Benefiting from the past: establishing in vitro culture of European beech (Fagus sylvatica L.) from provenance trial trees and seedlings

Abstract Background European beech (Fagus sylvatica L.) is distributed across diverse climate conditions throughout Europe. Local adaptations, such as drought tolerance, could become crucial for maintaining beech populations facing climate change. In vitro culture offers a promising tool for preserving and propagating valuable genotypes and provides a basis for biotechnological research, although establishing and propagating recalcitrant beech in vitro is difficult. To the best of our knowledge, this study is the first to use beeches from a provenance trial to establish in vitro cultures, aiming to capture a wide genetic spectrum and investigate provenance-specific suitability for in vitro cultivation. In addition, a high-throughput method using seedlings has been developed to increase the success of establishing in vitro cultures of a provenance. Results Actively growing shoots from 22 field-grown provenances were obtained for in vitro establishment. After 12 weeks, shoot formation on shoot tips and nodal segments was induced in 13 provenances (57%), with success rates ranging from 3 to 80%, significantly influenced by the provenance and sampling date of the branches. Combining one harvest each in February and May resulted in the highest shoot formation rate (18%). However, after two years, stable micropropagation was achieved for a single genotype. In the second approach, whole shoots or shoot tips from seedlings were used for in vitro establishment, achieving shoot formation rates between 38 and 94%. Bacterial contamination during establishment was controlled through antibiotic application. Using culture medium without phytohormones improved initial leaf flush on shoot tips within the first 8 weeks of in vitro culture. Phytohormone-supplemented media were needed for shoot multiplication and prolonged in vitro culture. Cultures of 25 genotypes were maintained for up to two years. The viability of in vitro shoots was maintained by supplementing the medium with FeNaEDTA, MgSO4, and glucose. Some genotypes showed enhanced performance on sugar-free media with increased light intensity, which reduced bacterial outgrowth. Conclusion With the technical approaches presented here, we provide starting points for the establishment of beech cultures from various types of starting material, as well as for further method improvement for establishment and long-term cultivation.