A submerged culture system for rapid micropropagation of the commercially important aquarium plant, ‘Amazon sword’ (Echinodorus ‘Indian Red’)

  • Sk Moquammel Haque
  • Biswajit GhoshEmail author
Plant Tissue Culture


Echinodorus ‘Indian Red’ is an underwater plant, used worldwide for aquarium ornamentation. An efficient method for in vitro propagation and plantlet acclimatization of this popular aquarium plant was standardized. Surface-disinfected shoot-tips were cultured in submerged conditions in a solid–liquid bilayer medium, consisting of an upper, liquid layer (sterile distilled water) and a lower, solid layer Murashige and Skoog (MS) basal medium supplemented with 3.0% (w/v) sucrose, 0.8% (w/v) agar-agar, and plant growth regulators (PGRs) in different combinations and concentrations. The combination of 2.5 mg L−1 6-benzylaminopurine and 1.0 mg L−1 α-naphthaleneacetic acid improved the multiplication rate to a maximum of 26.8 ± 0.51 shoots per explant after 60 d of culture. The number of multiplied shoots increased with each regeneration cycle, thus from only 26.8 ± 0.51 shoots per explant (first regeneration cycle), this number increased to 33.5 ± 0.58 (second regeneration cycle), and to 38.3 ± 0.62 for the third regeneration cycle with the same medium composition. The highest number of roots (8.3 ± 0.28) per shoot was induced in the presence of 1.0 mg L−1 indole-3-butyric acid, but further growth of these roots was stunted. The best rooting was achieved on PGR-free ½-strength MS medium, where 6.1 ± 0.21 roots per shoot were induced with 5.8 ± 0.35 cm length after 30 d of culture. The regenerated plantlets were successfully acclimatized to submerged underwater conditions, with 100% survival rate. The present protocol is suitable for the commercial propagation of Echinodorus ‘Indian Red’ for aquarium-industries.


Amazon sword Aquarium plant micropropagation Submerged aquatic plant Bilayer culture Echinodorus ‘Indian Red’ 



SMH acknowledges the Ministry of Minority Affairs and the University Grant Commission (UGC) of India for providing Maulana Azad National Fellowship (MANF). The authors acknowledge Swami Kamalasthananda, Principal, Ramakrishna Mission Vivekananda Centenary College, Rahara, Kolkata, India for the facilities provided for this study. Further, the authors are thankful to aquarium hobbyist Mr. Diptarup Das for gifting us initial plant materials and Mr. Anupam Das for providing consent to use his aquariums during acclimatization of the regenerated plants. The DST-FIST program is also acknowledged hereby for infrastructural facilities.


  1. Barth H (1998) Echinodorus plant named ‘Indian Red’, united state patent, patent number: plant 10726. Date of Patent: 15th December 1998Google Scholar
  2. Bolyard M (2018) In vitro regeneration of Artemisia abrotanum L. by means of somatic organogenesis. In Vitro Cell Dev Biol-Plant 54:127–130. CrossRefGoogle Scholar
  3. Brunel S (2009) Pathway analysis: aquatic plants imported in 10 EPPO countries. Eur Mediterr Plant Prot Org Bull 39:201–213. Google Scholar
  4. Dissanayake C, Hettiarachchi M, Iqbal MCM (2007) Sustainable use of Cryptocoryne wendtii and Echinodorus cordifolius in the aquaculture industry of Sri Lanka by micropropagation. Sri Lanka J Aquat Sci 12:89–101 CrossRefGoogle Scholar
  5. Duncan DB (1955) Multiple range and multiple F test. Biometrics 11:1–42CrossRefGoogle Scholar
  6. Haque SM, Ghosh B (2013a) Micropropagation, in vitro flowering and cytological studies of Bacopa chamaedryoides, an ethno-medicinal plant. Env Exp Biol 11:59–68 Google Scholar
  7. Haque SM, Ghosh B (2013b) High frequency microcloning of Aloe vera and their true-to-type conformity by molecular cytogenetic assessment of two years old field growing regenerated plants. Bot Stud 54:46 CrossRefPubMedPubMedCentralGoogle Scholar
  8. Haque SM, Ghosh B (2016) High-frequency somatic embryogenesis and artificial seeds for mass production of true-to-type plants in Ledebouria revoluta: an important cardioprotective plant. Plant Cell Tissue Organ Cult 127:71–83. CrossRefGoogle Scholar
  9. Haque SM, Paul S, Ghosh B (2016) High-frequency in vitro flowering, hand-pollination and fruit setting in ten different cultivars of Capsicum spp. (C. annuum, C. chinense, and C. frutescens): an initial step towards in vitro hybrid production. Plant Cell Tissue Organ Cult 127:161–173. CrossRefGoogle Scholar
  10. Jabir T, George S, Raj A, Sree Lakshmi S, Joseph A (2016) Micropropagation and in vitro flowering of an ornamental aquarium plant Lindernia antipoda L. (Alston). Int J Aquacult 6:8 Google Scholar
  11. Jenks MA, Kane ME, McConnell DB (2000) Shoot organogenesis from petiole explants in the aquatic plant Nymphoides indica. Plant Cell Tissue Organ Cult 63:1–8. CrossRefGoogle Scholar
  12. Kam MYY, Chai LC, Chin CF (2016) The biology and in vitro propagation of the ornamental aquatic plant, Aponogeton ulvaceus. SpringerPlus 5:1657. CrossRefPubMedPubMedCentralGoogle Scholar
  13. Kane ME, Gilman EF, Jenks MA, Sheehan TJ (1990) Micropropagation of the aquatic plant Cryptocoryne lucens. HortSci 25:687–689 CrossRefGoogle Scholar
  14. Lehtonen S, Falck D (2011) Watery varieties: aquarium plant diversity from aesthetic, commercial, and systematic prospective. In: Aquino JC (ed) Ornamental plants: types, cultivation and nutrition. Nova Science Publishers, New York, pp 1–46Google Scholar
  15. Lunardi RF, Wohlenberg M, Medeiros N, Agostini F, Funchal C, Dani C (2014) In vitro antioxidant capacity of tea of Echinodorus grandiforus, “leather hat,” in Wistar rat liver. An Acad Bras Ciênc 86:1451–1461. CrossRefPubMedGoogle Scholar
  16. Marques AM, Provance DW Jr, Kapla MAC, Figueiredo MR (2017) Echinodorus grandiflorus: Ethnobotanical, phytochemical and pharmacological overview of a medicinal plant used in Brazil. Food Chem Toxicol 109(Pt 2):1032–1047 CrossRefPubMedGoogle Scholar
  17. Murashige T, Skoog F (1962) A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol Plant 15:495–497. CrossRefGoogle Scholar
  18. Öztürk M, Khawar KM, Atar HH, Sancak C, Özcan S (2004) In vitro micropropagation of the aquarium plant Ludwigia repens. Asia Pacific J Mol Biol Biotechnol 12:21–25Google Scholar
  19. Ridings WH, Zettler FW (1973) Aphanomyces blight of Amazon sword plants. Phytopathology 63:289–295. CrossRefGoogle Scholar
  20. Rieder A, Figueiredo GC, Bonilla MG (2011) Plant known as “Leather hat” (Echinodorus spp.) (Alismataceae) and its medicinal use in southwestern Mato Grosso, Brazil. Planta Med 77:PF86. Google Scholar
  21. Sahai A, Shahzad A (2013) High frequency in vitro regeneration system for conservation of Coleus forskohlii: a threatened medicinal herb. Acta Physiol Plant 35:473–481. CrossRefGoogle Scholar
  22. Sheeja GE, Joseph A, Korath A (2015) In vitro propagation of an ornamental aquatic plant, Anubias barterii Var. Nana petite. Int J Curr Sci 18:E1–E12 Google Scholar
  23. Shekhawat MS, Manokari M, Revathi J (2017) In vitro propagation, micromorphological studies and ex vitro rooting of Alternanthera philoxeroides (Mart.) Griseb.: an important aquatic plant. Aquacult Int 25:423–435. CrossRefGoogle Scholar
  24. Shibayama Y, Kadono Y (2007) Reproductive success and genetic structure of populations of the heterostylous aquatic plant Nymphoides indica (L.) Kuntze (Menyanthaceae). Aquat Bot 86:1–8. CrossRefGoogle Scholar
  25. Winkelmann T, Geier T, Preil W (2006) Commercial in vitro plant production in Germany. Plant Cell Tissue Organ Cult 86:319–327. CrossRefGoogle Scholar

Copyright information

© The Society for In Vitro Biology 2018

Authors and Affiliations

  1. 1.Plant Biotechnology Laboratory, Post Graduate Department of BotanyRamakrishna Mission Vivekananda Centenary CollegeKolkataIndia

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