Establishment of the Fraxinus mandshurica gene speedy editing technology system by using the Agrobacterium-mediated transformation method and identification of the drought resistance function of the FmbHLH1 gene

The lack of an effective genetic transformation system for Fraxinus mandshurica restricts the study of its gene functions, and improving its traits through genetic engineering methods is challenging. We here employed an Agrobacterium-mediated efficient transient transformation system, with the growing points of F. mandshurica as explants. By using a combination of different Agrobacterium concentrations and infection times, we established an efficient genetic transformation system for the growing points. Furthermore, a tissue culture system was developed for clustered buds by adding different hormones with varying concentrations. Accordingly, FmbHLH1 gene-edited chimeric plants were generated through the CRISPR target cutting efficiency assay by using the developed genetic transformation system for growth points. Homozygote plants were screened using the developed clustered bud system. Among 100 randomly transformed growing points, 18 chimeras were obtained, which further yielded 12 homozygotes, indicating an editing efficiency of 18%, which confirmed that the fabricated gene editing technology system was efficient. On analyzing the phenotypes and drought resistance physiological indicators of FmbHLH1 knockout and wild-type lines, we inferred that FmbHLH1 is positively correlated with peroxidase and superoxide dismutase activities. In addition, we found that the gene positively regulates the drought resistance of F. mandshurica by adjusting its ability to scavenge reactive oxygen species and osmotic potential. This study developed an efficient Agrobacterium-mediated gene editing technology system for F. mandshurica, using which the drought resistance gene FmbHLH1 was identified. This study offers an effective method and candidate resistance genes for the molecular breeding of F. mandshurica.