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Pelargonium is a versatile genus mainly from the Cape Region, South Africa. The genus is divided into four subgenera and 16 sections characterized by several groups of chromosomes sizes and numbers. The DNA content of species from all subgenera and sections of Pelargonium, except for the sections Subsucculentia and Campylia was estimated using flow cytometry. Nuclei of Pelargonium samples (leaf or petal tissue) and an internal plant standard (leaf tissue) were isolated together and stained with propidium iodide. The DNA content was estimated providing that the 2C peaks of sample and standard be in linearity in the flow cytometer histograms. In total, 96 Pelargonium accessions of 60 species (22 Pelargonium species for the first time) were analyzed. The 2C DNA content ranged from 0.84 pg (P. longifolium, section Hoarea) to 6.69 pg (P. schizopetalum, section Magnistipulacea) and the corresponding 1Cx DNA content from 0.42 pg (P. longifolium) to 1.72 pg (P. transvaalense. This demonstrates the high plasticity within the genus Pelargonium. Some species, such as P. peltatum accessions revealed a pronounced endopolyploidization in leaves but not in petals underlining the importance to choose the right tissue as sample for the flow cytometry analysis. The reported genome sizes are a step forward towards the characterization of the Pelargonium collection within the German Gene Bank for Ornamental Plants and a valuable base for future sequencing programs of the Pelargonium genomes.

Modern Pelargonium crispum hybrids (section Pelargonium) show low genetic and phenotypic variation due to the domestication effect. Species of the sections Cortusina, Ligularia, and Pelargonium are potential breeding partners at the diploid level (2n = 2x = 22). Five P. × crispum cultivars were used as seed parents and pollinated with one genotype of P. grandiflorum (section Pelargonium) and three genotypes of P. fulgidum (section Ligularia). In both combinations, embryo rescue was necessary. Embryos were rescued and cultured on Murashige & Skoog medium supplemented with phytohormones. After callus and adventitious shoot regeneration 15 viable interspecific hybrids were obtained from crossbreeding with P. grandiflorum and 11 intersectional hybrids from crossings with P. fulgidum, respectively. The hybrids were cultivated in the greenhouse until flowering. Their hybrid character was evident due to the intermediate morphological traits. Molecular investigations using dp-RAPD analysis confirmed this. Within the F1 population P. × crispum with P. grandiflorum three hybrids and after crossing with P. fulgidum one hybrid possessed larger flowers and fully developed anthers, respectively. Their ploidy level was confirmed as tetraploid using flow cytometry. Therefore, a spontaneous polyploidisation occurred during in vitro regeneration. The tetraploid F1 hybrids are fertile and could be used for further breeding.

Seven Pelargonium × crispum cultivars, four breeding clones and the species P. crispum were analysed by flow cytometry. Tomato ‘Stupické’ (2C value = 1.96 pg) and cauliflower ‘Korso’ (2C value = 1.31 pg) were used as inter­nal standards to estimate the DNA content of the samples. As expected, the 2C values of the investigated diploid and tetraploid genotypes discriminated significantly. Overall, the mean 2C values ranged from 0.97 to 2.18 pg DNA and formed three significantly different groups. The estimation of the DNA content of the cultivars and breeding clones was independent of the standard used with one exception; for P. crispum the estim­ated DNA contents differed significantly. The standards ‘Stupické’ and ‘Korso’ are equally appropriate for flow cyto­metric investigation of genotypes with 2C values < 1.8 pg like diploid genotypes of the section Pelargonium. Tomato ‘Stupické’ is a rather impractical standard for geno­types with 2C values of about 2 pg, due to the overlapping positions of the 2C values of sample and standard.

Pelargonium acetosum L’Hér. is a wild species from South Africa with decorative bluish foliage. Only few reports describe crossings between P. acetosum and P. peltatum L’Hér. (or P. ×peltatum). Therefore, information about hybridization barriers is limited. In this study, two different genotypes of Pelargonium acetosum (AC1 and AC2) were crossed with the diploid P. ×peltatum ‘Tornado Fuchsia’ (PTF). Embryos and F1 hybrids from the combination AC1 × PTF were hampered by chlorophyll deficiencies. Embryos and seeds of the combination AC2 × PTF were underdeveloped. The reciprocal combination PTF × AC1 did not show any fruit set. The combination PTF × AC2 resulted in low numbers of seeds, which were normally developed. Hybrids from seeds were only obtained from the combinations AC1 × PTF and PTF × AC2. Embryo rescue of the combinations AC1 × PTF and AC2 × PTF resulted in few but viable hybrids. Flowers of all hybrids had shrivelled anthers and proved to be sterile. The occurrence of most hybridization barriers varied strongly between the different combinations and depended on both the genotype and the direction of cross-breeding. The bluish leaf colour did not appear among the F1. To overcome hybrid sterility a polyploidization is suggested.

Blue Angelonia × angustifolia flowers can show spontaneous mutations resulting in white/blue and white flower colourations. In such a white line, a loss of dihydroflavonol 4-reductase (DFR) activity was observed whereas chalcone synthase and flavanone 3-hydroxylase activity remained unchanged. Thus, cloning and characterization of a DFR of Angelonia flowers was carried out for the first time. Two full length DFR cDNA clones, Ang.DFR1 and Ang.DFR2, were obtained from a diploid chimeral white/blue Angelonia × angustifolia which demonstrated a 99% identity in their translated amino acid sequence. In comparison to Ang.DFR2, Ang.DFR1 was shown to contain an extra proline in a proline-rich region at the N-terminus along with two exchanges at the amino acids 12 and 26 in the translated amino acid sequence. The recombinant Ang.DFR2 obtained by heterologous expression in yeast was functionally active catalyzing the NADPH dependent reduction of dihydroquercetin (DHQ) and dihydromyricetin (DHM) to leucocyanidin and leucomyricetin, respectively. Dihydrokaempferol (DHK) in contrast was not accepted as a substrate despite the presence of asparagine in a position assumed to determine DHK acceptance. We show that substrate acceptance testing of DFRs provides biased results for DHM conversion if products are extracted with ethyl acetate. Recombinant Ang.DFR1 was inactive and functional activity could only be restored via exchanges of the amino acids in position 12 and 26 as well as the deletion of the extra proline. E. coli transformation of the pGEX-6P-1 vector harbouring the Ang.DFR2 and heterologous expression in E. coli resulted in functionally active enzymes before and after GST tag removal. Both the GST fusion protein and purified DFR minus the GST tag could be stored at -80°C for several months without loss of enzyme activity and demonstrated identical substrate specificity as the recombinant enzyme obtained from heterologous expression in yeast.