Cytokinins combined with activated charcoal do not impair in vitro rooting in Quercus robur L.: insights from morphophysiological and hormonal analyses.

Background: In vitro rooting remains a challenge for many woody species, including Quercus robur. While auxins combined with activated charcoal (AC) have traditionally been used, Q. robur explants exhibit a strong dependence on cytokinins (CKs). This study introduces a novel approach combining AC with either 6-benzylaminopurine (BAP) or meta-topolin (mT) during the rooting phase to evaluate morphophysiological and hormonal responses.

Methods: Eight treatments were tested: 0 µM CK + 2 g L AC; 2, 4, and 8 µM BAP + 2 g L AC; 2, 4, and 8 µM mT + 2 g L AC; and to isolate the effect of AC, a treatment without exogenous CK and AC (0 µM CK + 0 g L AC) was also included. Morphophysiological and hormonal analyses were performed after 40 days of cultivation.

Results: Plantlets grown without AC exhibited a high shoot-to-root biomass ratio, indicating reduced growth balance. Treatments with 0-8 µM BAP combined with 2 g L AC reduced the ratio of vascular cylinder area to total root cross-section by 16.5% compared to the other treatments. Notably, mT-treated plantlets developed root systems with improved anatomical features conducive to transport efficiency. CKs at 8 µM significantly enhanced stem thickening via stimulated cambial activity. Additionally, CK + AC treatments increased chlorophyll (Chl a + b) and carotenoid (Car) contents, while the absence of both CK and AC resulted in higher Car/Chl a + b ratios and an increased photosystem II closure rate (M), indicating stress. Hormonally, CK-free treatments led to elevated indole-3-acetic acid (IAA) and reduced jasmonic acid (JA) levels, suggesting an imbalance.

Conclusion: Overall, the combination of AC and 4-8 µM mT during rooting enhanced secondary growth and improved the hormonal balance and morphophysiological traits of Q. robur plantlets.