Establishment of a leaf regeneration system and its molecular basis in Poplar 741.

Poplar 741 has significant ecological and economic value and is widely used. This study developed an efficient regeneration system using sterile Poplar 741 leaves as explants to directly induce adventitious buds. The effects of exogenous hormone concentration (6-Benzylaminopurine: 0, 1.0, and 2.0 mg/L), type, Thidiazuron pre-treatment duration, culture medium type, and leaf position (upper, middle, or lower) on leaf regeneration were investigated. Endogenous hormone content, physiological indices, microscopic observations, and transcriptome sequencing analyses were conducted.Results showed that the highest adventitious bud induction was achieved in Murashige and Skoog medium with 1.0 mg/L 6-BA, 0.05 mg/L indole-3-acetic acid, and 0.01 mg/L TDZ, reaching an 85% adventitious bud adventitious bud induction rate. Upper leaves pre-treated with TDZ for 6 days were most effective. Endogenous hormone content and microscopic properties under different treatments matched the regeneration system. Transcriptomic analyses indicated that differentially expressed genes were enriched in pathways related to phenylpropanoid biosynthesis, starch and sucrose metabolism, and plant hormone signal transduction. Core genes identified were PTI5, LBD1, and WER for 6-BA, TDZ, and leaf position treatments, respectively. Cellular status during pre-culture showed the highest Synthesis phase cell proportion on day 3, with Gap 2/Mitosis phase cells appearing from day 4. Key cell cycle S-phase gene expression peaked on day 3. β-Glucuronidase staining was highest on days 0, 2, and 3. Day 3 of pre-culture was identified as the optimal time for genetic transformation, with a transient transformation efficiency of up to 68%. This study provides a theoretical and technical basis for poplar regeneration and genetic transformation, offering insights for future forestry biotechnology applications.