Micropropagation of Helleborus through Axillary Budding

  • Margherita Beruto
  • Serena Viglione
  • Alessandro Bisignano


Helleborus genus, belonging to the Ranunculaceae family, has 20 species of herbaceous perennial flowering plants. The commercial exploitation of this plant is dependent on the selection and propagation of appropriate lines. High propagation rate could be accomplished by using a suitable tissue culture method enabling the rapid introduction of valuable selections in the market. However, in vitro cultivation of Helleborus is still very difficult. Thereby the development of reliable in vitro propagation procedures is crucial for future production systems. Axillary buds cultured on agar-solidified Murashige and Skoog medium supplemented with 1 mg/L benzyladenine, 0.1 mg/L β-naphthoxyacetic acid, and 2 mg/L isopentenyl adenine develop shoots after 16 weeks of culture under 16 h light regime, 50–60 μmol/s/m2, and 19 ± 1°C. The multiplication rate ranges from 1.4 to 2.1. However, the genotype and the number of subcultures affect the efficiency of the micropropagation process. The rooting of shoots is about 80% in solidified MS medium containing 1 mg/L 1-naphthaleneacetic acid and 3 mg/L indole-3-butyric acid. The described protocol provides information which can contribute to the commercial production of Helleborus plants.

Key words

Helleborus Micropropagation Axillary bud stimulation Rooting 


  1. 1.
    Springer P (1995) DE/GA Ausstellungen—IPM. Dtsch Gartenbau 8:477Google Scholar
  2. 2.
    Dan Y, Rothrock RE (2007) Development of an efficient micropropagation system for Helleborus. In Vitro Biology Meeting Abstract Issue p. 2022Google Scholar
  3. 3.
    Lim CC, Kitto SL (1995) Micropropagation of Helleborus orientalis Lam. and Aconitum uncinatum Linn. (Ranunculaceae). HortSci 30:871Google Scholar
  4. 4.
    Seyring M (2002) In vitro cloning of Helleborus niger. Plant Cell Rep 20:895–900CrossRefGoogle Scholar
  5. 5.
    Poupet R, Cardin L, Henri A, Onesto JP (2006) Healthy in vitro propagation by meristem tip culture of Helleborus niger’s selected clone for cut flower. Acta Hortic 725:301–310Google Scholar
  6. 6.
    Dhooghe E, Van Labeke MC (2007) In vitro propagation of Helleborus species. Plant Cell Tissue Organ Cult 91(2):175–177CrossRefGoogle Scholar
  7. 7.
    Beruto M, Curir P (2009) Effects of chilling and hormonal supply on rooting and In vivo establishment of micropropagated plantlets of Helleborus spp. Acta Hortic 812:365–372Google Scholar
  8. 8.
    Colston Burrell C, Knott Tyler J (2006) Hellebores: a comprehensive guide. Timber press, PortlandGoogle Scholar
  9. 9.
    Murashige T, Skoog F (1962) A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol Plant 15:473–497CrossRefGoogle Scholar
  10. 10.
    Quoirin M, Lepoivre P (1977) Étude de milieux adaptés aux cultures in vitro de Prunus. Acta Hortic 78:437–442Google Scholar
  11. 11.
    Jay-Allemand C, Capelli P, Cornu D (1992) Root development of in vitro hybrid walnut microcuttings in a vermiculite-containing gelrite medium. Sci Hortic 15:335–342CrossRefGoogle Scholar
  12. 12.
    Lloyd G, Mc Cown B (1980) Commercially-feasible micropropagation of mountain laurel, Kalima latifolia, by use of shoot tip culture. Pro Intl Plant Prop Soc 30:421–427Google Scholar

Copyright information

© Springer Science+Business Media New York 2012

Authors and Affiliations

  • Margherita Beruto
    • 1
  • Serena Viglione
    • 1
  • Alessandro Bisignano
    • 1
  1. 1.Istituto Regionale per la Floricoltura (IRF)SanremoItaly

Personalised recommendations