Temporal Dynamics of Auxins and Strigolactones in the Root Emergence Zone of Chestnut Cuttings During Adventitious Root Formation

Adventitious root (AR) formation is essential for the vegetative propagation of woody plants, yet recalcitrant species within Castanea spp. exhibit strong genotype-dependent differences in rooting ability. Strigolactones (SLs) and auxins are key phytohormones regulating AR development, but their dynamic interplay remains poorly understood. We analysed three Castanea genotypes with contrasting rooting capacities—two hybrids, Castanea crenata × Castanea sativa (‘Maraval’ and ‘Marsol’), and one native accession, Castanea sativa (‘Kozjak’)—to investigate temporal changes in auxin and SL content following severance. Cuttings were sampled at multiple time points from 0 min to 3 weeks prior to visible root formation, and again at 12 weeks, when rooting outcomes were assessed. Individual tissues were analysed for active auxins, conjugated and oxidised auxin metabolites, and strigolactones. From these data, total auxin content (TAC), total auxin-related compounds (TCC), total strigolactone content (TSC), and the ratios TAC:TCC and TAC:TSC were calculated. Rooting success differed markedly among genotypes: ‘Maraval’ and ‘Marsol’ rooted successfully (75–79%), whereas ‘Kozjak’ failed to produce roots and instead formed extensive callus. Hormonal profiling indicated that rooting competence depends not only on auxin levels but also on the dynamic balance between active auxins, inactive metabolites, and strigolactones. ‘Kozjak’ showed an early predominance of free IAA and elevated SL levels, correlating with inhibited AR initiation, whereas the hybrid genotypes maintained more stable hormonal ratios, which supported successful rooting. These results highlight the importance of coordinated hormonal balance rather than absolute hormone concentrations in regulating AR formation. Our study provides new insights into hormone dynamics underlying rooting recalcitrance in chestnut and suggests potential strategies to improve clonal propagation of woody species.