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The genus Helleborus comprises 22 species, which are allocated to six Helleborus sections. Helleborus species show differences with regard to leaf and flower morphology, scent, and sensitivity to different pests and diseases. Breeding programs aiming at these traits require the inclusion of a broader spectrum of Helleborus species in addition to the most popular species H. niger (Christmas Rose) and H. A— hybridus (Lenten Rose), thus interspecific hybridisations need to be performed. Pollen viability was estimated for nine species by staining with thiazolyl blue (MTT) and varied between 67 and 99% for fresh pollen and between 74 and 99% for pollen dried for 24 h. Crossing barriers had previously been localized in Helleborus species as predominantly post-zygotic. Therefore, embryo rescue techniques via ovule culture were established to overcome these barriers. Ovules were isolated from the maternal plants five to seven weeks after pollination and then cultured in vitro. Overall, from 661 crosses involving 13 Helleborus species, 40,885 ovules were obtained. In a combination with parents of low genetic distances between the parental species, up to 26.08% of these ovules produced hybrid shoots, whereas in intersectional crosses this was the case in only 0.17 to 0.85% of the ovules. In total, 217 hybrid offspring were verified by flow cytometry and molecular marker analysis (random amplified polymorphic DNAs), whereof 16 were derived from parental species belonging to different Helleborus sections. Hybrid plantlets of some cross combinations were transferred to the greenhouse and expressed intermediate leaf and flower morphology when compared to the parental species.

Chromosome doubling is a frequently used strategy in breeding programmes. In ornamentals now mostly in vitro protocols are used for polyploidisation, as good micropropagation protocols have already been established for many plant species. Tissue culture protocols offer a broader pallet of methods that can be evaluated for polyploidisation as compared with the in vivo applications of antimotitotic agents. Here we give an overview on in vitro polyploidisation methods with practical examples on herbaceous and woody ornamental species: Helleborus, Ranunculus, Spathiphyllum, Buddleja and Rhododendron.

The genus Helleborus comprises 22 species which are allocated to six sections. Due to high intraspecific morphological variability, taxonomic and genetic relationships are ambiguous especially within section Helleborastrum. In order to investigate taxonomic and genetic relationships in the genus Helleborus, flow cytometry and amplified fragment length polymorphisms were used. Nuclear DNA contents of 21 analyzed species varied between 18.3 pg/2C for H. argutifolius and 32.7 pg/2C for H. thibetanus. Genome sizes between 26.8 and 31.3 pg/2C were determined for the majority of species. The genetic relationships within the genus were analyzed using ten AFLP primer combinations for 19 out of 22 Helleborus species. On the basis of 1109 polymorphic fragments across all species and the outgroup Pulsatilla vulgaris 'Violet', genetic distances were determined to conduct neighbor-joining cluster and bootstrap analysis to calculate a majority rule consensus tree. The six Helleborus sections were clearly definable in the phenogram that thereby supports the present genus division.

In order to establish protocols for embryo rescue techniques, in a first step this study aimed at the identification of culture conditions for ovules from intraspecific crosses. The effects of dissection date after pollination and temperature during ovule culture were examined in intraspecific crossings of Helleborus niger, H. argutifolius, H. × hybridus and H. foetidus. Ovaries were harvested 3-6 weeks after pollination, surface disinfected in 70% ethanol for 30 s, 2% sodium hypochlorite with one drop Tween for 10 min and rinsed in sterilised water three times. Ovules were dissected from ovaries and cultured on medium based on MS solidified with 0.4% Gelrite at a pH of 5.8. Two media supplemented with 2.5 or 5% sucrose were compared. Ovules were cultured in darkness at 24±1 or 16±1°C for 12 weeks. Thereafter, ovules of each temperature treatment were split and one half was incubated at 6±1°C for 11 weeks, while the other half remained in the initial temperature. Afterwards the ovules were placed back to their initial temperature. On average of all species, 2% of the ovules dissected 3 weeks and 1% dissected 4 weeks after pollination germinated, while preparation after 5 weeks resulted in 4% and after 6 weeks in 8% germination, respectively. The intermediate cold treatment with 6°C turned out to be very beneficial for later germination. Furthermore, no effect of the media supplemented with either 2.5 or 5% sucrose was observed.