PIN1a-mediated auxin release from rootstock cotyledon contributes to healing in watermelon as revealed by seeds soaking-VIGS and cotyledon grafting

Abstract

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.

Acknowledgements

This work was supported by the National Natural Science Foundation of China (31972434), the China Agriculture Research System of MOF and MORA (CARS-25), the Hubei Provincial Key Research and Development Program (2023BBB033), the Fundamental Research Funds for the Central Universities (2662024JC004), the Young Scientist Forstering Funds for the National Key Laboratory for Germplasm Innovation and Utilization of Horticultural Crops (11909920008), and the Huazhong Agricultural University-Agricultural Genomics Institute at Shenzhen, Chinese Academy of Agricultural Sciences Cooperation Fund (SZYJY2021005), Shannan Science and Technology Plan Project(SNSBJKJJHXM2023004).

Author contributions

Y.H., Z.B., and M.N. conceived the study and designed the experiments. X.W. measured the IAA content and conducted the qRT-PCR analyses. J.X., X.W., and A.K. performed the grafting experiments and microscopy analyses. T.Z. and X.W. carried out the VIGS experiments. X.W., M.X., and Y.H. drafted the manuscript. M.N. reviewed the manuscript and provided critical revisions. All authors approved the final manuscript.

Data availability

Data supporting the findings of this study are available within the paper and its supplementary materials. Additional data are available from the corresponding author upon reasonable request.

Conflict of interest statement

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Supplementary data

Supplementary data is available at Horticulture Research online.

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