Effect of Medium Composition and Culture Conditions on the In Vitro Multiplication, Rooting and Acclimatization of Helleborus ‘Molly’s White’ and ‘Anna’s Red’

The genus Helleborus belongs to the family Ranunculaceae and comprises about 22 species, which are distributed across Europe and West Asia. Hellebores are popular as garden perennials, pot plants, cut flowers, and medicinal plants. The present study aimed to evaluate the effects of sucrose level (20, 30, 40, and 50 g·L⁻¹), mineral salts content (50%, 100%, 150% of Murashige and Skoog 1962 medium – MS), growth regulators (BAP – 6-benzylaminopurine, 2iP – 6-(γ,γ-dimethylallylamino) purine, kinetin, GA₃ – gibberellic acid, IBA – indole-3-butyric-acid), and white light sources (fluorescent and LED) for multiplication and in vitro rooting Helleborus of ‘Molly’s White’ and ‘Anna’s Red’. Additionally, the post-effect of white, blue, and red light (LED) used during the storage of shoots at a temperature of 15°C for 3 months in glass jars and plastic vessels on the subsequent in vitro rooting and on ex vitro acclimatization was investigated. The results presented here showed that sucrose and nitrogen salts were more critical for axillary shoot branching and subsequent in vitro rooting in both hellebore genotypes than the concentration of growth regulators in the multiplication and rooting media. Low level of sucrose (20 g·L⁻¹) and standard content of mineral salts (100% in the MS medium) were optimal for the development of axillary shoots. The medium composition used in the multiplication experiments markedly affected the rooting ability of both cultivars. The sucrose/nitrogen salts ratio in the multiplication medium was more critical for subsequent rooting than the type and concentration of growth regulators or the composition of the rooting media. Genotype ‘Molly’s White’ exhibited the best rooting after multiplication on the medium with high (40 g·L⁻¹) and low (20 g·L⁻¹) sucrose levels, but ‘Anna’s Red’ only on the medium with low sucrose content (20 g·L⁻¹). The white LED increased the multiplication and rooting rates of both hellebores compared with white fluorescent light. Plastic vessels significantly increased root number in both hellebore genotypes. The post-effect of white, blue, and red light (LED) used during the storage of cultures had a weak influence on ex vitro acclimatization compared to in vitro rooting. Genotypic differences in multiplication rate, rooting ability, and ex vitro acclimatization efficiency were observed.