In Vitro Cellular & Developmental Biology - Plant

, Volume 55, Issue 6, pp 725–732 | Cite as

In vitro culture and micropropagation of the Baetic-Moroccan endemic plant Lapiedra martinezii Lag. (Amaryllidaceae)

  • Jorge Juan-VicedoEmail author
  • Atanas Pavlov
  • Segundo Ríos
  • Jose Luis Casas
Short Protocol Communication


Lapiedra martinezii Lag. is a potential medicinal and ornamental plant facing conservation challenges. Thus, this study was focused on determining the conditions for culture initiation and propagation using in vitro techniques. The optimal sterilization procedure combined thermotherapy at 54°C for 60 min and immersion in 7% (w/v) Ca(ClO)2 solution for 20 min. The most suitable medium to initiate bulb scales cultures was Gamborg B5 medium containing 500 mg L−1 casein, 2 mg L−1 adenine, 10 mg L−1 glutathione and 10 g L−1 sucrose. The most productive multiplication medium tested was Murashige and Skoog medium containing 30 g L−1 sucrose, 4.0 mg L−1 6-benzylaminopurine, and 0.12 mg L−1 1-naphtaleneacetic acid. Most plants developed in vitro rooted spontaneously in the multiplication phase. The vast majority of the plants (89%) were successfully transferred to ex vitro conditions, and 100% survived over 1 yr of cultivation outdoors. Sucrose at a concentration of 60 g L−1 was the most effective treatment to increase the biomass of bulblets. High auxin/cytokinin ratios produced the highest callus induction efficiency. The vast majority of callus developed in dark conditions, but none regenerated in the combinations of growth regulators previously tested. The plants obtained by micropropagation did not show significant differences in morphometric traits compared with the wild specimens, which supported the stability of the materials produced in vitro. This is the first report on cell cultures and micropropagation of L. martinezii, and the results can be applied to other Amaryllidaceae for industrial or conservation purposes.


Micropropagation Lapiedra Amaryllidaceae Medicinal plants Ornamentals 



The authors would also like to thank Tatiana Gocheva and Andrey Marchev (Institute of Microbiology-Bulgarian Academy of Sciences) for their help in the laboratory, Ana Marco Medina for her critical review of the manuscript draft, and John Paul Duran Rodgers for his linguistic assistance.

Author contribution

JJV developed the concept and performed the experiments, analysis and interpretation of data, as well as drafted the manuscript. AP helped in the work design, SR helped in the fieldwork and JLCM participated in experiments designing, data interpretation and in manuscript writing.

Funding information

This research was supported by a research grant from the University of Alacant program “Becas y Contratos para la formación de Doctores del Vicerrectorado de Investigación de la Universitat d’Alacant FPU2010.”

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflicts of interest.


  1. Aedo C (2010) Lapiedra. Flora Ibérica CLXXXIV (Amaryllidaceae). Accessed 25 Sep 2018
  2. Aslam F, Naz S, Tariq A, Ilyas S, Shahzadi K (2013) Rapid multiplication of ornamental bulbous plants. Pak J Bot 45:2051–2055Google Scholar
  3. Chandra S, Bandopadhyay R, Kumar V, Chandra R (2010) Acclimatization of tissue cultured plantlets: from laboratory to land. Biotechnol Lett 32:1199–1205CrossRefGoogle Scholar
  4. Chen LJ, Yi-Zhu X, Gu L, Wu J (2005) Efficient callus induction and plant regeneration from anther of Chinese narcissus (Narcissus tazetta L. var. chinensis Roem). Plant Cell Rep 24:401–407CrossRefGoogle Scholar
  5. De Klerk GJ (2002) Rooting of microcuttings: theory and practice. In Vitro Cell Dev Biol-Plant. 38:415–422CrossRefGoogle Scholar
  6. De Klerk GJ (2012) Micropropagation of bulbous crops: technology and present state. Floriculture Ornam Biotechnol 6:1–8Google Scholar
  7. Deb CR, Imchen T (2010) An efficient in vitro hardening of tissue culture raised plants. Biotechnol 9:79–83CrossRefGoogle Scholar
  8. Gamborg OL, Miller RA, Ojima K (1968) Nutrient requirements of suspension culture of soybean root cells. Exp Cell Res 50:15–158CrossRefGoogle Scholar
  9. Georgiev V, Ivanov I, Pavlov A (2010) Obtaining and selection of Pancratium maritimum L. in vitro cultures with acetylcholinesterase inhibitory action. Biotechnol Biotechnol Equip 24:149–154CrossRefGoogle Scholar
  10. González-Benito ME, Martin C (2011) In vitro preservation of Spanish biodiversity. In Vitro Cell Dev Biol-Plant 47:46–54CrossRefGoogle Scholar
  11. Hussey G (1982) In vitro propagation of Narcissus. Ann Bot 49:707–719CrossRefGoogle Scholar
  12. Juan-Vicedo J, Fernández-Pereira J, Ríos S, Casas JL, Martín I (2016) Seed germination and storage behaviour of Lapiedra martinezii (Amaryllidaceae). Seed Sci Technol 44:1–8CrossRefGoogle Scholar
  13. Koné M, Koné T, Kouakou HT, Konaté S, Ochatt JS (2013) Plant regeneration via direct shoot organogenesis from cotyledon explants of Bambara groundnut, Vigna subterranea (L.) Verdc. Biotechnol Agron Soc Environ 17:584–592 Google Scholar
  14. Langen-Gerrits MM, de Klerk GJ (1999) Micropropagation of flower bulbs: Lily and Narcissus. Methods Mol Biol 111:141–147Google Scholar
  15. Larsen MM, Adsersen A, Davis AP, Lledó MD, Jäger AK, Rønsted N (2010) Using a phylogenetic approach to selection of target plants in drug discovery of acetilcholinesterase inhibiting alkaloids in Amaryllidaceae tribe Galantheae. Biochem Syst Ecol 38:1026–1034CrossRefGoogle Scholar
  16. Murashige T, Skoog F (1962) A revised medium for rapid growth and bio assays with tobacco tissue cultures. Physiol Plant 15:473–497. CrossRefGoogle Scholar
  17. Pavlov A, Berkov S, Courot E, Gocheva T, Tuneva D, Pandova B, Georgiev M, Georgiev V, Yanev S, Burrus M, Ilieva M (2007) Galanthamine production by Leucojum aestivum in vitro systems. Process Biochem 42:734–739CrossRefGoogle Scholar
  18. Ríos S, Berkov S, Martínez-Francés V, Bastida J (2013) Biogeographical patterns and phenological changes in Lapiedra martinezii Lag. (Amaryllidaceae) related to its alkaloid diversity. Chem Biodivers 10:1220–1238CrossRefGoogle Scholar
  19. Sage DO, Lynn J, Hammatt N (1999) Somatic embryogenesis in Narcissus pseudonarcissus cvs Golden Harvest and St. Keverne. Plant Sci 150:209–216CrossRefGoogle Scholar
  20. Sellés M, Viladomat F, Bastida J, Codina C (1999) Callus induction, somatic embryogenesis and organogenesis in Narcissus confusus: correlation between the state of differentiation and the content of galanthamine and related alkaloids. Plant Cell Rep 18:646–651CrossRefGoogle Scholar
  21. Sochacki D, Orlikowska T (2005) Factors influencing micropropagation of Narcissus. In: Okubo H, Miller WB, Chastagner GA (eds) Proceedings of the IXth International Symposium on Flower Bulbs. Acta Hortic 673 International Society for Horticultural Science, Leuven, BelgiumGoogle Scholar
  22. Squires WM, Langton FA, Fenlon JS (1991) Factors influencing transplantation success of micropropagated Narcissus bulblets. J Hortic Sci 66:661–671CrossRefGoogle Scholar
  23. Telos Rare Bulbs (2018) Lapiedra martinezii. Accessed 20th Oct 2018
  24. Van Staden J, Zazimalova E, George EF (2008) Plant growth regulators II: cytokinins, their analogues and antagonists. In: George EF, Hall MA, de Klerk GJ (eds) Plant propagation by tissue culture, 3rd edn. The Netherlands, Springer, Dordrecht, pp 205–226Google Scholar
  25. Wang QM, Gao FZ, Gao X, Zou FY, Sui X, Wang M, Hui YJ, Wang L (2011) Regeneration of Clivia miniata and assessment of clonal fidelity of plantlets. Plant Cell Tissue Organ Cult 109:191–200CrossRefGoogle Scholar

Copyright information

© The Society for In Vitro Biology 2019

Authors and Affiliations

  • Jorge Juan-Vicedo
    • 1
    • 2
    Email author
  • Atanas Pavlov
    • 3
    • 4
  • Segundo Ríos
    • 1
  • Jose Luis Casas
    • 1
  1. 1.Research Institute of Biodiversity CIBIOUniversitat d’AlacantSant Vicent del RaspeigSpain
  2. 2.Instituto de Investigación en Medio Ambiente y Ciencia Marina IMEDMARUniversidad Católica de ValenciaValenciaSpain
  3. 3.Laboratory of Applied Biotechnologies, Institute of MicrobiologyBulgarian Academy of SciencesPlovdivBulgaria
  4. 4.University of Food TechnologiesPlovdivBulgaria

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