Indirect auxiliary organogenesis of Fraxinus excelsior L. as a tool for ash dieback control

The existence of European ash (Fraxinus excelsior L.) is threatened by fungus-induced ash dieback. It is essential to find effective methods to multiply ash genotypes resistant to ash dieback while preserving the genetic diversity of these tree populations. In this paper the efficient method for production of European ash seedlings using indirect auxiliary organogenesis with multi-factor analysis of its effectiveness is presented. Procedures for a dormancy breaking treatment of seeds and effective disinfection of F. excelsior primary explants, as well as appropriate composition of the culture media taking into account impact of growth regulators and physiological gradient on the micropropagation efficiency were developed. As primary explant for micropropagation of F. excelsior, leaf buds, megagametophytes and zygotic embryos were tested. The best-performing type of primary explant for micropropagation of European ash proved to be zygotic embryos, which were successfully used to regenerate seedlings via indirect auxiliary organogenesis. No statistically significant impact of population origin of F. excelsior explant donor trees was observed on the effectiveness of callus initiation. However, such difference was significant in regard to average productivity of acquired callus cultures (number of seedlings produced) and to average root length of regenerated seedlings. Health condition of explant donor trees and their seeds affects the callus initiation rate from zygotic embryos, but does not affect the productivity of callus lines derived from the seeds and the quality of regenerated seedlings. Indirect auxiliary organogenesis of F. excelsior, developed in our study, not only provides the acquisition of ash seedlings of different genotypes, but also enables rapid selection of desired genotypes already at the callus stage. In this way, the presented method benefits not only profit oriented forestry and wood industry, but also provide the effective and fully controllable tool for reintroduction of various resistant to ash-dieback F. excelsior genotypes without loss of variability and genetic identity of its populations.

Automated summary

This study presents an efficient method for producing European ash seedlings through indirect auxiliary organogenesis, with a multi-factor analysis of its effectiveness. The researchers developed procedures for breaking seed dormancy and disinfecting primary explants, as well as determining the appropriate composition of culture media. Zygotic embryos were found to be the best type of primary explant for micropropagation of European ash. This method not only allows for the acquisition of ash seedlings with different genotypes, but also facilitates the rapid selection of desired genotypes at the callus stage.