An efficient genetic transformation system is imperative for advancing gene functional studies and molecular breeding in horticultural tree species. Traditional Agrobacterium tumefaciens-mediated methods have encountered significant technical challenges when applied to tree species, particularly Liriodendron hybrids. The high costs, and extended transformation cycles associated with this method have substantially limited its utility in gene functional analysis and breeding applications. In response to these challenges, this study presents the development of a streamlined, rapid, and efficient Agrobacterium rhizogenes-mediated transformation system tailored specifically for Liriodendron hybrids, an important ornamental and timber tree species. Among the three Agrobacterium rhizogenes strains (K599, MSU440, and C58C1) evaluated in this study, K599 demonstrated the highest transformation efficiency, reaching 46.09 % for inducing hairy roots from apical bud incisions. Further optimization of the system revealed that 2 days and two-month-old seedlings were the most suitable co-culture duration and explants, yielding a peak transformation efficiency of 60.38 %, 53.12 %, respectively. The applicability of this transformation system was validated across various Liriodendron hybrids genotypes and Liriodendron tulipifera, with transformation efficiencies ranging from 15.47 % to 60.63 %. Additionally, the system was effectively employed for subcellular localization analysis, which confirmed that the aquaporin (AQP) protein. LhAQP1 is localized in the plasma membrane and exhibits enrichment in the vascular tissues of the hairy roots. Notably, this study marks the first application of the Agrobacterium rhizogenes-mediated system for CRISPR/Cas9-mediated gene editing in Liriodendron hybrids, successfully achieving targeted mutagenesis of the LhAQP1 gene and establishing the feasibility of gene editing within this species. In addition, the hairy root-based transformation system was employed to investigate the functional role of LhAQP1 in the improved Liriodendron variety 'Nanlin-Jinsen E1' by comparing wild-type, LhAQP1-overexpressing and gene-edited lines generated via the CRISPR/Cas9. LhAQP1-overexpression significantly promoted plant growth and drought tolerance, whereas pYLCRISPR-LhAQP1 increases dehydration sensitivity, underscoring the essential role of LhAQP1 in water stress adaptation. Optimized transformation platform represents a crucial advancement for functional genomics and molecular breeding efforts in woody plants.
