Regulation of the cytokinin metabolic spectrum and morphogenesis in Quercus robur (L.) in vitro by exogenous cytokinins and activated charcoal

The large-scale in vitro propagation of Quercus robur requires exogenous cytokinins (CKs); however, this process can cause physiological changes such as tissue and medium oxidation. Activated charcoal (AC) is often used to mitigate these effects. This study evaluated the de novo regenerative capacity, morphophysiological responses, and hormonal status of Q. robur shoots exposed to aromatic CKs—6-benzylaminopurine (BAP) and meta-topolin (mT)—either alone or in combination with AC. Shoots were cultured with four concentrations of BAP or mT (0, 2, 4, and 8 μM) and two AC concentrations (0 or 2 g L⁻¹) for 40 days, followed by morphophysiological and CK metabolic analyses. In the absence of AC, new shoots formed in media supplemented with CKs, whereas the H₂O₂ content was lower in CK-treated plants, regardless of the presence of AC. The presence of AC altered the morphogenetic response, promoting rooting even under BAP or mT exposure. Root formation was observed when CKs were absent (0 µM BAP or mT) and/or when 2 g L⁻¹ AC was added, irrespective of the CK concentration. The plants grown with AC presented increased levels of trans -zeatin (tZ) and its derivatives. In contrast, BAP-treated plants without AC accumulated N⁶-benzyladenine (BA) derivatives, although excess BA was converted into inactive forms. The anatomical status of the leaves affects the performance of the photosynthetic apparatus. Both BAP and mT effectively induced de novo shoot regeneration in Q. robur in vitro . However, AC inhibited BA-type CK accumulation while promoting t Z-type accumulation, highlighting its regulatory role in CK metabolism. • BAP and mT can trigger de novo regeneration. • AC and aromatic CK coexposure inhibits shoot induction and promotes rooting. • Exogenous CKs can reduce the H₂O₂ content and mitigate IAA degradation. • Exogenous CKs without AC induce less differentiation in leaf tissues and lower PSII efficiency. • BAP supplementation leads to an accumulation of inactivated BA-type derivatives.