Grafting is a propagation method extensively utilized in cucurbits. However, the mechanisms underlying graft healing remain poorly understood. This study employed self-grafted watermelon plants to investigate how rootstock cotyledon affects healing. The complete removal of rootstock cotyledons significantly hindered scion growth, as evidenced by reductions in scion fresh weight and the area of true leaves. Physiological assessments revealed reduced callus formation, weaker adhesion forces, a more pronounced necrotic layer, and decreased rates of xylem and phloem reconnection at the graft junction when rootstock cotyledons were completely removed. Additionally, auxin levels at the rootstock graft junction notably decreased following cotyledon removal. In contrast, the exogenous application of indole-3-acetic acid (IAA) notably enhanced graft healing. Moreover, gene expression analysis of the PIN auxin efflux carriers in the rootstock cotyledons indicated significant activation of ClPIN1a postgrafting. Furthermore, we developed an improved Virus-Induced Gene Silencing (VIGS) system for cucurbits using seeds soaking method. This method achieved an infection success rate of 83% with 60%–75% gene silencing efficiency, compared to the 37% success rate with 40%–60% efficiency seen with traditional cotyledon infection. Combining our novel VIGS approach with cotyledon grafting techniques, we demonstrated that rootstock cotyledons regulate callus formation through ClPIN1a-mediated endogenous auxin release, thus facilitating graft union development. These findings suggest potential strategies for enhancing watermelon graft healing by manipulating rootstock cotyledons.

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