Comparative anatomical and physiological characterization of adventitious root formation in Ilex integra Thunb. and Ilex verticillata ‘Oosterwijk’

Ilex (Aquifoliaceae) is a widely distributed genus of high ornamental, medicinal, and ecological value. Seeds of many Ilex species exhibit pronounced dormancy and poor germination, so vegetative propagation via cutting propagation is routinely employed to accelerate nursery turnover and enhance reproductive efficiency. This study employed optimized phytohormone combinations to cultivate Ilex integra Thunb. and Ilex verticillata ‘Oosterwijk’ cuttings, and comparatively characterized their rooting differences at morpho-anatomical and physio-biochemical levels. The results showed that the rooting type of I. verticillata was mainly bark rooting type, which may contribute to rapid adventitious root (AR) formation, whereas I. integra mainly relied on callus-derived rooting that contribute to slower AR formation. Anatomical examination revealed that AR of I. integra originated from vascular cambium or the parenchyma tissue in pith, or extend from callus-induced regions at the base, and this AR formation mode may explain the slow rooting. The content of endogenous phytohormones (IAA, GA 3 , ZR, ABA), related enzyme activities (IAAO, PPO, POD), total phenolic content and C/N ratio in cuttings showed complex dynamics during the rooting process. Overall, exogenous phytohormone treatment altered the temporal patterns of several physiological and biochemical variables compared with the water-treated control, and these changes differed between I. integra and I. verticillata , with responses in I. integra generally occurring later, consistent with its longer rooting process. The findings expand the theoretical framework for the cutting propagation of Ilex , and provide practical guidance for addressing the rooting difficulties of this genus.