Two-Stage Regulation of <i>trans</i> -Pterostilbene Biosynthesis in Blueberry Leaf-Derived Cell Cultures

<i>Trans</i>-pterostilbene (t-Pt), a methylated derivative of <i>trans</i>-resveratrol (t-R), exhibits potent pharmacological properties but accumulates minimally in plants, limiting its practical applications. This study establishes a proof-of-concept <i>in vitro</i> system for t-Pt production using blueberry (<i>Vaccinium corymbosum</i> L.) leaf-derived callus cultures. Calli were induced on a solid medium containing 0.5 mg/L 2,4-D and 0.2 mg/L BA, with red calli (RC) demonstrating superior stilbenoid accumulation. Suspension cultures of RC in liquid medium supplemented with 0.2 mg/L 2,4-D yielded about 89-fold higher t-Pt levels than control leaves without UV treatment. Furthermore, UV-A and UV-C treatments (600 J/m², followed by a 7-day recovery) produced modest increases in <i>ROMT1</i> expression: in leaves, ∼1.06-fold with UV-A and ∼1.10-fold with UV-C; in calli, ∼1.17-fold with UV-A and ∼1.26-fold with UV-C, with only UV-C reaching statistical significance. These transcriptional changes at a stabilized 7-day time point are consistent with a role for resveratrol O-methyltransferase 1 (<i>ROMT1</i>) in supporting t-Pt biosynthesis. Although conducted as separate experiments, the combined results from RC suspension cultures and UV-C elicitation demonstrate a two-stage proof-of-concept processRC induction followed by UV-C treatment, as a residue-free, controlled <i>in vitro</i> approach for enhancing t-Pt biosynthetic capacity.