A Combinational Phytomolecular-Mediated Assessment in Micropropagated Plantlets of Coelogyne ovalis Lindl.: A Horticultural and Medicinal Orchid

Abstract

Coelogyne ovalis Lindl. is an evergreen epiphytic, sympodial, ornamental orchid having medicinal properties due to the presence of bioactive compounds such as flavidin, flavidinin, coelogin. In the present study, nodal buds of Coelogyne ovalis, when cultured in Knudson C medium supplemented with meta-Topolin (mT), 6-benzyl aminopurine (BAP), kinetin (Kn) and α-naphthalene acetic acid (NAA), responded to the induction of protocorm-like bodies (PLBs) or shoot buds. The highest numbers of PLBs (22.73 ± 0.47) and shoot (14.53 ± 0.27) per explant were augmented in the medium containing a combination of mT (10 µM) and NAA (0.5 µM). Meta-Topolin in the medium was found to be superior in enhancing the response of explants as compared to BAP and Kn. The best rooting of the shoots was observed in the medium supplemented with 10 µM indole-3-acetic acid. Well-developed plantlets obtained after 20 weeks of culture were acclimatized and transferred to the net house. Genetic stability of the acclimatized plantlets was evaluated and contrasted with the mother plant using two molecular markers, viz. start codon targeted and inter-simple sequence repeats, wherein 5.15% clonal variability was observed. Phytochemical screenings revealed that the micropropagated tissues exhibited higher contents of secondary metabolites, antioxidant potentials and chlorophyll contents as compared to the mother plant. The present study could be of great significance for the conservation and commercial utilization of C. ovalis, an orchid facing threat due to its overexploitation from nature.

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References

  1. 1.

    White KJ, Sharma B (2000) Wild orchids in Nepal: the guide to the Himalayan orchids of the Tribhuvan Rajpath and Chitwan jungle. White Lotus Press, Bangkok, p 307

    Google Scholar 

  2. 2.

    Gogoi K, Kumaria S, Tandon P (2012) Ex situ conservation of Cymbidium eburneum Lindl.: a threatened and vulnerable orchid, by asymbiotic seed germination. 3 Biotech 2:337–343

    PubMed Central  Article  Google Scholar 

  3. 3.

    Abraham A, Vatsala P (1981) Introduction to orchids with illustrations and descriptions of 150 South Indian orchids. Tropical Botanic Garden and Research Institute Palode, Thiruvananthapuram, pp 285–287

    Google Scholar 

  4. 4.

    Kaur S, Bhutani KK (2014) In vitro conservation and asymbiotic propagation of Coelogyne flaccida Lindl.: a threatened orchid. Plant Biosyst 148:935–944

    Article  Google Scholar 

  5. 5.

    Sachdev K, Kulshreshtha DK (1986) Phenolic constituents of Coelogyne ovalis. Phytochemistry 25:499–502

    CAS  Article  Google Scholar 

  6. 6.

    Hossain MM, Sharma M, da Silva Teixeira, Ja Pathak P (2010) Seed germination and tissue culture of Cymbidium giganteum Wall. ex Lindl. Sci Hortic (Amst) 123:479–487

    CAS  Article  Google Scholar 

  7. 7.

    Bhattacharyya P, Kumaria S, Tandon P (2016) High frequency regeneration protocol for Dendrobium nobile: a model tissue culture approach for propagation of medicinally important orchid species. S Afr J Bot 104:232–243

    CAS  Article  Google Scholar 

  8. 8.

    Gentile A, Jaquez Gutierrez M, Martinez J, Frattarelli A, Nota P, Caboni E (2014) Effect of meta -Topolin on micropropagation and adventitious shoot regeneration in Prunus rootstocks. Plant Cell Tissue Organ Cult 118:373–381

    CAS  Article  Google Scholar 

  9. 9.

    Mata-Rosas M, Baltazar-Garcia RJ, Moon P, Hietz P, Luna-Monterrojo VE (2010) In vitro regeneration of Lycaste aromatica (Graham ex Hook) Lindl. (Orchidaceae) from pseudobulb sections. Plant Biotechnol Rep 4:157–163

    Article  Google Scholar 

  10. 10.

    Larkin PJ, Scowcroft WR (1981) Somaclonal variation: a novel source of variability from cell cultures for plant improvement. Theor Appl Genet 601:97–214

    Google Scholar 

  11. 11.

    Salvi ND, George L, Eapen S (2001) Plant regeneration from leaf base callus of turmeric and random amplified polymorphic DNA analysis of regenerated plants. Plant Cell Tissue Organ Cult 66:113–119

    CAS  Article  Google Scholar 

  12. 12.

    Sun S, Zhong J, Li S, Wang X (2013) Tissue culture-induced somaclonal variation of decreased pollen viability in torenia (Torenia fournieri Lind.). Bot Stud 54:36

    PubMed  PubMed Central  Article  Google Scholar 

  13. 13.

    Bose B, Kumaria S, Choudhury H, Tandon P (2016) Assessment of genetic homogeneity and analysis of phytomedicinal potential in micropropagated plants of Nardostachys jatamansi, a critically endangered, medicinal plant of alpine Himalayas. Plant Cell Tissue Organ Cult 124:331–349

    Article  Google Scholar 

  14. 14.

    Knudson L (1922) Non-symbiotic germination of orchid seeds. Bot Gaz 73:1–25

    Article  Google Scholar 

  15. 15.

    Murray MG, Thompson WF (1980) Rapid isolation of high molecular weight plant DNA. Nucleic Acids Res 8:4321–4326

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  16. 16.

    Rohlf FJ (1998) NTSYS-pc version 2.0. numerical taxonomy and multivariate analysis system. Exeter Software, Setauket

    Google Scholar 

  17. 17.

    Singleton VL, Rossi JA (1965) Colorimetry of total phenolics with phosphomolybdic-phosphotungstic acid reagents. Am J Enol Viticult 16:144–158

    CAS  Google Scholar 

  18. 18.

    Chang CC, Yang MH, Wen HM, Chern JC (2002) Estimation of total flavonoid content in propolis by two complementary colorimetric methods. J Food Drug Anal 10:178–182

    CAS  Google Scholar 

  19. 19.

    Sreevidya N, Mehrotra S (2003) Spectrophotometric method for estimation of alkaloids precipitable with Dragendorff’s reagent in plant materials. J AOAC Int 86:1124–1127

    CAS  PubMed  Article  Google Scholar 

  20. 20.

    Singleton VL, Orthofer R, Lamuela-Raventos RM (1999) Analysis of total phenols and other oxidation substrates and antioxidants by means of Folin–Ciocalteu reagent. Methods Enzymol 299:152–178

    CAS  Article  Google Scholar 

  21. 21.

    Aremu AO, Bairu MW, Szucova L, Dolezal K, Finnie JF, Van Staden J (2012) Assessment of the role of meta-topolins on in vitro produced phenolics and acclimatization competence of micropropagated “Williams” banana. Acta Physiol Plant 34:2265–2273

    CAS  Article  Google Scholar 

  22. 22.

    Brand-Williams W, Cuvelier ME, Berset CLWT (1995) Use of a free radical method to evaluate antioxidant activity. LWT-Food Sci Technol 28:25–30

    CAS  Article  Google Scholar 

  23. 23.

    Jagtap UB, Waghmare SR, Lokhande VH, Suprasanna P, Bapat VA (2011) Preparation and evaluation of antioxidant capacity of Jackfruit (Artocarpus heterophyllus Lam.) wine and its protective role against radiation induced DNA damage. Ind Crops Prod 34:1595–1601

    CAS  Article  Google Scholar 

  24. 24.

    Benzie IF, Strain JJ (1996) The ferric reducing ability of plasma (FRAP) as a measure of “antioxidant power”: the FRAP assay. Anal Biochem 239:70–76

    CAS  Article  PubMed  Google Scholar 

  25. 25.

    Dinis TC, Madeira VM, Almeida LM (1994) Action of phenolic derivatives (acetaminophen, salicylate, and 5-aminosalicylate) as inhibitors of membrane lipid peroxidation and as peroxyl radical scavengers. Arch Biochem Biophys 315:161–169

    CAS  PubMed  Article  Google Scholar 

  26. 26.

    Shibu BS, Wesley PS, Moin S, Devi BC (2014) In vitro regeneration of Coelogyne nervosa A. Rich. and Eria pseudoclavicaulis Blatt., threatened orchids of Western Ghats, India. Ind J Exp Biol 52:658–663

    Google Scholar 

  27. 27.

    Naing AH, Myint KT, Hwang YJ et al (2010) Micropropagation and conservation of the wild medicinal orchid, Coelogyne cristata. Hortic Environ Biotechnol 51:109–114

    CAS  Google Scholar 

  28. 28.

    Aremu AO, Bairu MW, Dolezal K, Finnie JF, Van Staden J (2012) b) Topolins: a panacea to plant tissue culture challenges? Plant Cell Tissue Organ Cult 108:1–16

    CAS  Article  Google Scholar 

  29. 29.

    Bhattacharyya P, Paul P, Kumaria S, Tandon P (2018) Transverse thin cell layer (t-TCL)-mediated improvised micropropagation protocol for endangered medicinal orchid Dendrobium aphyllum Roxb: an integrated phytomolecular approach. Acta Physiol Plant 40:137

    Article  CAS  Google Scholar 

  30. 30.

    Amoo SO, Aremu AO, Van Staden J (2012) In vitro plant regeneration, secondary metabolite production and antioxidant activity of micropropagated Aloe arborescens Mill. Plant Cell Tissue Organ Cult 111:345–358

    CAS  Article  Google Scholar 

  31. 31.

    Kumaria S, Tandon P (1994) Clonal propagation and establishment of plantlets of Dendrobium fimbriatum var. oculatum Hook.f. In: Advances in plant tissue culture in India, Pragati prakashan, Meerut, India, pp 218–231

  32. 32.

    Hronkova M, Zahradnickova H, Simkova M, Simek P, Heydova A (2003) The role of abscisic acid in acclimation of plants cultivated in vitro to ex vitro conditions. Biol Plant 46:535–541

    CAS  Article  Google Scholar 

  33. 33.

    Malabadi RB, Mulgund GS, Nataraja K (2004) Efficient regeneration of Vanda coerulea, an endangered orchid using thidiazuron. Plant Cell Tissue Organ Cult 76:289–293

    CAS  Article  Google Scholar 

  34. 34.

    Chandra S, Bandopadhyay R, Kumar V, Chandra R (2010) Acclimatization of tissue cultured plantlets: from laboratory to land. Biotechnol Lett 32:1199–1205

    CAS  Article  PubMed  Google Scholar 

  35. 35.

    Collard BC, Mackill DJ (2009) Start codon targeted (SCoT) polymorphism: a simple, novel DNA marker technique for generating gene-targeted markers in plants. Plant Mol Biol Rep 27:86–93

    CAS  Article  Google Scholar 

  36. 36.

    Xiong F, Zhong R, Han Z, Jiang J, He L, Zhuang W, Tang R (2011) Start codon targeted polymorphism for evaluation of functional genetic variation and relationships in cultivated peanut (Arachis hypogaea L.) genotypes. Mol Biol Rep 38:3487–3494

    CAS  PubMed  Article  Google Scholar 

  37. 37.

    Thakur J, Dwivedi MD, Sourabh P et al (2016) Genetic homogeneity revealed using SCoT, ISSR and RAPD markers in micropropagated Pittosporum eriocarpum Royle-an endemic and endangered medicinal plant. PLoS ONE 11(7):e0159050

    PubMed  PubMed Central  Article  CAS  Google Scholar 

  38. 38.

    Chavan JJ, Gaikwad NB, Umdale SD, Kshirsagar PR, Bhat KV, Yadav SR (2014) Efficiency of direct and indirect shoot organogenesis, molecular profiling, secondary metabolite production and antioxidant activity of micropropagated Ceropegia santapaui. Plant Growth Regul 72:1–15

    CAS  Article  Google Scholar 

  39. 39.

    Sherif NA, Benjamin JF, Kumar TS, Rao MV (2018) Somatic embryogenesis, acclimatization and genetic homogeneity assessment of regenerated plantlets of Anoectochilus elatus Lindl., an endangered terrestrial jewel orchid. Plant Cell Tissue Organ Cult 132:303–316

    CAS  Article  Google Scholar 

  40. 40.

    Kumar S, Mangal M, Dhawan AK, Singh N (2011) Assessment of genetic fidelity of micropropagated plants of Simmondsia chinensis (Link) Schneider using RAPD and ISSR markers. Acta Physiol Plant 33:2541–2545

    CAS  Article  Google Scholar 

  41. 41.

    Pietta PG (2000) Flavonoids as antioxidants. J Nat Prod 63:1035–1042

    CAS  PubMed  Article  Google Scholar 

  42. 42.

    Vasanthi HR, ShriShriMal N, Das DK (2012) Phytochemicals from plants to combat cardiovascular disease. Curr Med Chem 19:2242–2251

    CAS  PubMed  Article  Google Scholar 

  43. 43.

    Bhattacharyya P, Kumaria S, Diengdoh R, Tandon P (2014) Genetic stability and phytochemical analysis of the in vitro regenerated plants of Dendrobium nobile Lindl., an endangered medicinal orchid. Meta Gene 2:489–504

    PubMed  PubMed Central  Article  Google Scholar 

  44. 44.

    Xanthopoulou MN, Fragopoulou E, Kalathara K, Nomikos T, Karantonis HC, Antonopoulou S (2010) Antioxidant and anti-inflammatory activity of red and white wine extracts. Food Chem 120:665–672

    CAS  Article  Google Scholar 

  45. 45.

    Bahadoran Z, Mirmiran P, Azizi F (2013) Dietary polyphenols as potential nutraceuticals in management of diabetes: a review. J Diabetes Metab Disord 12:43

    PubMed  PubMed Central  Article  CAS  Google Scholar 

  46. 46.

    Kolodziej H, Kiderlen AF (2005) Antileishmanial activity and immune modulatory effects of tannins and related compounds on Leishmania parasitised RAW 264.7 cells. Phytochemistry 66:2056–2071

    CAS  PubMed  Article  Google Scholar 

  47. 47.

    Gentile A, Jaquez Gutierrez M, Martinez J, Frattarelli A, Nota P, Caboni E (2014) Effect of meta -Topolin on micropropagation and adventitious shoot regeneration in Prunus rootstocks. Plant Cell Tissue Organ Cult 118:373–381

    CAS  Article  Google Scholar 

  48. 48.

    Brewer MS (2011) Natural antioxidants: sources, compounds, mechanisms of action, and potential applications. Compr Rev Food Sci Food Saf 10:221–247

    CAS  Article  Google Scholar 

  49. 49.

    Srinivasan K (2014) Antioxidant potential of spices and their active constituents. Crit Rev Food Sci Nutr 54:352–372

    CAS  PubMed  Article  Google Scholar 

  50. 50.

    Bhattacharyya P, Kumaria S, Job N, Tandon P (2015) Phyto-molecular profiling and assessment of antioxidant activity within micropropagated plants of Dendrobium thyrsiflorum: a threatened, medicinal orchid. Plant Cell Tissue Organ Cult 122:535–550

    CAS  Article  Google Scholar 

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Acknowledgements

The financial support received from the University Grants Commission (UGC), North-Eastern Hill University (NEHU), Shillong (F24-25/DSW/2014/864), and Department of Biotechnology (DBT), Government of India, New Delhi (BT/PR16385/NER/95/124/2015), is gratefully acknowledged.

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Correspondence to Suman Kumaria.

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Significance Statement

An efficient in vitro propagation protocol was developed through nodal buds of Coelogyne ovalis, and phytomolecular-mediated assessments were studied from in vitro raised plantlets. The present work provides the baseline data for further investigation of possible novel bioactive compound(s) responsible for medicinal properties.

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Singh, N., Kumaria, S. A Combinational Phytomolecular-Mediated Assessment in Micropropagated Plantlets of Coelogyne ovalis Lindl.: A Horticultural and Medicinal Orchid. Proc. Natl. Acad. Sci., India, Sect. B Biol. Sci. 90, 455–466 (2020). https://doi.org/10.1007/s40011-019-01118-5

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Keywords

  • Orchid propagation
  • Meta-Topolin
  • Molecular markers
  • Phytochemicals
  • Antioxidants