A stable transformation platform in pomegranate uncovers PgMYB10 as a key regulator of anthocyanin biosynthesis

An efficient genetic transformation platform enables functional validation of PgMYB10, identifying it as a master regulator governing anthocyanin biosynthesis in pomegranate. Limited availability of stable genetic transformation systems restricts functional genomics research in pomegranate. Here, we established efficient in vitro regeneration and Agrobacterium tumefaciens-mediated transformation systems for 'Taishanhong' pomegranate using stem segment explants. Optimized medium combinations produced high-frequency regeneration: a 93.3% shoot-induction rate on MS medium with 1.5 mg/L 6-benzylaminopurine (BAP), 0.6 mg/L 1-naphthaleneacetic acid (NAA) and 30.0 mg/L adenine sulfate (ADS); a proliferation coefficient of 5.4 on MS medium supplemented with 0.8 mg/L BAP and 0.3 mg/L indole‑3‑butyric acid (IBA); effective shoot-strengthening on MS medium containing 1.2 mg/L BAP, 0.3 mg/L NAA and 0.2 mg/L gibberellic acid (GA₃); and a rooting rate of 95.3% on half-strength MS medium with 1.5 mg/L IBA and 0.5 mg/L NAA. For transformation, precultured explants were immersed with A. tumefaciens suspension (OD₆₀₀ = 0.8) containing 20.0 mg/L acetosyringone (AS) for 30 min. After four days of dark co-culture, sequential antibiotic screening with 30 mg/L kanamycin and bacteriostatic treatment with 400 mg/L timentin yielded a stable average transformation efficiency of 17.5% in 'Taishanhong' pomegranate. Subsequent functional analysis revealed that overexpression of PgMYB10 induced pigment accumulation in leaves and stems. In three independent transgenic lines, maximum anthocyanin content and PgMYB10 transcript levels were 5.4-fold and 27.2-fold higher than in wild-type plants, respectively. Six anthocyanin biosynthetic genes (PgCHS, PgCHI, PgF3H, PgDFR, PgANS, and PgUFGT) were markedly upregulated, demonstrating that PgMYB10 positively controls anthocyanin biosynthesis. This transformation system provides a reliable technical platform for functional genomic studies in pomegranate, and PgMYB10 represents as a promising candidate gene for molecular breeding aimed at improving fruit pigmentation.