J Biotechnol 8:3519–3523
Sevon N, Oksman-Caldentey KM (2002) Agrobacterium rhizogenes mediated transformation: root cultures as a source of alkaloids. Planta Med 68:859–868
Sheikholeslam SN, Weeks DP (1987) Acetosyringone promotes high efficiency transformation of Arabidopsis thaliana explants by Agrobacterium tumefaciens. Plant Mol Biol 8:291–298
Sivanandhan G, Kapil Dev G, Jeyaraj M, Rajesh M, Arjunan A, Muthuselvam M, Selvaraj N, Manickavasagam M, Ganapathi A (2013) Increased production of withanolide A, withanone and withaferin A in hairy root culture of Withania somnifera (L.) Dunal elicited with methyl jasmonate and salicylic acid. Plant Cell Tissue Organ Cult 114:121–129
Stachel SE, Messens E, Van Montagu M, Zambryski P (1985) Identification of the signal molecules produced by wounded plant cells that activate T-DNA transfer in Agrobacterium tumefaciens. Nature 318:624–629
Stevens JF, Reed RL, Morré JT (2008) Characterization of phytoecdysteroid glycosides in meadow foam (Limnanthes alba) seed meal by positive and negative ion LC-MS/MS. J Agric Food Chem 56:3945–3952
Sujatha G, Zdravkovic-Korac S, Calic DC, Flaminic G, Ranjitha Kumari BD (2013) High-efficiency Agrobacterium rhizogenes mediated genetic transformation in Artemisia vulgaris: hairy root production and essential oil analysis. Ind Crops Prod 44:643–652
Vergauwe A, Van Geldre E, Inze D, Van Montagu M, Van Den Eeckhout E (1998) Factors influencing A. tumefaciens mediated transformation of Artemisia annua L. Plant Cell Rep 18:105–110
Zehra M, Banerjee S, Sharma S, Kumar S (1999) Influence of Agrobacterium rhizogenes strains on biomass and alkaloid productivity in hairy root lines of Hyoscyamus muticus and H. albus. Planta Med 65:60–63
Acknowledgements
This study was carried out at School of Biosciences, Mahatma Gandhi University, Kerala, India, with the financial support of University Grants Commission, New Delhi (F. no- 41-552/2012 SR) and the instrumentation support by Department of Biotechnology, India, MSUB programme (BT/PR4800/INF/22/152/2012 dated 03/22/2012).
Author information
Authors and Affiliations
Corresponding author
Ethics declarations
Conflict of interest
Authors have no conflict of interest in relation with this work.
Additional information
Communicated by J. Van Staden.
Electronic supplementary material
Below is the link to the electronic supplementary material.
Rights and permissions
About this article
Cite this article
John, R., Shajitha, P.P., Devassy, A. et al. Hairy-root cultures of Achyranthes aspera Linn. as a novel route for the production of 20-hydroxyecdysone. Acta Physiol Plant 39, 255 (2017). https://doi.org/10.1007/s11738-017-2555-x
Received:
Revised:
Accepted:
Published:
DOI: https://doi.org/10.1007/s11738-017-2555-x