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Dianthus spiculifolius is a perennial herbaceous flower with strong environmental adaptability and is an important oamental ground cover plant. In this study, seeds of D. spiculifolius were used as explants for callus induction, adventitious bud differentiation, and rooting by adding different concentrations of 2,4-dichlorophenoxyacetic acid (2,4-D), 6-benzyl aminopurine (6-BA), and naphthaleneacetic acid (NAA) to Murashige and Skoog medium. The calli generated were co-cultured with Agrobacterium tumefaciens EHA105 containing pBI121-GUS or pBI121-GFP plasmids for 30 min, and transgenic regenerated plants were obtained by kanamycin (30 mg · L? 1) screening. RT-PCR confirmed the stable expression of the exogenous GUS and GFP genes in the D. spiculifolius. Theβ-glucuronidase (GUS) histochemical staining confirmed GUS gene expression in transgenic calli, adventitious buds, and regenerated plants of D. spiculifolius. The green fluorescent protein (GFP) visual analysis showed GFP gene expression in transgenic calli. Furthermore, subcellular localization analysis showed that the three organelle marker proteins were not only successfully expressed but also accurately localized to their corresponding organelles in D. spiculifolius callus cells. These results indicated a successful establishment of a reliable and efficient A. tumefaciens-mediated genetic transformation system, which will contribute to functional gene research and genetic improvement of D. spiculifolius.