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Optimization of factors affecting in vitro regeneration, flowering, ex vitro rooting and foliar micromorphological studies of Oldenlandia corymbosa L.: a multipotent herb

  • J. Revathi
  • M. Manokari
  • Mahipal S. Shekhawat
Original Article

Abstract

The conditions were optimized for efficient in vitro regeneration of shoots and roots of Oldenlandia corymbosa L. using nodal shoot explants. Murashige and Skoog’s (MS) medium augmented with additives and 2.0 mg L−1 BAP was recorded optimum for shoot bud induction from the nodal meristems. The shoots were proliferated by subsequent subcultures on half strength MS medium fortified with 1.0 mg L−1 BAP and 0.5 mg L−1 kinetin + additives. This media combination yielded maximum number of shoots (223 ± 4.12 shoots/culture bottle) with 13.4 cm average length. Flower buds were induced (4.2 ± 0.28 flowers) from the in vitro multiplied shoots on MS medium contained 1.0 mg L−1 BAP and 0.5 mg L−1 of kinetin and IAA under 50 µmol m−2 s−1 SFPD light intensity for 12 h/day photoperiod. In vitro regenerated shoots were rooted on half strength MS medium conjunct with IAA, IBA and NAA singly at different concentrations. The best rooting response was observed on half strength MS medium containing IBA at 2.0 mg L−1 with activated charcoal. Roots were also induced from the cut ends of the shoots using ex vitro rooting techniques in O. corymbosa by pulse treating the shoots with 300 mg L−1 IBA for 4 min. Better roots were achieved in this method than the in vitro roots in terms of numbers (14.7 ± 0.21) and firmness. The foliar micromorphological studies could help to understand the structural adaptations of micropropagated O. corymbosa plantlets towards field environments. The in vitro induced anomalies in stomatal apparatus were repaired during hardening of plantlets in the greenhouse and after field transfer. Decrease in stomatal density (from 70.78 ± 0.55 to 55.6 ± 0.10), and increase in veins, trichomes and crystals/raphides densities revealed the developments of structural changes in the leaves to withstand in the harsh field conditions. The acclimatized plantlets with well developed root systems were successfully shifted to the natural soils with 98% survival rate.

Keywords

In vitro regeneration Oldenlandia corymbosa Flower induction Ex vitro rooting Acclimation Micromorphological studies 

Abbreviations

BAP

6-Benzylaminopurine

IAA

Indole-3-acetic acid

IBA

Indole-3-butyric acid

Kin

Kinetin

MS

Murashige and Skoog’s basal medium

NAA

α-Naphthalene acetic acid

RH

Relative humidity

SPFD

Spectral photon flux density

Notes

Acknowledgements

Authors are grateful to the Department of Science, Technology and Environment, Government of Puducherry, India for providing financial support to their laboratory under the Grant-In-Aid Scheme.

Author contributions

JR performed the experiments and analyzed data. MM designed the experiment and contributed to writing the manuscript. MSS conceived the idea, and contributed to writing and editing the manuscript.

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.

Ethical approval

The authors declare that the study was carried out following accepted professional conduct. However no ethical approval was needed for the study as it did not involve the use of animals or human subjects.

References

  1. Adeniran AA, Sonibare MA, Rajacharya GH, Kumar S (2017) Assessment of genetic fidelity of Dioscorea bulbifera L. and Dioscorea hirtiflora Benth. and medicinal bioactivity produced from the induced tuberous roots. Plant Cell Tiss Organ Cult.  https://doi.org/10.1007/s11240-1334-0 Google Scholar
  2. Ahmad R, Ali AM, Israf DA, Ismail NH, Shaari K, Lajis NH (2006) Antioxidant, radical-scavenging, anti-inflammatory, cytotoxic and antibacterial activities of methanolic extracts of some Hedyotis species. J Ethnopharmacol 106(2):245–249CrossRefGoogle Scholar
  3. Attims Y, Come D (1978) Dormance des graines d’une plante tropicale (Oldenlandia corymbosa L, Rubiacees): Selection de deux types de plantes. CR Acad Sci Ser D 286:1669–1672Google Scholar
  4. Baskaran P, Van Staden J (2013) Rapid in vitro micropropagation of Agapanthus praecox. S Afr J Bot 86:46–50CrossRefGoogle Scholar
  5. Begum F, Islam KM, Paul RN, Mehedi M, Rani S (2003) In vitro propagation of emetic nut Randia dumetorum (Lamb.). Indian J Exp Biol 41:1479–1481PubMedGoogle Scholar
  6. Behera SK, Rajasekaran C, Payas S, Fulzele DP, Doss CGP, Siva R (2017) In vitro flowering in Oldenlandia umbellata L. J Ayurveda Integr Med.  https://doi.org/10.1016/j.jaim.2017.02.011 PubMedGoogle Scholar
  7. Benmahioul B, Dorion N, Kaid-Harche M, Daguin F (2012) Micropropagation and ex vitro rooting of pistachio (Pistacia vera L.). Plant Cell Tiss Organ Cult 108:353–358CrossRefGoogle Scholar
  8. Blonder B, Enquist BJ (2014) Inferring climate from angiosperm leaf venation networks. New Phytol 204(1):116–126.  https://doi.org/10.1111/nph.12780 CrossRefPubMedGoogle Scholar
  9. Carvalho LC, Osorio ML, Chaves MM, Amancio S (2001) Chlorophyll inflorescences as an indicator of photosynthetic functioning of in vitro grapevine and chestnut plantlets under ex vitro acclimatization. Plant Cell Tiss Organ Cult 67:271–280CrossRefGoogle Scholar
  10. Chen JY, Linn CC, Namba T (1992) Development of natural crude drug resources from Taiwan (X). Parmacognostical studies on Chinese crude drug “han-lian-cao”. Am J Chin Med 20:51–64CrossRefPubMedGoogle Scholar
  11. Chen W, Zou SQ, Li KQ (2005) Extracting and identifying of ursolic acid from Hedyotis corymbosa Lam. Jiangxi Shifan Daxue Xuebao, Ziran Kexueban 29:126–128Google Scholar
  12. Cheruvathur MK, Abraham J, Thomas TD (2015) In vitro micropropagation and flowering in Ipomoea sepiaria Roxb. An important ethanomedicinal plant. Asian Pac J Reprod 4(1):49–53.  https://doi.org/10.1016/S2305-0500(14)60050-6 CrossRefGoogle Scholar
  13. Compton ME, Mize CW (1999) Statistical considerations for in vitro research: I—birth of an idea to collecting data. In Vitro Cell Dev Biol-Plant 35:115.  https://doi.org/10.1007/s11627-999-0020-2 CrossRefGoogle Scholar
  14. Corbineau F, Come D (1982) Effect of the intensity and duration of light at various temperatures on the germination of Oldenlandia corymbosa L. seeds. Plant Physiol 70:1518–1520.  https://doi.org/10.1104/pp.79.2.411 CrossRefPubMedPubMedCentralGoogle Scholar
  15. Croxdale JL (2000) Stomatal patterning in angiosperms. Am J Bot 87:1069–1080CrossRefPubMedGoogle Scholar
  16. Deng ZC, Jin H, He H (2015) An efficient micropropagation system for Morinda officinalis How. (Rubiaceae), an endangered medicinal plant. J Agric Sci Tech 17:1609–1618Google Scholar
  17. Ding XM, Bai DS, Qian JJ (2014) Novel cyclotides from Hedyotis biflora inhibit proliferation and migration of pancreatic cancer cell in vitro and in vivo. J Med Chem Res 23(3):1406–1413.  https://doi.org/10.1007/s00044-013-0746-6 CrossRefGoogle Scholar
  18. Do Cao T, Attims Y, Corbineau F, Come D (1978) Germination des graines produites par les plantes de deux lignees d’Oldenlandia corymbosa L. (Rubiacees) cultiviees dans des conditions controlees. Physiol Veg 16:521–531Google Scholar
  19. Durkovic J, Lengyelova A, Kurja D, Hlad ka D (2009) Photosynthetic performance and stomatal characteristics during ex vitro acclimatization of true service tree (Sorbus domestica L.). J Hortic Sci Biotechnol 84:223–227CrossRefGoogle Scholar
  20. Dutta R, Deb DB (2004) Taxonomic revision of Hedyotis L. (Rubiaceae) in Indian subcontinent. Botanical Survey of India, Kolkata, pp 1–211Google Scholar
  21. Gamble JS (1921) Flora of the Presidency of Madras, vol 1, London. Repr edn. (1995) Bishen Singh, Mahendra pal singh, DehradunGoogle Scholar
  22. Gaspar T, Franck T, Bisbis B, Kevers C, Jouve L, Hausman JF, Dommes J (2002) Concepts in plant stress physiology. Application to plant tissue cultures. Plant Growth Regul 37:263–285CrossRefGoogle Scholar
  23. Ghani A (2003) Medicinal plants of Bangladesh, 2nd edn. Asiatic society of Bangladesh, DhakaGoogle Scholar
  24. Ghatge S, Kudale S, Dixit G (2011) An improved plant regeneration system for high frequency multiplication of Rubia cordifolia L.: a rare medicinal plant. Asian J Biotechnol 3:397–405CrossRefGoogle Scholar
  25. Gupta RK, Singh RK, Swain SR, Hussain T, Rao CV (2012) Anti-hepatotoxic potential of Hedyotis corymbosa against D-galactosamine-induced hepatopathy in experimental rodents. Asian Pac J Trop Biomed 2(3):S1542–S1547.  https://doi.org/10.1016/S2221-1691(12)60450-X CrossRefGoogle Scholar
  26. Hanley ME, Lamont BB, Fairbanks MM, Rafferty CM (2007) Plant structural traits and their role in anti-herbivore defence. Perspect Plant Ecol Evol Syst 8:157–178CrossRefGoogle Scholar
  27. Hickey LJ, Wolfe JA (1975) The bases of angiosperm phylogeny. Vegetative morphology. Ann Missouri Bot Gard 62:538–589CrossRefGoogle Scholar
  28. Jansen S, Dessein S, Piesschaert F, Robbrecht E, Smets E (2000) Aluminium accumulation in leaves of Rubiaceae: systematic and phylogenetic implications. Ann Bot 85:91–101CrossRefGoogle Scholar
  29. Jiang W, Kuang LS, Hou AJ, Qian M, Li JZ (2007) Iridoid glycosides from Hedyotis corymbosa. J Helv Chim Acta 90:1296–1301.  https://doi.org/10.1002/hlca.200790130 CrossRefGoogle Scholar
  30. Johansen DA (1940) Plant microtechnique, 1st edn. McGraw Hill Book Co, New York, pp 182–197Google Scholar
  31. Joshi A, Mathur N (2015) In vitro propagation and conservation of Anthocephalus cadamba through apical bud and nodal explants—a valuable medicinal plant. CIBTech J Biotechnol 4(3):8–18Google Scholar
  32. Kanechi M, Ochi M, Abe M, Inagaki N, Maekawa S (1998) The effects of carbon dioxide enrichment, natural ventilation, and light intensity on growth, photosynthesis, and transpiration of cauliflower plantlets cultured in vitro photoautotrophically and photomixotrophically. J Am Soc Hortic Sci 123:176–181Google Scholar
  33. Kostman TA, Tarlyn NM, Loewus FA, Franceschi VR (2001) Biosynthesis of L-ascorbic acid and conversion of carbons 1 and 2 of L-ascorbic acid to oxalic acid occurs within individual calcium oxalate crystal idioblasts. Plant Physiol 125:634–640CrossRefPubMedPubMedCentralGoogle Scholar
  34. Krishnan SRS, Siril EA (2015) Enhanced in vitro shoot regeneration in Oldenlandia umbellata L. by using quercetin: a naturally occurring auxin-transport inhibitor. Proc Natl Acad Sci India Sect B.  https://doi.org/10.1007/s40011-015-0672-0 Google Scholar
  35. Lai KD, Tran VS, Pham GD (2002) Two anthraquinones from Hedyotis corymbosa and Hedyotis diffusa. Tap Chi Hoa Hoc 40:66–68Google Scholar
  36. Lodha D, Rathore N, Kataria V, Shekhawat NS (2014) In vitro propagation of female Ephedra foliata Boiss. & Kotschy ex Boiss.: an endemic and threatened Gymnosperm of the Thar Desert. Physiol Mol Biol Plant 20:375–383.  https://doi.org/10.1007/s12298-014-0232-8 CrossRefGoogle Scholar
  37. Manetas Y (2003) The importance of being hairy: the adverse effects of hair removal on stem photosynthesis of Verbascum speciosum are due to solar U-VB radiation. New Phytol 158:503–508.  https://doi.org/10.1046/j.1469-8137.2003.00768.x CrossRefGoogle Scholar
  38. Manokari M, Shekhawat MS (2017) Comprehensive analysis of in vitro to field transition of micromorphology and leaf architecture in Passiflora edulis Sims. f. flavicarpa Deg. Ind J Plant Physiol 22(2):240–246.  https://doi.org/10.1007/s40502-017-0290-3 CrossRefGoogle Scholar
  39. Miller IM (1990) Bacterial leaf nodule symbiosis. Adv Bot Res 17:163–234CrossRefGoogle Scholar
  40. Murashige T, Skoog F (1962) A revised medium for rapid growth and bioassays with tobacco tissue culture. Physiol Plant 15:473–497CrossRefGoogle Scholar
  41. Naidoo G, Kaliamoorthy S, Naidoo Y (2009) The secretory apparatus of Xerophyta viscosa (Velloziaceaea): epidermis anatomy and chemical composition of the secretory product. Flora 204:561–568CrossRefGoogle Scholar
  42. Noiarsa P, Ruchirawat S, Otsuka H, Kanchanapoom T (2008) Chemical constituents from Oldenlandia corymbosa L. of Thai origin. J Nat Med 62:249–250CrossRefPubMedGoogle Scholar
  43. Patel AK, Agarwal T, Phulwaria M, Kataria V, Shekhawat NS (2014) An efficient in vitro plant regeneration system from leaf of mature plant of Leptadenia reticulata (Jeewanti): a life giving endangered woody climber. Ind Crop Prod 52:499–505CrossRefGoogle Scholar
  44. Patel AK, Lodha D, Ram K, Shekhawat S, Shekhawat NS (2016) Evaluation of physiochemical factors affecting high-frequency plant regeneration of Blyttia spiralis (synonym: Pentatropis spiralis), a threatened climber of medicinal value. In Vitro Cell Dev Biol-Plant.  https://doi.org/10.1007/s11627-015-9738-1 Google Scholar
  45. Permadi A (2006) Urine facilitating medicinal Plants. Sower Self Reliance, LondonGoogle Scholar
  46. Prabhakar M (2004) Structure, delimitation, nomenclature and classification of stomata. Acta Bot Sin 46:242–252Google Scholar
  47. Rai MK, Kalia RK, Singh R, Gangola MP, Dhawan AK (2011) Developing stress tolerant plants through in vitro selection—an overview of the recent progress. Environ Exp Bot 71:89–98CrossRefGoogle Scholar
  48. Rathore NS, Rathore N, Shekhawat NS (2013) In vitro propagation and micromorphological studies of Cleome gynandra: a C4 model plant closely related to Arabidopsis thaliana. Acta Physiol Plant.  https://doi.org/10.1007/s11738-013-1301-2 Google Scholar
  49. Rezali NI, Sidik NJ, Saleh A, Osman NI, Adam NAM (2017) The effects of different strength of MS media in solid and liquid media on in vitro growth of Typhonium flagelliforme. Asian Pac J Trop Biomed 7(2):151–156.  https://doi.org/10.1016/j.apjtb.2016.11.019 CrossRefGoogle Scholar
  50. Roja G (2008) Micropropagation and production of Camptothecin from in vitro plants of Ophiorrhiza rugosa var. decumbens. Nat Prod Res 22:1017–1023.  https://doi.org/10.1080/14786410802006165 CrossRefPubMedGoogle Scholar
  51. Sadasivan S, Latha PG, Sasikumar JM, Rajashekaran S, Shyamal S, Shine VJ (2006) Hepatoprotective studies on Hedyotis corymbosa (L.) Lam. J Ethnopharmacol 106:245–249CrossRefPubMedGoogle Scholar
  52. Salisbury EJ (1932) The interrelations of soil climate and organisms and the use of stomatal frequency as an integrating index of relation of the plant. Bech Bot Zbl 99:402–420Google Scholar
  53. Shekhawat MS, Manokari M (2016) In vitro regeneration frequency, micro-morphological studies and ex vitro rooting of Hemidesmus indicus (L.) R. Br.: a multipotent endangered climber. Ind J Plant Physiol 21(2):151–160.  https://doi.org/10.1007/s40502-016-0216-5 CrossRefGoogle Scholar
  54. Shekhawat MS, Manokari M (2017) In vitro multiplication, micromorphological studies and ex vitro rooting of Hybanthus enneaspermus (L.) F. Muell.—a rare medicinal plant. Acta Bot Croat.  https://doi.org/10.1515/botcro-2017-0012 Google Scholar
  55. Shekhawat MS, Kannan N, Manokari M, Revathi J (2012) In vitro propagation of Oldenlandia umbellata L.—a highly medicinal & dye-yielding plant of coromandel coast. Int J Recent Sci Res 3(9):758–761Google Scholar
  56. Shekhawat MS, Manokari M, Kannan N (2017) Micromorphological response towards altered environmental conditions in subsequent stages of in vitro propagation of Morinda coreia. Environ Exper Biol 15:37–46Google Scholar
  57. Tisserat B, Galletta PD (1995) In vitro flowering and fruiting of Capsicum frutescens L. Hortic Sci 30:130–132Google Scholar
  58. Van der Krieken WM, Breteler H, Visser MHM, Mavridou D (1993) The role of the conversion of IBA into IAA on root regeneration in apple: introduction of a test system. Plant Cell Rep 12(4):203–206.  https://doi.org/10.1007/BF00237054 CrossRefPubMedGoogle Scholar
  59. Vanu MR, Palanivelu S, Panchanatham S (2006) Immunomodulatory and anti-inflammatoy effects of Semecarpus anacardium Linn. Nut milk extract in experimental inflammatory conditions. Biol Pharm Bull 29:693–700.  https://doi.org/10.1248/bpb.29.693 CrossRefGoogle Scholar
  60. Vaz APA, Figueiredo-Ribeiro RCL, Kerbauy GB (2004) Photoperiod and temperature effects on in vitro growth and flowering of Psygmorchis pusilla, an epiphytic orchid. Plant Physiol Biochem 42:411–415CrossRefPubMedGoogle Scholar
  61. Walter HL (1964) Oldenlandia corymbosa (Rubiaceae). Grana Palynol 5(3):330–341.  https://doi.org/10.1080/00173136409430024 CrossRefGoogle Scholar
  62. Waman AA, Bohra BNP, Sathyanarayana K, Umesha GK, Mukunda TH, Gowda AB (2015) Optimization of factors affecting in vitro establishment, ex vitro rooting and hardening for commercial scale multiplication of silk banana (Musa AAB). Erwerbs-Obstbau 57(3):153–164CrossRefGoogle Scholar
  63. Xiao XB, Lin YX, Xu GB, Gong XB, Gu Y, Tong JF, Yang J (2011) Two new cytotoxic naphthoquinones from Didymocarpus hedyotideus. J Helv Chim Acta 94(3):404–409.  https://doi.org/10.1002/hlca.201000199 CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media B.V., part of Springer Nature 2018

Authors and Affiliations

  • J. Revathi
    • 1
  • M. Manokari
    • 1
  • Mahipal S. Shekhawat
    • 2
  1. 1.Department of BotanyKanchi Mamunivar Center for Postgraduate StudiesPuducherryIndia
  2. 2.Department of Plant ScienceM.G.G.A.C.Mahe, PuducherryIndia

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