Title: Transcriptome-assisted optimization of adventitious bud induction from field-grown stem segments to enhance genetic transformation of Poplar 107 (Populus × euramericana cv. Neva)

Efficient genetic transformation of poplar is a prerequisite for gene function studies and variety improvement. Poplar 107 ( Populus × euramericana cv. Neva ), widely planted in the North China Plain, is hampered by poor regeneration, long transformation cycles, and low efficiency. To address this, our study first optimized adventitious bud induction from field-grown, semi-lignified stems. Optimal adventitious bud induction and rooting were achieved using disinfected, 0.5–1.0 cm semi-lignified middle stem segments (SLMS) on Murashige and Skoog (MS) medium with 0.5 mg/L 6-Benzylaminopurine (6-BA) and 0.15 mg/L Indole-3-Acetic Acid (IAA) and on 1/2 MS medium with 0.5 mg/L Indole-3-Butyric Acid (IBA), respectively. Transcriptome analysis revealed that growth-regulating factors (GRFs) synergized with brassinosteroid (BR) biosynthesis to drive active cell division in the CM group (SLMS treated with 0.5 mg/L 6-BA and 0.15 mg/L IAA). This molecular mechanism was consistent with the adventitious bud induction phenotype, which underpinned the successful regeneration of transformed stems. Based on these findings, we established a protocol for SLMS that achieved the highest resistant bud induction rate through a 1-day pre-culture on MS medium with 0.5 mg/L 6-BA and 0.15 mg/L IAA, followed by 15-min infection (OD 600 = 0.6), 3-day co-culture, and screening on MS differentiation medium with 40 mg/L kanamycin. Application of this protocol to three independent transformations with different vectors yielded a transformation efficiency of 6.67 %–13.33 % by polymerase chain reaction (PCR) validation. The established system constitutes a robust platform for genetic manipulation in Poplar 107, paving the way for accelerated gene function studies and molecular breeding in woody plants. • 0.5–1.0 cm semi-lignified middle stem segments (SLMS) show optimal adventitious bud induction. • SLMS treated with 0.5 mg/L 6-BA and 0.15 mg/L IAA exhibit strong cellular activity. • GRF-brassinosteroid module drives adventitious bud induction. • A robust Agrobacterium -mediated transformation protocol for semi-lignified field-grown Poplar 107 was developed. • A novel transformation framework enables accelerated functional genomics and breeding.