Advertisement

Augmentation of wedelolactone through in vitro tetraploid induction in Eclipta alba (L.) Hassk.

  • Umme Salma
  • Suprabuddha Kundu
  • Alok Kumar Hazra
  • Md. Nasim Ali
  • Nirmal Mandal
Original Article

Abstract

A complete protocol for the in vitro induction of Eclipta alba tetraploids has been optimized to enhance the wedelolactone content, an anti-cancerous compound. The effects of different concentrations of colchicine (0, 0.01, 0.05, 0.1, 0.2 and 0.3%; w/v) along with treatment durations (12, 24, 36 and 48 h) were investigated on shoot tip (ST) and nodal segment (NS). The treated explants were then incubated on Murashige and Skoog (MS) medium having 1.5 mg L−1 N6-benzylaminopurine and 0.5 mg L−1 α-napthalene acetic acid for shoot regeneration and afterward root was induced on 1.0 mg L−1 indole-3-acetic acid enriched ½MS medium. The tetraploids of E. alba were proficiently induced by the treatment of 0.1% colchicine for 24 h. The highest tetraploid induction efficiency was obtained from ST (30.56%) in comparison to the NS (22.22%). Analysis by spectrophotometry and flow cytometry showed that colchicine treated plants contained higher quantity of DNA than diploid plants. Cytological studies demonstrated doubled the chromosome number in tetraploids (2n = 4x = 44) than diploids (2n = 2x = 22). The ploidy level enhancement lead to alteration of other traits, like increased plant height, stem diameter, leaf size, stomatal size and chlorophyll content. As determined through high performance thin-layer chromatography, the ultimate achievement of this technique is the higher accumulation of wedelolactone in tetraploid plants (300.32 µg g−1 dry weight) in evaluation to in vitro diploid (131.31 µg g−1 dry weight) and in vivo diploid mother plants (93.26 µg g−1 dry weight), thus improving the pharmaceutical value of E. alba.

Keywords

Colchicine Chromosome counting Eclipta alba Tetraploid Wedelolactone 

Abbreviations

BAP

N6-benzylaminopurine

FCM

Flow cytometry

HPTLC

High performance thin-layer chromatography

IAA

Indole-3-acetic acid

MS

Murashige and Skoog (1962)

NAA

α-Napthalene acetic acid

NS

Nodal segment

RT

Root tip

ST

Shoot tip

Notes

Acknowledgements

Authors acknowledge the laboratory as well as library assistance from the Bidhan Chandra Krishi Viswavidyalaya, West Bengal, India.

Author contributions

US, SK, MNA and NM conceived the idea; US and SK performed the experiments; US and SK surveyed the literature and wrote the manuscript; AKH assisted in the phytochemical assessment; MNA and NM edited the manuscript. All the authors approved the final version of the manuscript prior to submission.

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflicts of interest.

Supplementary material

11240_2018_1381_MOESM1_ESM.docx (16 kb)
Supplementary material 1 (DOCX 16 KB)

References

  1. Abdoli M, Moieni A, Badi HN (2013) Morphological, physiological, cytological and phytochemical studies in diploid and colchicine-induced tetraploid plants of Echinacea purpurea (L.). Acta Physiol Plant 35:2075–2083CrossRefGoogle Scholar
  2. Acanda Y, Martinez O, Gonzalez MV, Prado MJ, Rey M (2015) Highly efficient in vitro tetraploid plant production via colchicine treatment using embryogenic suspension cultures in grapevine (Vitis vinifera cv. Mencía). Plant Cell Tissue Organ Cult 123:547–555CrossRefGoogle Scholar
  3. Amiri S, Kazemitabaar S, Ranjbar G, Azadbakht M (2010) The effect of trifluralin and colchicine treatments on morphological characteristics of jimsonweed (Datura Stramonium L.). Trakia J Sci 8:47–61Google Scholar
  4. Arnon DI (1949) Copper enzymes in isolated chloroplasts polyphenoloxidase in Beta vulgaris. Plant Physiol 24:1–15CrossRefPubMedPubMedCentralGoogle Scholar
  5. Bagheri M, Mansouri H (2015) Effect of induced polyploidy on some biochemical parameters in Cannabis sativa L. Appl biochem biotechnol 175:2366–2375CrossRefPubMedGoogle Scholar
  6. Baskaran P, Jayabalan N (2005) An efficient micropropagation system for Eclipta alba - a valuable medicinal herb. In vitro Cell Dev Biol Plant 41:532–539CrossRefGoogle Scholar
  7. Chen Z, Sun X, Shen S, Zhang H, Ma X, Liu J, Kuang S, Yu Q (2013) Wedelolactone a naturally occurring coumestan, enhances interferon-G signaling through inhibiting STAT1 protein dephosphorylation. J Biol Chem 288:14417–14427CrossRefPubMedPubMedCentralGoogle Scholar
  8. Cheniclet C, Rong WY, Causse M, Frangne N, Bolling L, Carde JP, Renaudin JP (2005) Cell expansion and endoreduplication show a large genetic variability in pericarp and contribute strongly to tomato fruit growth. Plant Physiol 139:1984–1994CrossRefPubMedPubMedCentralGoogle Scholar
  9. Duncan DB (1955) Multiple range and multiple F test. Biometrics 11:1–42CrossRefGoogle Scholar
  10. Elyazid DMA, El-Shereif AR (2014) In vitro induction of polyploidy in Citrus reticulata Blanco. Am J Plant Sci 5:1679–1685CrossRefGoogle Scholar
  11. Galbraith DW, Harkins KR, Maddox JM, Ayres NM, Sharma DP, Firoozabady E (1983) Rapid flow cytometric analysis of the cell cycle in intact plant tissues. Science 220:1049–1051CrossRefPubMedGoogle Scholar
  12. Gantait S, Mandal N, Bhattacharyya S, Das PK (2011) Induction and identification of tetraploids using in vitro colchicine treatment of Gerbera jamesonii Bolus cv Sciella. Plant Cell Tissue Organ Cult 106:485–493CrossRefGoogle Scholar
  13. Gomes SSL, Saldanha CW, Neves CS, Trevizani M, Raposo NRB, Notini MM, de Oliveira Santos M, Campos JMS, Otoni WC, Viccini LF (2014) Karyotype, genome size, and in vitro chromosome doubling of Pfaffia glomerata (Spreng.) Pedersen. Plant Cell Tissue Organ Cult 118:45–56Google Scholar
  14. Hannweg K, Sippel A, Bertling I (2013) A simple and effective method for the micropropagation and in vitro induction of polyploidy and the effect on floral characteristics of the South African iris, Crocosmia aurea. S Afr J Bot 88:367–372CrossRefGoogle Scholar
  15. Heping H, Shanlin G, Lanlan C, Xiaoke J (2008) In vitro induction and identification of autotetraploids of Dioscorea zingiberensis. Vitro Cell Dev Biol Plant 44:448–455CrossRefGoogle Scholar
  16. Javadian N, Karimzadeh G, Sharifi M, Moieni A, Behmanesh M (2017) In vitro polyploidy induction: changes in morphology, podophyllotoxin biosynthesis, and expression of the related genes in Linum album (Linaceae). Planta 245:1165–1178CrossRefPubMedGoogle Scholar
  17. Kobori M, Yang Z, Gong D, Heissmeyer V, Zhu H, Jung YK, Gakidis MA, Rao A, Sekine T, Ikegami F, Yuan C, Yuan J (2004) Wedelolactone suppresses LPS-induced caspase-11 expression by directly inhibiting the IKK complex. Cell Death Differ 11:123–130CrossRefPubMedGoogle Scholar
  18. Kundu S, Salma U, Ali MN, Mandal N (2017) Factors influencing large scale micropropagation of Sphagneticola calendulacea (L.) Pruski and clonality assessment using RAPD and ISSR markers. In Vitro Cell Dev Biol Plant 53:167–177CrossRefGoogle Scholar
  19. Li W, Sixiang Z, Zhijian G (2002) In vitro culture of tetraploids of Aloe vera L. Acta Hortic Sin 29:176–178Google Scholar
  20. Madani H, Hosseini B, Dehghan E, Rezaei-chiyaneh E (2015) Enhanced production of scopolamine in induced autotetraploid plants of Hyoscyamus reticulatus L. Acta Physiol Plant 37:55CrossRefGoogle Scholar
  21. Majdi M, Karimzadeh G, Malboobi MA, Omidbaigi R, Mirzaghaderi G (2010) Induction of tetraploidy to feverfew (Tanacetum parthenium Schulz-Bip.): morphological, physiological, cytological, and phytochemical changes. HortScience 45:16–21Google Scholar
  22. Malladi A, Hirst PM (2010) Increase in fruit size of a spontaneous mutant of ‘Gala’ apple (Malus × domestica Borkh.) is facilitated by altered cell production and enhanced cell size. J Exp Bot 61:3003–3013CrossRefPubMedPubMedCentralGoogle Scholar
  23. Moghbel N, Borujeni MK, Bernard F (2015) Colchicine effect on the DNA content and stomata size of Glycyrrhiza glabra var. glandulifera and Carthamus tinctorius L. cultured in vitro. J Genet Eng Biotechnol 13:1–6CrossRefGoogle Scholar
  24. Murashige T, Skoog FA (1962) A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol Plant 15:473–497CrossRefGoogle Scholar
  25. Nilanthi D, Yang YS (2013) In vitro induction of octaploid from colchicine-treated tetraploid petiole explants of purple coneflower (Echinacea purpurea L.). Trop Agric Res Ext 16:25–30Google Scholar
  26. Nilanthi D, Chen XL, Zhao FC, Yang YS, Wu H (2009) Induction of tetraploids from petiole explants through colchicine treatments in Echinacea purpurea L. BioMed Res Int.  https://doi.org/10.1155/2009/343485 Google Scholar
  27. Noori SAS, Norouzi M, Karimzadeh G, Shirkool K, Niazian M (2017) Effect of colchicine induced polyploidy on morphological characteristics and essential oil composition of ajowan (Trachyspermum ammi L.). Plant Cell Tissue Organ Cult 1–9Google Scholar
  28. Omidbaigi R, Mirzaee M, Hassani ME, Sedghi Moghadam M (2010) Induction and identification of polyploidy in basil (Ocimum basilicum L.) medicinal plant by colchicine treatment. Int J Plant Prod 4:87–98Google Scholar
  29. Pansuksan K, Sangthong R, Nakamura I, Mii M, Supaibulwatana K (2014) Tetraploid induction of Mitracarpus hirtus L. by colchicine and its characterization including antibacterial activity. Plant Cell Tissue Organ Cult 117:381–391CrossRefGoogle Scholar
  30. Pôças ES, Lopes DV, da Silva AJ, Pimenta PH, Leitão FB, Netto CD, Buarque CD, Brito FV, Costa PR, Noël F (2006) Structure-activity relationship of wedelolactone analogues: structural requirements for inhibition of Na, K-ATPase and binding to the central benzodiazepine receptor. Bioorg Med Chem 14:7962–7966CrossRefPubMedGoogle Scholar
  31. Praça MM, Carvalho CR, Clarindo WR (2009) A practical and reliable procedure for in vitro induction of tetraploid tomato. Sci Hortic 122:501–505CrossRefGoogle Scholar
  32. Ray A, Bhattacharya S (2010) Storage and conversion of Eclipta alba synseeds and RAPD analysis of the converted plantlets. Biol Plant 54:547–550CrossRefGoogle Scholar
  33. Salma U, Kundu S, Ali MN, Mandal N (2017a) An efficient micropropagation protocol for Eclipta alba (L.) Hassk.: an endangered, medicinally important plant. J Crop Weed 13:49–54Google Scholar
  34. Salma U, Kundu S, Mandal N (2017b) Artificial polyploidy in medicinal plants: advancement in the last two decades and impending prospects. J Crop Sci Biotechnol 20:9–19CrossRefGoogle Scholar
  35. Shao JZ, Chen CL, Deng XX (2003) In vitro induction of tetraploid in pomegranate (Punica granatum). Plant Cell Tissue Organ Cult 75:241–246CrossRefGoogle Scholar
  36. Sharma A, Bhansali S, Kumar A (2013) In vitro callus induction and shoot regeneration in Eclipta alba (l.) Hassk. Life Sci 3:43–46Google Scholar
  37. Tavan M, Mirjalili MH, Karimzadeh G (2015) In vitro polyploidy induction: changes in morphological, anatomical and phytochemical characteristics of Thymus persicus (Lamiaceae). Plant Cell Tissue Organ Cult 122:573–583CrossRefGoogle Scholar
  38. Thakur VD, Mengi SA (2005) Neuropharmacological profile of Eclipta alba (Linn.) Hassk. J Ethnopharmacol 102:23–31CrossRefPubMedGoogle Scholar
  39. Verma AK, Mishra M, Mallick L, Bharati KA, Dash SS, Singh H, Singh P (2016) Database on chromosome counts of plants of Acharya Jagadish Chandra Bose Indian Botanic Garden, Howrah, India. Nelumbo 58:57–78CrossRefGoogle Scholar
  40. Widoretno W (2016) In vitro induction and characterization of tetraploid Patchouli (Pogostemon cablin Benth.) plant. Plant Cell Tissue Organ Cult 125:261–267CrossRefGoogle Scholar
  41. Yan HJ, Xiong Y, Zhang HY, He ML (2016) In vitro induction and morphological characteristics of octaploid plants in Pogostemon cablin. Breed Sci 66:169–174CrossRefPubMedPubMedCentralGoogle Scholar
  42. Yun-Soo K, Eun-Joo H, Hosakatte NM, Kee-Yoeup P (2004) Effect of polyploidy induction on biomass and ginsenoside accumulations in adventitious roots of ginseng. J Plant Biol 47:356–360CrossRefGoogle Scholar
  43. Zhang XY, Hu CG, Yao JL (2010) Tetraploidization of diploid Dioscorea results in activation of the antioxidant defense system and increased heat tolerance. J Plant Physiol 167:88–94CrossRefPubMedGoogle Scholar
  44. Zhang Q, Zhang F, Li B, Zhang L, Shi H (2016) Production of tetraploid plants of Trollius chinensis Bunge induced by colchicine. Czech J Genet Plant Breed 52:34–38CrossRefGoogle Scholar

Copyright information

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

Authors and Affiliations

  1. 1.Department of Agricultural Biotechnology, Faculty of AgricultureBidhan Chandra Krishi ViswavidyalayaMohanpur, NadiaIndia
  2. 2.Faculty Centre for Integrated Rural Development and ManagementRamakrishna Mission Vivekananda University, Ramakrishna Mission AshramaNarendrapur, KolkataIndia

Personalised recommendations