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Transgenic technology is a potent tool for verifying gene functions, and poplar serves as a model system for genetically transforming perennial woody plants. However, the current poplar genetic transformation system is limited to a few genotypes. In this study, we developed an efficient transformation system based on the <i>Agrobacterium</i>-mediated transformation of <i>Populus wulianensis</i>, a rare and endangered tree species endemic to Shandong Province. Aseptic seedlings of <i>P. wulianensis</i> were used as experimental materials, and the optimal medium for inducing adventitious buds was explored as 1/2(NH₄NO₃) MS + 0.05 mg/L naphthalene acetic acid (NAA) + 0.5 mg/L 6-benzylaminopurine (6-BA), resulting in up to 35 adventitious buds. The selection resistance critical pressure of 300 mg/L for timentin can effectively inhibit the growth of <i>Agrobacterium</i> while promoting the induction of adventitious buds in leaves. The critical screening pressure for kanamycin for producing resistant adventitious buds and inducing resistant rooting seedlings was 100 mg/L. We optimized several independent factors, which significantly enhanced the efficiency of genetic transformation. The leaves were infected with <i>Agrobacterium</i> suspension diluted twice by adding 100 μmol/L acetylsyringone (β-AS) (OD₆₀₀ = 0.6) for 15 min, followed by co-culture in the dark for 3 d. Using this improved transformation system, we obtained transgenic <i>P. wulianensis</i> clones overexpressing the enhanced green fluorescent protein (<i>EGFP</i>) gene through direct organogenesis. Among the 112 resistant buds obtained, 17 developed resistant rooting in seedlings. Eight positive plants were identified through DNA, RNA, and protein level analyses, with a positivity rate of 47.06%. This study provides a foundation for developing and utilizing <i>P. wulianensis</i> germplasm resources and lays the groundwork for resource improvement.