1,3-di(benzo[d]oxazol-5-yl)urea acts as either adventitious rooting adjuvant or xylogenesis enhancer in carob and pine microcuttings depending on the presence/absence of exogenous indole-3-butyric acid

Ceratonia siliqua L. (carob) is a woody angiosperm of commercial value but, as often it happens with many tree species, its asexual propagation remains difficult because of adventitious rooting recalcitrance. In an attempt to improve the adventitious root (AR) formation in cuttings of this species, previous results obtained in Pinus radiata, a distantly-related woody gymnosperm, have been considered. In particular, adventitious rooting of pine hypocotyl cuttings has been enhanced by two urea derivatives, 1,3-di(benzo[d]oxazol-5-yl)urea (5-BDPU) and 1,3-di(benzo[d]oxazol-6-yl)urea (6-BDPU), combined with the auxin indole-3-butyric acid (IBA) (Brunoni et al., 2014, Plant Cell Tiss Organ Cult 118: 111). This research is the first attempt to utilize these two unusual chemicals, besides the auxin (IBA), to improve adventitious rooting in carob cuttings, with the aims to define their role in AR process and to identify morphogenic roles in carob, but also in pine, even when used alone. To the aims, results in carob were compared histologically with those obtained from the cuttings of pine. Different protocols were applied in order to enhance adventitious rooting in carob, using different chemicals application/dark exposition times and/or hormones (IBA, kinetin) combinations and concentrations. The results show that 5-BDPU (10 M) highly promoted AR formation in combination with IBA (1 M) when applied for 3 days, followed by a transfer onto hormone free medium (HF) up to culture end (four weeks). IBA was more effective to enhance AR-forming capability of the cuttings when applied alone than when combined with kinetin (Kin, 10 nM), a synthetic adenine derivative with cytokinin-like activity, which is frequently used to induce adventitious rooting in combination with this auxin (e.g., Della Rovere et al., 2013, Ann Bot 112: 1395). Kin alone and 5-BDPU alone were not AR-inductive. The histological analysis showed that the cambial cells initiated the ARs, and similar numbers of AR primordia were visible at day 12 after different AR-inductive treatments (i.e., IBA alone, IBA plus 5-BDPU, IBA plus Kin). No cutting treated with Kin alone, and rare HF (with or without 5-BDPU)-treated cuttings, showed AR primordia at the same day. The number of explants forming AR-primordia increased under IBA with 5-BDPU. By contrast, the cambial cells were stimulated to initiate deuteroxylem instead of ARs under the treatment with 5-BDPU alone. The histological analysis in pine microcuttings treated with IBA and/or 5-BDPU at the same concentrations confirmed that 5-BDPU applied alone enhanced xylogenesis, highlighting that this urea derivative exhibits a dual morphogenic role in both species, being involved in the switching between adventitious rooting and xylogenesis depending on the presence/absence of exogenous auxin.