Effects of Exogenous Naphthylacetic Acid Application on the Graft Union Healing of Oriental Melon Scion Grafted onto Squash Rootstock and the Qualities of Grafted Seedlings

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The article “Effects of exogenous naphthylacetic acid application on the graft union healing of oriental melon scion grafted onto squash rootstock and the qualities of grafted seedlings” by Wu et al. investigates how exogenous application of naphthylacetic acid (NAA) affect graft union healing and seedling quality in oriental melon scions grafted onto squash rootstocks.  Specifically, the study aims to determine the physiological, biochemical, and molecular mechanisms underlying NAA-induced improvements in graft success and seedling vigor.

Grafting is a widely adopted practice in cucurbit production, yet the optimization of healing conditions, especially at the hormonal level, remains an area of active research. This study addresses a critical gap by exploring the exogenous application of synthetic auxin (NAA) as a tool to enhance graft success, a topic not yet fully explored in oriental melon–squash combinations. The investigation into auxin biosynthesis pathways and specific gene expression profiles during graft healing is particularly relevant, offering mechanistic insights that extend beyond horticultural application into fundamental plant developmental biology.

Compared to previous studies, which often describe general morphological outcomes of grafting or the role of auxins in isolation, this paper integrates a multi-layered analysis: physiological (seedling growth), biochemical (enzyme activities), and molecular (gene expression of auxin biosynthesis and wound healing markers). The authors provide a comprehensive dataset linking NAA application to both anatomical vascular reconnection and upregulation of key genes such as CmoWOX4, CmoVND7, and CmoWIND1. This systems-level approach represents a notable advancement over more narrowly focused reports.

While the study deals with a very interesting topic, several improvements could enhance its rigor, the main one is the way the data was analyzed. The key point of the study is to evaluate the effect of NAA over time, and this should be done by comparing it to the control. In Figs. 2, 3, 4, and 5, it is not clear why the control and NAA treatments are plotted one above the other and not on the same graph, where they should be compared as in Figs. 6 and 7. Thus, the statistical analysis should be redone, presenting the data in a way that is more consistent with the objectives of the study.

The conclusions are largely consistent with the presented data. The upregulation of auxin biosynthesis-related enzymes and genes, in conjunction with the increased IAA content, supports the assertion that NAA influences hormonal regulation favorably during graft union healing. Additionally, the expression of WIND, WOX, and VND family genes ties well into known wound response and vascular regeneration pathways. However, the assertion that NAA provides a “highly effective strategy” for grafting efficiency would benefit from multi-season, field-scale validation to strengthen translational applicability. Also, the statistical reevaluation mentioned above is crucial for a more correct presentation of the results.

The reference list is adequate.

There is a large overlap of text with the work already published in the Int. J. Mol. Sci – MDPI “Exogenous Melatonin Application Accelerated the Healing Process of Oriental Melon Grafted onto Squash by Promoting Lignin Accumulation” (https://doi.org/10.3390/ijms25073690) and this should be resolved by reducing these redundancies in the text.

This manuscript presents a valuable and timely contribution to the field of horticultural science, particularly for those studying grafting, hormonal regulation, and seedling quality in cucurbits. With improvements in the way the results are presented and analysed and the reductions of similarity with previous published paper, the findings could have strong implications for commercial grafting protocols and fundamental understanding of wound healing in plants.

Author Response

Dear reviewer,

Thanks for your generous comments. We have revised the figures and some content.

Prof. Chuanqiang Xu

On behalf of all authors.

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

Dear Authors,
In this study, the authors investigated the effects of exogenous naphthylacetic acid on graft union healing and seedling quality in oriental melon grafted onto squash rootstock. The authors measured anatomical, physiological, and gene expression changes induced by NAA to assess its impact on graft success and plant vigor. However, despite the scientific merit and relevance of the topic, I regret to inform you that the manuscript cannot be accepted for publication in its current form due to several critical issues outlined below. Substantial revisions are required before my final decision can be considered.
Line 17–35 (Abstract): The abstract is overly descriptive and lacks clarity in its structure. It should more explicitly present the hypothesis, key findings with statistical strength, and specific conclusions rather than listing numerous outcomes. Revise the abstract to present a clear problem statement, followed by the hypothesis, major quantitative results, and a concise conclusion highlighting the novelty of the findings.
Line 66–79 (Introduction): The knowledge gap is poorly defined. While auxin's role in graft healing is discussed broadly, the novelty of using NAA specifically in oriental melon is not convincingly distinguished from existing studies (e.g., references 29–30 already suggest similar findings). Clarify the specific novelty of this study and justify why NAA’s role needs to be re-evaluated despite similar existing reports.
Line 88–97 (Materials and Methods): The description of grafting conditions lacks crucial environmental parameters (light, humidity, temperature) in the healing chamber, which significantly influence graft success. Provide detailed environmental conditions during healing to enable reproducibility and assess interaction effects.
Line 98–106: Root analysis lacks a sufficient sample size and does not mention how five plants were selected (randomization or replication). Clarify replication strategy and justify the number of biological replicates used in root morphology analysis.
Line 113–122 (qRT-PCR): Primer sequences, efficiency validations, and reference gene selection (for normalization) are missing, which are essential for the reliability of qRT-PCR results. Include primer sequences in supplementary material and report validation metrics (efficiency, melt curves, reference gene stability).
Line 130–138 (Section 3.1): The conclusion that NAA accelerates vascular connectivity is based solely on a visual acid fuchsin assay with no quantification or histological validation. Quantify vascular reconnection (e.g., % of seedlings with full dye uptake) or complement with histological data.
Line 159–188 (Section 3.3 & 3.4): The gene expression results are inconsistently interpreted. For instance, changes in YUCCA gene expression are not always statistically significant but are still discussed as impactful. Avoid overinterpretation of non-significant differences and explicitly indicate which comparisons are statistically meaningful.
Line 190–216 (Section 3.4): It is unclear whether the reported gene expression changes directly relate to functional healing improvement. No functional validation (e.g., through inhibitors or mutants) is presented. Temper the conclusions or include future directions for functional validation of these candidate genes.
Line 218–239 (Section 3.5): Although root system data is statistically supported, there is a lack of information on physiological parameters such as photosynthesis or water use efficiency, which could strengthen claims of improved vigor.  Consider including or discussing complementary physiological parameters to reinforce the vigor claims.
Line 240–344 (Discussion): The discussion extensively reiterates results rather than offering deeper critical interpretation. In addition, many references cited support general auxin roles but do not directly relate to the specific NAA concentrations or experimental context used here. Reorganize the discussion to address limitations (e.g., only one NAA concentration, short-term outcomes) and compare with more contextually relevant studies.
Line 350–359 (Conclusions): The conclusion overstates the practical application of NAA without long-term field validation or cost-benefit analysis. Qualify the practical implications more cautiously and acknowledge that results are limited to early-stage healing and controlled conditions.
In light of the above issues, I recommend major revision. If the authors address the methodological gaps, add necessary validations, and revise their interpretations accordingly, the manuscript could be reconsidered for publication. I will be glad to re-evaluate the revised version and provide a final decision based on the extent and quality of revisions.

Author Response

Dear reviewer,

Thanks for your generous comments. We have revised some content according to your suggestions.

Prof. Chuanqiang Xu

On behalf of all authors.

Author Response File: Author Response.pdf

Round 2

Reviewer 2 Report

Comments and Suggestions for Authors

Dear Authors,

I have reviewed your responses to the reviewer comments and am pleased to see your willingness to undertake revisions. However, I believe more detailed and concrete corrections are needed on several critical issues. First, you need to provide a clearer explanation regarding the novelty of your study - you must more explicitly demonstrate how it differs from your previous work and clearly articulate your contribution to the literature. I recommend being more cautious in interpreting statistically non-significant results and addressing the lack of functional validation not only in the discussion section but also from a methodological perspective. Your positive approach to adding methodological details (primer sequences, environmental conditions, sampling strategy) is commendable. Finally, I request that you present your results regarding practical applications more cautiously and clearly state the limitations of your study. I believe that with these corrections, your work will make a valuable contribution, and I will make my final decision after the revision.

 

Author Response

Dear reviewer,

Thanks for your generous comments again. We have revised some content according to your suggestions.

Prof. Chuanqiang Xu

On behalf of all authors.

Author Response File: Author Response.pdf

Round 3

Reviewer 2 Report

Comments and Suggestions for Authors

Accept in present form.