Overexpression of AtMYB12 transcription factor simultaneously enhances quercetin-dependent metabolites in radish callus.

The study aimed to enhance quercetin production in radish by optimizing Agrobacterium tumefaciens -mediated in-planta transformation. This protocol involved infecting radish seed embryo axis with A. tumefaciens EHA105 strain carrying the 35S::AtMYB12 . Radish seeds were infected with the Agrobacterium suspension (0.8 OD600 ) for 30 min, followed by sonication for 60 s and vacuum infiltration for 90 s at 100 mm Hg. A 3-day co-cultivation in Murashige and Skoog medium with 150 μM acetosyringone yielded a transformation efficiency of 59.6% and a transgenic callus induction rate of 32.3%. Transgenic plant and callus lines were confirmed by GUS histochemical assay, PCR, and qRT-PCR. The transgenic lines showed an increased expression of flavonoid pathway genes ( AtMYB12, CHS, F3H, and FLS ) and antioxidant genes ( GPX, APX, CAT, and SOD ) compared to WT plants. Overexpression of AtMYB12 in transgenic callus increased enzyme activity of phenylalanine ammonia lyase, catalase, and ascorbate peroxidase. In half-strength MS medium with 116.8 mM sucrose, the highest growth index (7.63) was achieved after 20 days. In AtMYB12 overexpressed callus lines, phenolic content (357.31 mg g-1 dry weight), flavonoid content (463 mg g-1 dry weight), and quercetin content (48.24 mg g-1 dry weight) increased significantly by 9.41-fold. Micro-wounding, sonication, and vacuum infiltration improved in-planta transformation in radishes. These high-quercetin-content transgenic callus lines hold promise as valuable sources of flavonoids.