Stable in vitro propagation of Fagus sylvatica using micro-cuttings established from seedlings

The objective of this study was to establish a reliable protocol for the in vitro multiplication of Fagus sylvatica L. shoots. This species is increasingly threatened by pathogen pressure and drought stress, making in vitro propagation a potentially valuable tool for breeding and resistance research. Although protocols for in vitro culture of F. sylvatica have been reported, they do not ensure stable propagation, which is an essential prerequisite for studies on resistance to biotic and abiotic stress. In our study, mature seeds from seven provenances in western and southern Poland were germinated in soil, sterilized, and established in vitro on semi-solid Woody Plant Medium. During the establishment phase, cultures contained a mixture of genotypes from the different provenances, which were later identified by microsatellite analysis and assigned to single multilocus genotypes. For the multiplication phase, shoots were cultured with different phytohormones and carbon sources (sucrose or glucose), and shoot length and multiplication rates were assessed. A second experiment compared semi-solid medium in Erlenmeyer flasks with liquid medium in Temporary Immersion System bioreactors. Multiplication rates varied among provenances, with the highest rates achieved when shoots were cultured on Woody Plant Medium supplemented with zeatin and propagated in Temporary Immersion System bioreactors. These findings demonstrate that stable in vitro propagation of F. sylvatica is possible and provide a foundation for future breeding and resistance studies. This study documents successful Fagus sylvatica establishment and stable propagation in vitro. We maintained consistent multiplication rates in vitro, marking a significant advancement in the sustainable cultivation of F. sylvatica.