Azalea (Rhododendron simsii hybrids) is the most important flowering pot plant produced in Belgium, being exported world-wide. In the breeding program, flower color is the main feature for selection, only in later stages cultivation related plant quality traits are evaluated. As a result, plants with attractive flowering are kept too long in the breeding cycle. The inheritance of flower color has been well studied; information on the heritability of cultivation related quality traits is lacking. For this purpose, QTL mapping in diverse genetic backgrounds appeared to be a must and therefore 4 mapping populations were made and analyzed.

Flower colour variation is one of the most crucial selection criteria in the breeding of a flowering pot plant, as is also the case for azalea (Rhododendron simsii hybrids). Flavonoid biosynthesis was studied intensively in several species. In azalea, flower colour can be described by means of a 3-gene model. However, this model does not clarify pink-coloration. The last decade gene expression studies have been implemented widely for studying flower colour. However, the methods used were often only semi-quantitative or quantification was not done according to the MIQE-guidelines. We aimed to develop an accurate protocol for RT-qPCR and to validate the protocol to study flower colour in an azalea mapping population. An accurate RT-qPCR protocol had to be established. RNA quality was evaluated in a combined approach by means of different techniques e.g. SPUD-assay and Experion-analysis. We demonstrated the importance of testing noRT-samples for all genes under study to detect contaminating DNA. In spite of the limited sequence information available, we prepared a set of 11 reference genes which was validated in flower petals; a combination of three reference genes was most optimal. Finally we also used plasmids for the construction of standard curves. This allowed us to calculate gene-specific PCR efficiencies for every gene to assure an accurate quantification. The validity of the protocol was demonstrated by means of the study of six genes of the flavonoid biosynthesis pathway. No correlations were found between flower colour and the individual expression profiles. However, the combination of early pathway genes (CHS, F3H, F3'H and FLS) is clearly related to co-pigmentation with flavonols. The late pathway genes DFR and ANS are to a minor extent involved in differentiating between coloured and white flowers. Concerning pink coloration, we could demonstrate that the lower intensity in this type of flowers is correlated to the expression of F3'H. Currently in plant research, validated and qualitative RT-qPCR protocols are still rare. The protocol in this study can be implemented on all plant species to assure accurate quantification of gene expression. We have been able to correlate flower colour to the combined regulation of structural genes, both in the early and late branch of the pathway. This allowed us to differentiate between flower colours in a broader genetic background as was done so far in flower colour studies. These data will now be used for eQTL mapping to comprehend even more the regulation of this pathway.

Celeriac F₁ hybrid seed production is currently complicated due to the instability of cytoplasmic male sterile lines. To develop alternative alloplasmic CMS lines, an asymmetric protoplast fusion and hybrid screening methodology was established. Celeriac suspension cells protoplasts were used as the acceptor and carrot, coriander and white celery mesophyll protoplasts as the donor for protoplast fusion experiments. Acceptor cytoplasmic inheritance was inhibited by iodoacetamide treatment and donor nuclear genome inheritance was prevented by UV exposure. Protoplasts were selectively stained and fused using electroporation and polyethylene glycol, and candidate hybrid shoots were obtained. One chloroplast and three mitochondrial markers that could distinguish acceptor and donors organelles were used to characterize over 600 plants obtained after fusion events, without identifying any cybrid. In order to increase the testing efficiency a high number of micro plantlets were pooled and hence the presence of the carrot specific Atp1 marker in one of the pooled samples was detected. We demonstrated that fusion took place between celeriac and a carrot indicating that the creation of viable hybrids is possible although at a very low frequency. These findings open the path for new cytoplasmic hybridization and the isolation of novel CMS lines of celeriac.