The friability of callus is closely associated with its genetic transformation efficiency, and optimizing induction and transformation conditions is essential for establishing an efficient transformation system. In this study, we developed a high-efficiency friable callus induction and Agrobacterium-mediated transformation system for Chrysanthemum ‘Jimba’. Three plant growth regulator (PGR) combinations—6-Benzylaminopurine (6-BA) + Naphthaleneacetic Acid (NAA), 6-BA + 2,4-Dichlorophenoxyacetic Acid (2,4-D), and Thidiazuron (TDZ) + 2,4-D—were evaluated for their effects on callus morphology, proliferation, and transformation efficiency. The optimal PGR combination was identified as 1.0 mg/L 6-BA + 0.4 mg/L NAA, which produced highly friable calli with a loose structure, rapid proliferation, and the highest nuclear-to-cytoplasmic ratio. The optimal subculture time for maintaining friability and high proliferation was the 7th week, while the best Agrobacterium infection conditions were OD600 = 0.5 with a 10 min infection period, which achieved a transformation efficiency of 91%. This optimized protocol provides an efficient and rapid transformation method for future gene function studies using callus transformation.

The anthocyanin biosynthesis pathway has been investigated as a candidate for visual reporting in plants, due to its pigment-producing capacity. Activation of this pathway is predominantly regulated by MYB-bHLH regulatory complexes. In a previous study, two chrysanthemum transcription factors, CmMYB6 and CmbHLH2 , were shown to induce anthocyanin accumulation when co-expressed. However, the stability and reliability of the visible pigmentation mediated by this complex under varying environmental conditions remain unclear. In this study, the visible anthocyanin phenotype was assessed under different environmental conditions, including darkness, high temperature, and drought stress. Our results show that anthocyanin accumulation mediated by the 35S::CmMYB6-CmbHLH2 complex is highly dependent on light and temperature. High temperature reduced anthocyanin accumulation mainly by decreasing transgene expression efficiency, whereas darkness suppressed pigmentation primarily at the post-transcriptional level, while drought stress moderately enhanced anthocyanin accumulation. Collectively, these findings indicate that environmental factors regulate anthocyanin accumulation mediated by the CmMYB6-CmbHLH2 complex through distinct regulatory mechanisms, thereby suggesting that stable visible pigmentation mediated by the MYB-bHLH complex requires sufficient light intensity and a moderate temperature condition.