Main research question/goal
Can we succesfylly integrate the cytoplasmic male sterility (CMS) technique in the development of new cultivars in model crops celery (fam. Apiaceae) and leek (fam. Liliaceae), with the final aim to produce F1 hybrids? For breeding in both vegetables, hybrid seeds are only partially used, but CMS has not yet been introduced. For breeders and growers, this technique can represent a tool for easier development of stable and uniform races. To obtain CMS plants we need to perform asymmetric fusion of protoplasts (cells without a cell wall). The specific questions that need to be answered are: Can we apply this technique in two major vegetable crop families, namely Apiaceae and Liliaceae? Is it possible to regenerate protoplasts of at least one of the model crops? Can we combine this with either chemical or electrical fusion with a donor protoplast (with fragmented nuclear DNA), and can we establish an efficient screening technique for possible fusion products?
First, we look into the regeneration potential of celery and leek protoplasts. We evaluate the effect of different culture methods, genotypes, source materials and medium compositions. After obtaining these preliminary results, we focus on just one family to actually realize asymmetric somatic hybridization, unless the regeneration capacity of both crops is sufficiently high to justify fusion experiments in both groups. Donor and acceptor protoplast DNA fragmentation will be optimized with UV irradiation and chemicals, respectively. Following, we test all necessary fusion parameters, depending on whether fusion is performed electrically or chemically, and perform the asymmetric fusions. Finally, we control the presence of CMS in all regenerants with phenotypical and genotypical markers.Relevance/Valorisation
We have successfully regenerated protoplasts of green celery, white celery and celeriac. Leek protoplast regeneration did not succeed and therefore we focused on celery. The morphological effects of protoplast regeneration were evaluated. Protocols for protoplast fragmentation (irradiation, chemicals) and protoplast fusion were elaborated. We also developed a molecular screening technique to be able to demonstrate the hybrid character of a plant resulting from fusion. The relevance of these results is important in a specialized marketplace: regeneration of celery protoplasts can be valorized in other research projects (e.g. use of CRISPR in celery) or to introduce variation in the commercial celery assortment. The developed technology also enables the creation of fusion products on a larger scale, which is needed to screen the potential of protoplast fusion for CMS introduction.