Grafting in watermelon using traditional methods often causes rootstock regrowth, increasing labor demand and production costs. Although cotyledon-less splice grafting eliminates regrowth by excising meristem tissue, its success rate has consistently been lower. Here, we developed a novel cotyledon-less splice grafting methodology that achieved high survival rates by modulating pre-grafting light intensities from 100 to 300 μmol·m−2·s−1 for scion and rootstock, generating four experimental groups: high-light intensity scion/high-light intensity rootstock (HS/HR), high-light intensity scion/low-light intensity rootstock (HS/LR), low-light intensity scion/high-light intensity rootstock (LS/HR), and low-light intensity scion/low-light intensity rootstock (LS/LR). The results demonstrated that HS/HR and LS/HR exhibited the highest survival rates, nearly 98%, and displayed high seedling quality, markedly enhanced graft-union adhesion, and accelerated vascular reconnection. Pretreatment of high light intensity increased starch accumulation in rootstock hypocotyls, enhancing tolerance to carbon starvation after grafting especially in the cotyledon-less grafts. Metabolomic analysis identified elevated levels of key metabolites, including auxins, cytokinins, D-galactose, galactinol, starch, cinnamic acid, M-coumaric acid, and vanilloloside. Transcriptomic profiling revealed significant enrichment of plant hormone signal, starch and sucrose metabolism, and phenylpropanoid biosynthesis pathways in scion and rootstock tissues underpinning hormonal regulation, carbohydrate metabolism, and lignin biosynthesis under high-light conditions. WGCNA identified key co-expression modules associated with graft healing traits and key metabolites. Furthermore, graft healing related genes (PXY, NAC086, CALS7, and TMO6) were upregulated. In conclusion, our findings underscore the critical role of light intensity in orchestrating transcriptional and metabolic networks to optimize graft healing, providing a physiological and molecular foundation for improving cotyledon-less grafting efficiency.

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