An Efficient Agrobacterium-Mediated Transient Transformation System Using In Vitro Embryo-Derived Seedlings for Gene Function Elucidation in Paeonia ostii

<i>Paeonia ostii</i> is an economically significant species serving as an ornamental, medicinal herb, and woody oilseed crop. Gene function elucidation and molecular breeding are hindered by the lack of efficient, stable transformation methods due to tissue culture challenges. To enable year-round functional studies without material constraints, we established a novel transient transformation system mediated by Agrobacterium using in vitro embryo-derived seedlings (TTAES) in <i>P. ostii</i>. By optimizing embryo germination media, we achieved consistent seedling production. Orthogonal experiments with a GUS reporter identified optimal conditions: OD₆₀₀ = 1.0, 200 μM of acetosyringone, six negative-pressure treatments, and 2 h infection. Under this optimized system, maximum transformation efficiency was achieved at 35 days after germination. With this system, we demonstrated its application in investigating transcription factor-mediated regulation of target gene promoters using GUS as a reporter gene. To achieve non-destructive identification of transiently transformed plants, we employed GFP as a reporter gene. Using transient expression of VIGS (knockdown) and 35S constructs (overexpression), we characterized gene functions, thereby confirming the system's effectiveness for functional analysis. This system facilitates the acquisition of plant experimental materials and significantly improves research efficiency for year-round gene function elucidation in <i>P. ostii</i>.