Embryogenic cultures of Cyclamen coum were established on solid media and in suspension, and their growth characteristics in response to different concentrations of plant growth regulators (PGRs) were evaluated. Embryogenic cultures exhibited a high regeneration capacity of 876 somatic embryos per gram fresh mass. Up to 4.24 9 10 5 protoplasts per gram of fresh mass were isolated from somatic embryos and embryogenic suspension cultures. Protoplasts derived from both embryos and suspension cultures were successfully cultured in vitro and regenerated into plants via somatic embryogenesis. Phenotypic analyses and flow cytometric measurements revealed that some regenerated plants were tetraploid. About 20% of the protoplast-derived calluses used for regeneration were tetraploid, while tetraploidy was found in 0.9% of the plants regenerated from the embryogenic cultures.

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By applying polyethylene glycol (PEG)-mediated protoplast fusion, the first somatic hybrids were obtained between Cyclamen persicum (2n = 2x = 48) and C. coum (2n = 2x = 30)-two species that cannot be combined by cross breeding. Heterofusion was detected by double fluorescent staining with fluorescein diacetate and scopoletin. The highest heterofusion frequencies (of about 5%) resulted from a protocol using a protoplast density of 1 × 10(6)/mL and 40% PEG. The DNA content of C. coum was estimated for the first time by propidium iodide staining to be 14.7 pg/2C and was 4.6 times higher than that of C. persicum. Among 200 in vitro plantlets regenerated from fusion experiments, most resembled the C. coum parent, whereas only 5 plants showed typical C. persicum phenotypes and 46 had a deviating morphology. By flow cytometry, six putative somatic hybrids were identified. A species-specific DNA marker was developed based on the sequence of the 5.8S gene in the ribosomal nuclear DNA and its flanking internal transcribed spacers ITS1 and ITS2. The hybrid status of only one plant could be verified by the species-specific DNA marker as well as sequencing of the amplification product. RAPD markers turned out to be less informative and applicable for hybrid identification, as no clear additivity of the parental marker bands was observed. Chromosome counting in root tips of four hybrids revealed the presence of the 30 C. coum chromosomes and 2-41 additional ones indicating elimination of C. persicum chromosomes.

Somatic embryogenesis in Cyclamen persicum was first reported in 1984 and has potential applications for propagation and breeding of this economically important ornamental crop. This in vitro regeneration system can be used for vegetative propagation of parental lines of F 1 hybrids and elite plants, production of artificial seeds, Agrobacterium tumefaciens-mediated genetic transformation, long-term cryopreservation, protoplast to plant regeneration and somatic hybridization. Somatic embryogenesis was shown to be a powerful propagation system for some C. persicum genotypes, but commercial application in large scale so far is hindered by several limitations, i.e., asynchronous development, malformations or secondary somatic embryogenesis. However, recent molecular approaches by transcriptomic and proteomic analyses were undertaken in order to better understand and control this in vitro regeneration system and to overcome these problems. Our studies aim at comparing somatic embryos to their zygotic counterparts regarding their proteomes. Protein separation by two dimensional isoelectric focusing -sodium do-decyl sulfate polyacrylamide gel electrophoresis led to a resolution of about 1000 protein spots per gel, of which the first 253 were identified by mass spectrometry. Most were found to be involved in glycolysis/gluconeogenesis and stress response pathways. A proteome reference map of zygotic embryos will be publicly released soon and may serve as a basis for further investigations and improvements of somatic embryogenesis.