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In vitro propagation, clonal fidelity and phytochemical analysis of Rhodiola imbricata Edgew: a rare trans-Himalayan medicinal plant

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Abstract

The present study focuses on development of a micropropagation protocol for true to type plants of Rhodiola imbricata, an endangered medicinal plant found in trans-Himalayan Leh-Ladakh region of India. It also aims at analyzing the pharmaceutically important secondary metabolites and antioxidant potential of in vitro and in vivo plants. Various cytokinins and auxins were tested for shoot proliferation and in vitro rooting of the microshoots, respectively. Random primers were used for checking genetic uniformity at different stages of micropropagation. Pharmaceutically important secondary metabolites of R. imbricata such as Rosavin, total polyphenols and free radical scavenging activity were analyzed by HPLC. Among different cytokinins used, BAP (5 µM) and TDZ (1 µM) were found to perform better in terms of shoot proliferation, shoot length and number of leaves as compared to other concentrations. For rooting of microshoots, a lower concentration of NAA (0.5 µM) yielded more efficient rooting of micro shoots (17.33 roots per micro shoot). In vitro rooted microshoots were hardened and showed 60% survival rate. The content of gallic acid, chlorogenic acid and 4-hydroxybenzoic acid was higher in the in vivo plant. The amount of ferulic acid was higher in the in vitro raised plant when compared to field grown plant. Furthermore, caffeic acid and p-coumaric acid were higher in the in vitro raised plants as compared to field grown plants. This work will facilitate in conservation of this endangered herb and provide necessary plant materials for various biotechnological and pharmaceutical applications.

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Abbreviations

ANOVA:

Analysis of variance

BAP:

6-Benzyl amino purine

CTAB:

Cetyl trimethyl ammonium bromide

DPPH:

2,2-Diphenyl-1-picrylhydrazyl

FDA:

Fluorescein diacetate

GA:

Gibberellic acid

HPLC:

High performance liquid chromatography

IAA:

Indole-3-acetic acid

IBA:

Indole-3-butyric acid

KN:

Kinetin

NAA:

Naphthalene acetic acid

RAPD:

Random amplified polymorphic DNA

TDZ:

Thidiazuron

TPC:

Total polyphenolic content

TTC:

Triphenyl tetrazolium chloride

References

  • Ahmad S, Garg M, Tamboli ET, Abdin MZ, Ansari SH (2013) In vitro production of alkaloids: factors, approaches, challenges and prospects. Pharmacogn Rev 7:27–33

    Article  Google Scholar 

  • Ahmed MR, Anis M (2012) Role of TDZ in the quick regeneration of multiple shoots from nodal explant of Vitex trifolia L.—an important medicinal plant. App Biochem Biotechnol 168(5):957–966

    Article  CAS  Google Scholar 

  • Aygun A, Dumanoglu H (2015) In vitro shoot proliferation and in vitro and ex vitro root formation of Pyrus elaeagrifolia Pallas. Front Plant Sci 6:225

    Article  Google Scholar 

  • Bhardwaj AK, Kapoor S, Naryal A, Bhardwaj P, Warghat AR, Kumar B, Chaurasia OP (2016) Effect of various dormancy breaking treatments on seed germination, seedling growth and seed vigour of medicinal plants. Trop Plant Res 3(3):508–516

    Article  Google Scholar 

  • Chaurasia OP, Ballabh B, Singh B (2003) Himalyan rose root, Rhodiola spp., potential health drink and drug plant. Nat Prod Rad 2:71–92

    Google Scholar 

  • Chawla R, Jaiswal S, Kumar R, Arora R, Sharma RK (2010) Himalayan bioresource Rhodiola imbricata as a promising radioprotector for nuclear and radiological emergencies. J Pharma Bioallied Sci 2:213–219

    Article  CAS  Google Scholar 

  • Debnath SC (2009) Zeatin and TDZ-induced shoot proliferation and use of bioreactor in clonal propagation of medicinal Herb, Roseroot (Rhodiola rosea L). J Plant Biochem Biotechnol 18(2):245–248

    Article  CAS  Google Scholar 

  • De Klerk KG, Geert J, Hanecakova J, Jasik J (2008) Effect of medium-pH and MES on adventitious root formation from stem disks of apple. Plant Cell Tiss Organ Cult 95(3):285

    Article  Google Scholar 

  • Dimpfel W, Schombert L, Panossian AG (2018) Assessing the quality and potential efficacy of commercial extracts of Rhodiola rosea L. by analyzing the salidroside and rosavin content and the electrophysiological activity in Hippocampal long-term potentiation, a synaptic model of memory. Front Pharmacol 9:425

    Article  Google Scholar 

  • Fatima N, Anis M (2012) Role of growth regulators on in vitro regeneration and histological analysis in Indian ginseng (Withania somnifera L.) Dunal. Physiol Mol Biol Plants 18(1):59–67

    Article  CAS  Google Scholar 

  • Finch-Savage WE, Metzger GL (2006) Seed dormancy and the control of germination. New Phytol 171:501–523

    Article  CAS  Google Scholar 

  • Finkelstein R, Reeves W, Ariizumi T, Steber C (2008) Molecular aspects of seed dormancy. Annu Rev Plant Biol 59:387–415

    Article  CAS  Google Scholar 

  • Fogaca CM, Fett-Neto AG (2005) Role of auxin and its modulators in the adventitious rooting of Eucalyptus species differing in recalcitrance. Plant Growth Regul 45(1):1–10

    Article  CAS  Google Scholar 

  • Frankland B (1961) Effect of gibberellic acid, kinetin and other substances on seed dormancy. Nature 192:678–679

    Article  CAS  Google Scholar 

  • Galambosi B, Galambosi Z, Slacanin I (2007) Comparison of natural and cultivated roseroot (Rhodiola rosea L.) roots in Finland. Zeitschrift für Arznei-& Gewürzpflanzen 12(3):141–147

    Google Scholar 

  • Galambosi B, Galambosi Z, Hethelyi E, Szöke E, Volodin V, Poletaeva I, Iljina I (2010) Importance and quality of roseroot (Rhodiola rosea L.) growing in the European North. Zeitschrift für Arznei-& Gewürzpflanzen 15(4):160–169

    CAS  Google Scholar 

  • Ganzera M, Yayla Y, Khan I (2001) Analysis of the marker compounds of Rhodiola rosea L. (Golden Root) by reversed phase high performance liquid chromatography. Chem Pharm Bull 49(4):465–467

    Article  CAS  Google Scholar 

  • Germano C, Ramazanov Z, Bernal SM (1999) Arctic root (Rhodiola rosea): the powerful new Ginseng alternative. Kensington Publishing Corporation, New York

    Google Scholar 

  • Grech-Baran M, Sykłowska-Baranek K, Pietrosiuk A (2015) Biotechnological approaches to enhance salidroside, rosin and its derivatives production in selected Rhodiola spp. in vitro cultures. Phytochem Rev 14(4):657–674

    Article  CAS  Google Scholar 

  • Gupta A, Kumar R, Upadhyay NK, Pal K, Kumar R, Sawhney RC (2007) Effects of Rhodiola Imbricata on dermal wound healing. Planta Med 73(8):774–777

    Article  CAS  Google Scholar 

  • Gupta V, Lahiri S, Sultana S, Tulsawani R, Kumar R (2010) Anti-oxidative effect of Rhodiola Imbricata root extract in rats during cold, hypoxia and restraint (C–H–R) exposure and post-stress recovery. Food Chem Toxicol 48(4):1019–1025

    Article  CAS  Google Scholar 

  • Gupta S, Bhoyar MS, Kumar J, Warghat AR, Bajpai PK, Rasool M, Srivastva RB (2012) Genetic diversity among natural populations of Rhodiola Imbricata edgew. From trans himalyan cold arid desert using random amplified polymorphic DNA (RAPD) and inter simple sequence repeat (ISSR) Markers. J Med Plant Res 6(3):405–415

    CAS  Google Scholar 

  • Hare PD, Van Staden J (1994) Inhibitory effect of thidiazuron on the activity of cytokinin oxidase isolated from soybean callus. Plant Cell Physiol 35:1121–1125

    Article  CAS  Google Scholar 

  • Huetteman CA, Preece JE (1993) Thidiazuron: a potent cytokinin for woody plant tissue culture. Plant Cell Tiss Organ Cult 33:105–119

    Article  CAS  Google Scholar 

  • Ishaque S, Shamseer L, Bukutu C, Vohra S (2012) Rhodiola rosea for physical and mental fatigue: a systematic review. BMC Complement Altern Med 12:70

    Article  Google Scholar 

  • Jasrai YT, Wala BB (2000) Curculigo orchioides Gaertn. (Kali musli): an endangered medicinal herb. In: Khan IA, Khanum A (eds) Role of biotechnology in medicinal and aromatic plants, vol 4. Ukauz Publications, Hyderabad, pp 89–95

    Google Scholar 

  • Kanupriya PD, Ram MS, Kumar R, Sawhney R, Sharma S, Ilavazhagan G, Banerjee P (2005) Cytoprotective and antioxidant activity of Rhodiola Imbricata against tert-butyl hydroperoxide induced oxidative injury in U-937 human macrophages. Mol Cell Biochem 275(1–2):1–6

    Article  CAS  Google Scholar 

  • Kosman VM, Shikov AN, Makarov VG, Galambosi B, Dragland S, Vender C (2004) Comparative analysis of European and Russian Rhodiola rosea samples. In: Proceedings of the congress, actual problems of creation of new medicinal preparations of natural origin. The 8th international congress Phytopharma, 21–23 June, Mikkeli, pp 268–274

  • Kumar GP, Kumar R, Chaurasia OP (2011) Conservation status of medicinal plants in Laclakh: cold arid zone of Trans-Himalayas. Res J Med Plants 5:685–694

    Article  Google Scholar 

  • Lager IDA (2010) Changes in external pH rapidly alter plant gene expression and modulate auxin and elicitor responses. Plant Cell Environ 33(9):1513–1528

    CAS  PubMed  PubMed Central  Google Scholar 

  • Leifert C (1992) Effect of medium acidity on growth and rooting of different plant species growing in vitro. Plant Cell Tiss Organ Cult 30(3):171–179

    Article  Google Scholar 

  • Liu H, Xu Y, Liu Y, Liu C (2006) Plant regenration form explants of Rhodiola fastgiata. In Vitro Cell Dev Biol Plant 42:345–347

    Article  CAS  Google Scholar 

  • Makarov VG, Zenkevich IG, Shikov AN, Pimenov AI, Pozhariskaya ON, Ivanova SA, Galambosi B (2003) Comparative analyses of Rhodiola rosea of Scandinavian and Russian origin. In: Proceedings of the Congress Actual problems of creation of new medicinal preparations of natural origin. The 7th international congress Phytopharm, 3–5 July 2003, pp 390–396

  • Martins M, Sarmento D, Oliveira MM (2004) Genetic stability of micropropagated almond plantlets, as assessed by RAPD and ISSR markers. Plant Cell Rep 23(7):492–496

    Article  CAS  Google Scholar 

  • Minano HS, Gonzalez-Benito ME, Martín C (2010) Molecular characterization and analysis of somaclonal variation in chrysanthemum cultivars using RAPD markers. Sci Hort 122(2):238–243

    Article  Google Scholar 

  • Mishra K, Padwad Y, Dutta A, Ganju L, Sairam M, Banerjee P, Sawhney R (2008) Aqueous extract of Rhodiola imbricata rhizome inhibits proliferation of an erythroleukemic cell line K-562 by inducing apoptosis and cell cycle arrest at G2/M phase. Immunobiol 213(2):125–131

    Article  CAS  Google Scholar 

  • Mishra KP, Ganju L, Chanda S, Karan D, Sawhney RC (2009) Aqueous extract of Rhodiola imbricata rhizome stimulates Toll-like receptor 4, granzyme-B and Th1 cytokines In vitro. Immunobiol 214(1):27–31

    Article  CAS  Google Scholar 

  • Mishra KP, Ganju L, Singh SB (2012) Anti-cellular and immunomodulatory potential of aqueous extract of Rhodiola imbricata rhizome. Immunopharmacol Immunotoxicol 34:513–518

    Article  CAS  Google Scholar 

  • Murashige T, Skoog F (1962) A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol Plant 15:473–497

    Article  CAS  Google Scholar 

  • Overvoorde P, Fukaki H, Beeckman T (2010) Auxin control of root development. Cold Spring Harb Perspect Biol 2(6):a001537

    Article  Google Scholar 

  • Panossian A, Wikman G, Sarris J (2010) Rosenroot (Rhodila rosea): traditional use, chemical composition, pharmacology and clinical efficacy. Phytomedicine 17(7):481–493

    Article  CAS  Google Scholar 

  • Patial V, Sharma M, Bhattacharya A (2015) Potential of thidiazuron in improved micropropagation of Picrorhiza kurroa—an endangered medicinal herb of alpine Himalaya. Plant Biosyst 151(4):1–8

    Google Scholar 

  • Pierret A, Christopher CJ, Doussan MC (2005) Conventional detection methodology is limiting our ability to understand the roles and functions of fine roots. New Phytol. https://doi.org/10.1111/j.1469-8137.2005.01389.x

    Article  PubMed  Google Scholar 

  • Pop TI, Pamfil D, Bellini C (2011) Auxin control in the formation of adventitious roots. Not Bot Horti Agrobo Cluj-Napoca 39(1):307–316

    Article  CAS  Google Scholar 

  • Pritchard HW (1985) Determination of orchid seed viability using fluorescein diacetate. Plant Cell Environ 8(9):727–730

    Google Scholar 

  • Ramage CM, Williams RR (2002) Mineral nutrition and plant morphogenesis. In Vitro Cell Dev Biol Plant 38:116

    Article  CAS  Google Scholar 

  • Ramakrishna A, Ravishankar GA (2011) Influence of abiotic stress signals on secondary metabolites in plants. Plant Sig Behav 6:1720–1731

    Article  Google Scholar 

  • Ruzic D, Vujovic T (2008) The effects of cytokinin types and their concentration on in vitro multiplication of sweet cherry cv.Lapins (Prunus avium L.). J Hortic Sci 35:12–21

    Article  CAS  Google Scholar 

  • Saghai-Maroof MA, Soliman KM, Jorgensen RA, Allard RW (1984) Ribosomal DNA spacer-length polymorphisms in barley: mendelian inheritance, chromosomal location, and population dynamics. Proc Natl Acad Sci USA 81:8014–8018

    Article  CAS  Google Scholar 

  • Savita BA, Pati PK, Virk GS, Nagpal A (2012) An efficient micropropagation protocol for Citrus jambhiri Lush. and assessment of clonal fidelity employing anatomical studies and RAPD markers. In Vitro Cell Dev Biol Plant 48:512

    Article  CAS  Google Scholar 

  • Shehu UI, Sani LA, Ibrahim AB (2016) Auxin induced rooting of cactus pear (Opuntia ficus-indica L. Miller) cladodes for rapid on-farm propagation. Afr J Agric Res 11(10):898–900

    Article  CAS  Google Scholar 

  • Singh V, Singh B, Sharma A, Kaur K, Gupta AP, Salar RK, Pati PK (2016) Leaf spot disease adversely affects human health-promoting constituents and withanolide biosynthesis in Withania somnifera (L.) Dunal. J App Microbiol 122(1):153–165

    Article  Google Scholar 

  • Thorpe T, Stasolla C, Yeung EC, De Klerk GJ, Roberts A, George EF (2008) The components of plant tissue culture media II: organic addition, osmotic and pH effects, and support systems. In: George EF et al (ed) Plant propagation by tissue culture, vol 1, 3rd edn. (Springer, Dordrecht, pp 115–173

    Google Scholar 

  • Tolonen A, Pakonen M, Hohtola A, Jalonen J (2003) Phenylpropanoid glycosides from Rhodila rosea. Chem Pharm Bull 51(4):467–470

    Article  CAS  Google Scholar 

  • Tomsone S, Gertnere D, Novikova D (2004) The influence of thidiazuron on shoot regeneration and proliferation of rhododendrons in vitro. Acta Univ Latviensis ser Biol 676:239–242

    Google Scholar 

  • Waes JM, Debergh PC (1986) Adaptation of the tetrazolium method for testing the seed viability, and scanning electron microscopy study of some Western European orchids. Physiol Plant 66:435–442

    Article  Google Scholar 

  • Yamauchi Y, Ogawa M, Kuwahara A, Hanada A, Kamiya Y, Yamaguchi S (2004) Activation of gibberellin biosynthesis and response pathways by low temperature during imbibition of Arabidopsis thaliana seeds. Plant Cell. https://doi.org/10.1105/tpc.018143

    Article  PubMed  PubMed Central  Google Scholar 

  • Yan SP, Yang RH, Wang F, Sun LN, Song XS (2017) Effect of auxins and associated metabolic changes on cuttings of hybrid aspen. Forests 8(4):117

    Article  Google Scholar 

  • Yousef GG, Grace MH, Cheng DM, Belolipov IV, Raskin I, Lila MA (2006) Comparative phytochemical characterization of three Rhodila species. Phytochemistry 67(21):2380–2391

    Article  CAS  Google Scholar 

  • Zeliang PK, Pattanayak A, Iangrai B, Khongwir EA, Sarma BK (2010) Fertile plant regeneration from cryopreserved calli of Oryza rufipogon Griff. and assessment of variation in the progeny of regenerated plants. Plant Cell Rep 29:1423–1433

    Article  CAS  Google Scholar 

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Acknowledgements

The authors are thankful to Defence Research and Development Organization (DRDO), Ministry of Defence, Government of India for providing financial assistance for the research.

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Authors and Affiliations

  1. Department of Biotechnology, Defence Institute of High Altitude Research, Defence Research & Development Organization, Leh, 194101, India

    Ashwani Kumar Bhardwaj, Avilekh Naryal, Shweta Saxena & Om Prakash Chaurasia

  2. Department of Biotechnology, Guru Nanak Dev University, Amritsar, Punjab, 143005, India

    Baldev Singh, Kulwinder Kaur, Poonam Roshan, Anu Sharma, Dechen Dolker & Pratap Kumar Pati

Authors
  1. Ashwani Kumar Bhardwaj
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  2. Baldev Singh
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  3. Kulwinder Kaur
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  4. Poonam Roshan
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  5. Anu Sharma
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  6. Dechen Dolker
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  7. Avilekh Naryal
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  8. Shweta Saxena
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  9. Pratap Kumar Pati
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  10. Om Prakash Chaurasia
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Contributions

AKB, PKP developed the micropropagation protocol. AKB, BS, KK, PR wrote the manuscript. AKB, BS, KK, PR, AS, DD and AN performed the experiments. PKP, SS, OPC supervised the experimental tasks and the writing of the manuscript. KK, BS, PR and PKP have done English editing.

Corresponding authors

Correspondence to Pratap Kumar Pati or Om Prakash Chaurasia.

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The authors declare that they have no conflict of interest.

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Communicated by Wagner Campos Otoni.

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Bhardwaj, A.K., Singh, B., Kaur, K. et al. In vitro propagation, clonal fidelity and phytochemical analysis of Rhodiola imbricata Edgew: a rare trans-Himalayan medicinal plant. Plant Cell Tiss Organ Cult 135, 499–513 (2018). https://doi.org/10.1007/s11240-018-1482-x

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