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.

During the last century, plant breeding of crops and ornamentals has become an important business. Mendel’s principles of inheritance of traits laid the foundation of today’s modern genetics. Although intra- and interspecific cross breeding, chromosome doubling and mutation breeding still form the cornerstone of ornamental plant breeding, the speed of plant breeding has significantly increased since Mendel’s time. New genomic tools now offer possibilities for advanced precision breeding and selection towards more difficult to assess or quantitative traits. Today’s ornamental plant breeding is a complicated exercise, characterized by new and rapidly-changing challenges. Partnerships between industry and research will be essential to implement available technologies in minor crops.