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In vitro polyploidisation of Helleborus species


Helleborus species are members of the family of the Ranunculaceae. These popular perennials are all diploids (2n = 2x = 32). This study investigates polyploidy induction by different antimitotic agents. Colchicine, oryzalin and trifluralin were tested in vitro on shoots of Helleborus niger, H. orientalis and H. × nigercors. Furthermore the effect of the antimitotic agents on the viability and the multiplication rate of cultured plantlets were analyzed. Flow cytometry demonstrated that polyploidisation was genotype dependent: using H. niger, tetraploids were obtained using either oryzalin (3 μM) or trifluralin (3 or 10 μM), whereas for H. × nigercors only trifluralin (3 or 10 μM) induced polyploidisation. For H. orientalis neither treatment was effective to produce tetraploids or mixoploids. For these three species, colchicine (100 μM) was ineffective. The polyploidisation events in H. niger and H. × nigercors were confirmed by chromosome counts of mounted nuclei derived from root tips (2n = 4x = 64).

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Indole-3-butyric acid


1-Naphtaleneacetic acid


Naphthoxyacetic acid


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The authors thank the research center ILVO - Plant Unit (Melle-Belgium) and especially H. Carlier for the flow cytometric measurements. Special thanks also go to T. Versluys for the technical supports and to Dr. Ryan Whitford for critical reading of the manuscript.

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Correspondence to Emmy Dhooghe.

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Dhooghe, E., Grunewald, W., Leus, L. et al. In vitro polyploidisation of Helleborus species. Euphytica 165, 89–95 (2009).

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  • Colchicine
  • Flow cytometry
  • Helleborus
  • Oryzalin
  • Polyploidisation
  • Ranunculaceae
  • Trifluralin