Establishment of a CRISPR/Cas9 gene editing system based on growth points transformation method in Fraxinus...

The lack of an effective gene editing technology system for Fraxinus mandshurica makes it challenging to improve its traits through genetic engineering methods. In this study, an effective CRISPR/Cas9 gene editing system targeting plant growth points was established through the optimization of Agrobacterium tumefaciens concentration and infection duration. Furthermore, a tissue culture system for clustered buds was developed by supplementing the media with hormones at different concentrations. FmbHLH1-edited chimeric plants were successfully generated using the developed CRISPR/Cas9 gene editing system. Homozygous plants were induced and screened using the developed clustered bud system. Among 100 randomly transformed growing points, 18% of the induced clustered buds were gene-edited, which confirmed that the established CRISPR/Cas9 gene editing system was effective. Phenotypic analysis and evaluation of drought tolerance-related physiological indicators in FmbHLH1 knockout and wild-type lines revealed that FmbHLH1 positively regulated the drought tolerance of F. mandshurica by adjusting its ability to scavenge reactive oxygen species and to regulate osmotic potential. In summary, we developed an effective CRISPR/Cas9 gene editing system for F. mandshurica, providing an effective method for the molecular breeding of F. mandshurica.