A RUBY-Based Visual Hairy Root Transformation and CRISPR-PTG Genome-Editing System in Eucommia ulmoides

Eucommia ulmoides Oliv. is a woody plant with significant medicinal and industrial value. However, the lack of an efficient genetic transformation and gene-editing system has seriously hindered the research on its functional genes and the progress of molecular breeding. To break through this technical bottleneck, this study successfully established an efficient Agrobacterium-mediated genetic transformation system for E. ulmoides hairy roots based on the RUBY visual reporter gene. Through systematic optimization, the optimal transformation conditions were identified as infecting hypocotyl explants with Agrobacterium strain K599 at an OD600 of 0.6 for 20 min. Based on this optimized system, a RUBY-based CRISPR-PTG (polycistronic tRNA-gRNA) construct was further generated. With squalene synthase (EuSQS) as the target gene, Sanger sequencing and ICE (Inference of CRISPR Edits) analysis provided preliminary evidence for CRISPR-PTG-mediated editing at the EuSQS locus in transgenic hairy roots, although the potentially chimeric nature of hairy roots may contribute to variation in the observed editing profiles. In this study, a RUBY-based visual hairy root genetic transformation system was established, which provided preliminary evidence that CRISPR-PTG-mediated targeted mutagenesis can occur in E. ulmoides hairy roots. These results provide a useful basis for further optimization and application of genome editing in subsequent gene function analysis and metabolic engineering research of E. ulmoides, and also offer a reference for genetic transformation studies of other woody plants.