12759 10.30848/PJB2025-4(5) <p> Optimization of hairy root transformation system for gene functional studies in poplar (<em>Populus</em> species)</p> Mei Han, Junhu Feng, Shuyue Sun, Yujia Si, Xiaoning Liu, Mingyue Xu and Tao Su <p> Poplars (Populus spp.) are one of the most widespread ligneous plants in the world, which have a variety of applications ranging from landscape, agriculture, and industry to research. Although <em>Agrobacterium tumefaciens</em>-mediated gene transformation method has been widely utilized for poplar genetic manipulation, many defective technique defects such as the time-consuming procedures, and the occurrence of chimeric transformant have emerged. To address these problems, a simple, fast, straightforward and effective gene transformation method by using <em>Agrobacterium rhizogenes</em> to induce hairy roots in poplars was developed, and regenerated-transgenic poplar plantlets were obtained within a period of two to three months. The optimal procedure comprised the usage of poplar apex buds as explants, infection of <em>A. rhizogenes</em> by directly pricking colonies with the cuttings on the agar plates, and improving hairy-root initiation by application of indole-3-butyric acid (IBA) in combination with hairy-root morphological identification. Moreover, the GFP marker gene was applied to track record the expression level of the introduced genes using a portable fluorescence lamp. Further quantitative real-time PCR (qRT-PCR) and the enzyme activity assay showed that in comparison with the non-transformed control, the transcript abundance and enzyme activity of our target gene (i.e., <em>aspartate aminotransferase</em>) were significantly elevated in the transformed plant lines regenerated from the hairy roots, confirming that the current <em>A. rhizogenes</em>-mediated poplar transformation method is effective and applicable for poplar genetic manipulation and functional genomic study</p> Published 2025 57 4 1357 1368