Optimal rooting substrates and hormonal regulation via a multi-omics analysis during Vitis davidii cutting rooting

• Examination of multiple rooting substrates revealed that T12 (perlite) is the most suitable substrate for the growth of V.davidii cuttings, achieving the highest rooting rate (90%), followed by T1 (rice husk biochar + coarse river sand 1:1) and T2 (rice husk biochar + perlite 1:1). Additionally, grape rooting takes place through a mixed process, which does not rely on callus formation. • Vermiculite facilitates bud germination and leaf expansion of cuttings. Substrates with large, hard particles, such as perlite and coarse river sand, are optimal for rooting of V.davidii cuttings. • Auxin and cytokinin play key roles in sprouting and leaf expansion of cuttings, while SA is critical for callus and root formation. JA and GA are primarily involved in direct rooting, with GA4 identified as the main gibberellin that promotes bud germination. • A total of 17 transcription factors were identified as key hormonal regulators during rooting, with TIFY10A emerging as the most significant. Spine grapes ( Vitis davidii Foex), a typical wild grape species native to China, is primarily propagated through cutting. However, successful rooting remains a significant challenge in production. Thus, one aim of this study is to identify an optimal substrate for rooting of V.davidii cuttings and explore the hormonal regulation under the rooting process. Among 13 substrates tested, T12 (perlite) produced the highest rooting rate (90%) and a 100% callus formation rate, followed by T1 (rice husk biochar + coarse river sand 1:1) and T2 (rice husk biochar + perlite 1:1). Rooting materials with large, hard particles, such as perlite and coarse river sand, improved rooting. Electron microscopy showed that V. davidii exhibited mixed-type rooting, and there was no direct relationship between callus formation and rooting success. Transcriptome and metabolome analyses indicated that significant differences in auxin and cytokinin were observed between P1 vs P2, suggesting their important roles in bud germination and leaf expansion. Salicylic acid (SA) was essential for callus and root formation, while jasmonic acid (JA) and gibberellins (GA) were more closely associated with direct rooting of cuttings rather than callus formation. Integrated Gene Ontology (GO) and KEGG analyses screened 17 crucial hormone regulatory transcription factors during rooting in cuttings. Among these, ARR18 and RR26 were mainly expressed at the P1 stage, while TIFY6B was predominant in P2, and GAI1 was highly active in the P1 and P4 stages. Remarkably, TIFY10A expression was 2.93 times higher in P3 and P4 compared to P1 and P2 and was highly correlated with various hormones. TIFY10A expression increased sharply under exogenous JA treatment and exhibited tissue-specificity. These findings suggest an important role of TIFY10A in the rooting process of grape V. davidii cuttings, particularly in callus and adventitious root formation.