Differential morphophysiological and epigenetic responses during in vitro multiplication of Quercus robur depending on donor age and plant growth regulators

Abstract In the in vitro cloning of specific individuals of woody species, a challenge can be the low viability of explants collected from old mature donor trees due to the recalcitrance of their plant material. Other factors, such as the auxin–cytokinin balance, must also be considered. We investigated how in vitro conditions and donor age can modulate the regeneration capacity, morphophysiology, and DNA methylation of Quercus robur . Explants from two different donors (70- and 600-year-old trees) were subjected to 8 different treatments, which consisted of 0, 1.25, 3.5, or 7.0 µM 6-benzylaminopurine (BAP) combined with two concentrations of 0 or 0.5 µM 1-naphthaleneacetic acid (NAA). In the absence of BAP, there was no shoot induction, and root formation was achieved only in the shoots of the 70-year-old clone. Shoot number was lower in the shoots of the 600-year-old clone. Both clones showed increased regeneration capacity under BAP exposure, which was positively correlated with 8-hydroxydeoxyguanosine (8-oxo-dG) levels and the antioxidant capacity of total phenolics. In contrast, shoot induction was negatively correlated with 5-methylcytosine (m 5 C) levels. Treatments with increased BAP concentrations induced the development of anatomical and biochemical characteristics such as underdeveloped tissues and/or delayed tissue development, and this effect was less intense in the 600-year-old clone. Global hypomethylation strongly correlates with guanine hyperoxidation and the de novo regeneration response. Our results suggest that explants collected from fully mature trees may have different growth regulator supplementation needs due to the ‘donor age’ factor. Older clones may require a higher concentration of cytokinins to ensure a regenerative response.