Optimisation of somatic hybridisation in Cichorium species

Improving the inulin production in the root of industrial chicory by conventional breeding has reached its limit. Therefore, we aimed to increase genetic variability in industrial chicory (Cichorium intybus) to enhance its inulin production. To this end, we executed symmetric protoplast fusions between industrial chicory on the one hand and endive or a wild type Cichorium intybus foliosum on the other hand. In order to produce plantlets, efficient protocols needed to be established for protoplast fusion and regeneration, and for the screening of the putative hybrids.
We created a regeneration protocol by which we can produce plantlets out of protoplasts using low melting point agarose beads surrounded by liquid medium. Two weeks after protoplast isolation, 60% of the initial incubated protoplasts showed microcolony development in the beads. After transfer to solid medium, containing 0.5 mg L-1 IAA and 0.5 mg L-1 BAP, shoots were induced on the developing calli. Three months after protoplast isolation, the first plants were recovered. Protoplasts from different genotypes of C. intybus sativum and C. intybus foliosum could be regenerated.
Symmetric protoplast fusion was performed using PEG-induced, chemical fusion. Industrial chicory (C. intybus sativum), endive (C. endivia crispum) and wild chicory (C. intybus foliosum) were used as fusion partners. Up to 30% of the regenerants after fusion between industrial chicory and endive were hybrids. The fusion of industrial chicory with wild chicory produced on average 4% hybrid regenerants. Microsatellite markers confirmed the hybridity of the putative fusion products.
These findings enable us to produce, fuse and regenerate protoplasts of different Cichorium species in a rapid and efficient way. Moreover, the methods for symmetric protoplast fusion and regeneration presented in this study, can directly contribute to further research on asymmetric protoplast fusion experiments between these three species. This way, CMS could possibly be introduced by combining the nucleus of industrial chicory with the cytoplasm of other Cichorium types.