BACKGROUND: The trifoliate orange is used as rootstock in the Citrus genus
nevertheless, propagation from cuttings is challenging due to low rooting ability. The underlying mechanism for low rooting ability is still unclear. This study aims to investigate hormonal biosynthesis and signaling cascades during Adventitious Root (AR) formation. METHODS AND RESULTS: We systematically compared trifoliate orange and Micro-Tom tomato cuttings grown in water. The results indicated Micro-Tom tomato cuttings produced ARs within 3 days, whereas trifoliate orange developed ARs at 21 days after cuttings (DAC). The key endogenous hormones contents: indole-3-acetic acid (IAA), Jasmonic acid (JA), and salicylic acid (SA), were significantly lower in trifoliate orange than Micro-Tom tomato cuttings at earlier stages 1, 2, and 3 DAC. Moreover, IAA-related and ethylene biosynthesis genes demonstrated differential expression patterns during different developmental stages between trifoliate orange and Micro-Tom tomato. Specially, the expression levels of auxin biosynthesis genes (YUCCA6/4, GH3.3/3.6) and response factor (ARF8) were significantly elevated in Micro-Tom tomato cuttings, while lower in trifoliate orange cuttings at the earliest stages. Additionally, transcripts related to auxin signaling and transport (PIN2, LAX2, AUX1, TR2a, IAA11/12) and ethylene-related transcripts (ACS3/7, ASA1, ERF003/109) were lower in trifoliate orange than Micro-Tom tomato cuttings during the earliest stages. CONCLUSIONS: Our results confirmed AR formation in trifoliate orange was delayed compared to Micro-tom tomato. The low level IAA hormones, significantly lower expression IAA and ethylene-related genes at the earliest stages could be attributed to the delay of AR formation in trifoliate orange cuttings.