Effects of IBA Combined with NAA/Melatonin/Citric Acid on Rooting Characteristics of Grape Rootstocks

Efficient rooting is essential for the propagation of grape rootstocks. This study aimed to identify an effective rooting formulation for hardwood cuttings and to investigate its underlying physiological and molecular mechanisms. Seven grape rootstocks were treated with indole-3-butyric acid (IBA) combined with melatonin (MLT) or citric acid (CA), with IBA plus naphthaleneacetic acid (NAA) and water serving as controls. Rooting performance was comprehensively evaluated using morphological traits, physiological characteristics, endogenous hormone contents, and transcriptome analysis. Among all treatments, 400 mg/L IBA combined with 1.2 mM MLT exhibited the best rooting performance. This treatment significantly enhanced root activity, soluble sugar, and soluble protein contents. It also increased IAA and GA3 levels and improved the IAA/ABA and GA3/ABA ratios compared with the controls. Transcriptome analysis of ‘110R’ revealed that MLT-responsive genes were mainly enriched in plant–pathogen interaction, hormone signal transduction, and MAPK signaling pathways. Transcription factor families including MYB, ERF, and NAC were identified as potential regulators. Collectively, these findings demonstrate that IBA–MLT combined application promotes rooting by regulating physiological metabolism, hormone balance, and gene expression, providing a theoretical basis for improving grape propagation efficiency.