Dear Dr.  Liu,

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Additional Editor Comments:

Thank you for your submission on hydroponic alfalfa production. While the topic is timely and of potential value, the manuscript has major shortcomings in study purpose clarity, methodological detail, experimental design, statistical analysis, and consistency of data presentation. Key methodological information is missing, several results are ambiguously reported or misinterpreted, and statistical approaches require reconsideration. Please address all points outlined in the detailed reviewer comments to ensure the study meets publication standards.

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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. Is the manuscript technically sound, and do the data support the conclusions?

Reviewer #1: No

Reviewer #2: Partly

Reviewer #3: No

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2. Has the statistical analysis been performed appropriately and rigorously? -->?>

Reviewer #1: N/A

Reviewer #2: Yes

Reviewer #3: No

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3. Have the authors made all data underlying the findings in their manuscript fully available??>

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.-->

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: No

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4. Is the manuscript presented in an intelligible fashion and written in standard English??>

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: No

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Reviewer #1: Recently, studies on hydroponic farming methods have become quite popular among researchers. . Although I think that such studies should be done, I think that the issue I mentioned below is the biggest handicap of this article.

The purpose of the study is unclear. Is the purpose of the study the possibilities of growing alfalfa in hydroponic environment or the effectiveness of the hydroponic medium used?

The rationale for the proposed study is not clear and valid. The protocol is not technically sound. The methodology is not feasible and detailed enough to make the work replicable

The analysis of the changes occurred during the harvesting periods of alfalfa in hydroponic medium is extremely important because it can contribute to the optimization of the obtaining cultivation. However, more attention needs to be paid to experimental design so that the relevance of the data can be justified and applicable at the agricultural level. In the presented study there is no clear justification of the reason for choosing the hydroponic medium, therefore it would be useful to complete the motivation.

It is seen that the alfalfa in the study has higher protein content and lower NDF content compared to those grown in the field. This may be due to the Hoagland nutrient solution grown, the content of this solution should be given.

The experimental design should be explained. Six cuttings were made, but the interval between cuttings and growing conditions are not given.

High levels of concentrate feed (such as alfalfa) in the ration cause digestive disorders, bloat and metabolic diseases such as acidosis in animals. Therefore, forage crops such as alfalfa are planted in mixtures and added to the ration. It should be stated what kind of suggestion hydroponic cultivation brings to this subject.

Reviewer #2: 1-The purpose of the study should be emphasized at the end of the introduction. This study should be distinguished from other studies. However, this has not been done in this study.

2-The terms “variety” and “cultivar” are used interchangeably in some places. One of these terms should be chosen.

3-The methods section is quite inadequate. Where was the experiment conducted? When was it conducted? When was the harvest carried out? What is the composition of the nutrient solution provided? Answers to many such questions are not found in the materials and methods section.

4-In the tables, varieties are numbered as A, B, C, etc., but in the text, variety names are used instead of numbers, which makes it difficult to understand the text.

5-Although there is no parameter called DDM in Table 2, it has been interpreted in the text.

6-Although the unit of the DMI parameter is %, it has not been used in the text.

7-A protein content of 30% or higher in alfalfa is not considered normal. Such results can be obtained in very early stages. Alfalfa is generally harvested at the 10% flowering stage, and during this period, the crude protein content ranges between 15% and 25%. How often was the harvest conducted?

8-It is incorrect to present forage yield as a percentage in Table 2.

9-Relationship analysis and PCA analysis were conducted in the text. However, no comments were made about these in the results section.

Alfalfa cultivation under hydrponic conditions has been considered a topical issue. However, for the reasons mentioned above, it was not deemed appropriate to publish this study in its current form.

After the necessary corrections have been made, it would be more appropriate to have the article re-evaluated (by other reviewers, if necessary).

Reviewer #3: General Comments

This manuscript examines the impact of six mowing cycles on the yield and nutritional quality of five alfalfa varieties (Zhongcao No. 13, WL440HQ, WL525HQ, WL903, and WL712) under hydroponic conditions. Employing principal component analysis (PCA) and membership function analysis, the study identifies WL903 as superior, with a yield of 4888.31 Kg·hm⁻² and nutritional quality metrics (e.g., 24.37% ADF, 29.92% NDF, 217.94 RFV). The research is novel, addressing a gap in the literature by comparing multiple cultivars across six cuttings in a hydroponic system, which is highly relevant for sustainable forage production in resource-constrained regions like China. However, the manuscript has critical flaws that render it unsuitable for publication in PLOS ONE in its current form. These include an incomplete introduction, misrepresentation of “cutting frequency,” inappropriate statistical design, failure to address significant interaction effects, inadequate methodological detail, inconsistent terminology, limited engagement with prior research, and overstated conclusions. Below, I outline the major and minor issues, and recommendations for improvement.

Major issues

Incomplete introduction

The introduction fails to connect the background on alfalfa and hydroponics to the study’s focus. It discusses global alfalfa production and China’s import trends (2008–2016) but does not link these to hydroponic cultivation or the study’s objectives. Additionally, no objectives or hypotheses are stated, leaving readers unclear about the study’s purpose and expected outcomes. This violates PLOS ONE’s requirement for a clear rationale and objective, hindering assessment of the study’s relevance and novelty. Recommendation: Revise the introduction to link the background to hydroponic alfalfa cultivation, emphasizing its relevance to China’s forage needs. Clearly state the objectives (e.g., to compare yield and quality of five alfalfa varieties across six consecutive cuttings under hydroponic conditions) and hypotheses (e.g., specific cultivars will outperform others due to genetic differences, or quality will vary across cuttings).

Misrepresentation of “cutting frequency” and inappropriate statistical design

The manuscript refers to “cutting frequency” as a factor but does not test different frequencies of cutting (e.g., cutting twice vs. six times over a fixed period). Instead, it examines the evolution of response variables (yield, nutritional quality, leaf-to-stem ratio) over six consecutive cuttings applied uniformly to all cultivars. The experimental design is presented as a factorial design, analysed with two-way ANOVA, but this is inappropriate because the cuttings are repeated measures on the same experimental units (plants), not independent units for each cutting event. A repeated measures analysis is needed to account for the correlation between consecutive cuttings on the same subjects. The misuse of “cutting frequency” misrepresents the study’s design, and the factorial ANOVA violates statistical assumptions, potentially leading to invalid conclusions about main and interaction effects. This undermines the study’s credibility and interpretability. Recommendation: Rename “cutting frequency” to “consecutive cuttings” or “cutting cycles” to accurately reflect the design. Reanalyse the data using a repeated measures ANOVA or a mixed-effects model, treating cuttings as a within-subject factor and varieties as a between-subject factor. Report the model structure, including covariance assumptions, and update results and discussion to reflect the repeated measures analysis.

Failure to address significant interaction effects

Table 2 indicates significant variety-by-cutting interaction effects (P < 0.001) for all nutritional variables (ADF, NDF, CP, RFV, DMI) and yield. DDM is not presented in table 2! However, the results and discussion focus only on main effects (variety and cutting), ignoring how specific combinations of variety and cutting influence outcomes (e.g., WL903’s performance across cuttings). Additionally, no p-values are reported for main or interaction effects for the leaf-to-stem ratio, despite its discussion (Fig 1a). Ignoring significant interactions misrepresents the study’s findings, as the effect of cutting depends on the variety. The lack of statistical analysis for the leaf-to-stem ratio further reduces rigor. Recommendation: Analyse and discuss variety-by-cutting interaction effects using post-hoc tests or interaction plots to clarify specific treatment combinations. Report p-values and statistical details for the leaf-to-stem ratio, ensuring consistency with other variables.

Inconsistent terminology and labelling

The manuscript inconsistently labels varieties as A, B, C, D, and E in the experimental design section while using their names (e.g., Zhongcao No. 13, WL903) elsewhere, creating ambiguity. This reduces clarity and undermines confidence in data presentation. Recommendation: Use variety names consistently throughout the text, tables, and figures, removing A–E labels or defining their correspondence explicitly.

Inadequate methodological detail

The experimental design lacks details critical for reproducibility, including:

- Hydroponic system type (e.g., Nutrient Film Technique, Deep Water Culture).

- Rationale for selecting the five cultivars or their genetic/agronomic traits.

- Light treatment specifics (3.5 h/3.5 h/3 h with 10 h intervals), including intensity, source, and whether it represents light/dark cycles.

- Nutrient solution details (e.g., composition, pH, electrical conductivity for 1.5x Hoagland solution).

- Sample size (e.g., replicates per variety per cutting), plant density, and physical setup (e.g., tray size, growing area).

These omissions prevent replication and assessment of robustness, violating PLOS ONE’s standards. Recommendation: Provide a comprehensive description of the hydroponic system, nutrient solution, light treatment, plant density, and replication. Justify cultivar selection based on genetic or agronomic traits. Clarify the light schedule’s relevance to alfalfa growth.

Questionable data reporting and analysis

- Yield units: The use of Kg·hm⁻² (e.g., 4888.31 Kg·hm⁻² for WL903) is inappropriate for hydroponic systems, which typically use kg·m⁻² due to smaller growing areas.

- Statistical transparency: The two-way ANOVA (inappropriate analysis) and Tukey’s test are reported, but degrees of freedom, F-values, and exact P-values are missing. PCA results lack loading values, and the membership function methodology (e.g., D-value calculations) is not explained.

These issues reduce transparency and interpretability. Recommendation: Justify or convert yield units to kg·m⁻². Include detailed statistical outputs (e.g., Repeated Measurements ANOVA tables, PCA loadings) in the text or supplementary materials. Describe the membership function methodology, including equations and assumptions.

Limited engagement with prior research

The manuscript cites relevant studies (e.g., Feedipedia, Al-Karaki and Al-Momani, 2011) but does not compare its findings to prior hydroponic alfalfa research. The novelty of six cuttings is not contextualised. This limits the study’s ability to demonstrate its contribution. Recommendation: Compare yield, nutritional quality, and cutting effects with prior hydroponic alfalfa studies. Highlight the novelty of the multi-cultivar, six-cutting approach.

Overstated conclusions and lack of limitations

The conclusions advocate WL903 as the “primary germplasm resource” without discussing limitations, such as system specificity, scalability, or performance under varying conditions. The claim that hydroponics is “feasible” for countries with limited arable land lacks cost or scalability data. This overstates the study’s implications and reduces rigor. Recommendation: Revise conclusions to reflect context-specific findings and add a limitations section addressing system specificity, scalability, and condition variability.

Minor Issues

Language and clarity

Awkward phrasing and grammatical errors (e.g., “alfalfa, also known as alfalfa,” “stubble times” instead of “cutting cycles,” “super alfalfa” as an informal term) hinder readability. Conduct a thorough language edit, using scientific terms and replacing vague or informal terms.

Introduction context

The introduction’s focus on China’s alfalfa imports trends (2008–2016) is tangential and not linked to hydroponics. Streamline the introduction to emphasise hydroponic alfalfa research and its relevance to China’s forage needs.

Citation formatting

Citations are inconsistent, and the reference list is not provided, preventing source verification. Provide a complete reference list in PLOS ONE’s format and ensure consistent in-text citations.

Tables and figures

Tables titles and figures captions should be self-explanatory, i.e., readers should not need to screen through the main text to understand what is reported in the tables.

Use Table 2 to present the Repeated Measurements ANOVA (degrees of freedom, F-values, and exact P-values) for the treatment factor (variety) and the time effect factor (cutting number) for all the variables: ADF, NDF, CP, RFV, DMI, DDM, leaf-to-stem ratio and yield Retain figures 1 and 2, which present the means of the combination of all the combinations of variety by cutting number, but assign the letters comparing the 30 different means whenever the interaction effect variety by cut is significant.

Recommendation

The manuscript addresses a novel and timely research question but is unsuitable for publication in PLOS ONE due to critical flaws: an incomplete introduction, misrepresentation of “cutting frequency,” inappropriate use of factorial ANOVA instead of repeated measures analysis, failure to address significant variety-by-cutting interaction effects, inadequate methodological detail, inconsistent terminology, limited engagement with prior research, and overstated conclusions.

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what does this mean?). If published, this will include your full peer review and any attached files.). If published, this will include your full peer review and any attached files.). If published, this will include your full peer review and any attached files.). If published, this will include your full peer review and any attached files.

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Reviewer #1: No

Reviewer #2: Yes:Dr. Erdal CacanDr. Erdal CacanDr. Erdal CacanDr. Erdal Cacan

Reviewer #3: Yes:Jordana RiveroJordana RiveroJordana RiveroJordana Rivero

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Dear Dr. Liu,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

Please submit your revised manuscript by Feb 12 2026 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

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If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: https://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols. Additionally, PLOS ONE offers an option for publishing peer-reviewed Lab Protocol articles, which describe protocols hosted on protocols.io. Read more information on sharing protocols at . Additionally, PLOS ONE offers an option for publishing peer-reviewed Lab Protocol articles, which describe protocols hosted on protocols.io. Read more information on sharing protocols at . Additionally, PLOS ONE offers an option for publishing peer-reviewed Lab Protocol articles, which describe protocols hosted on protocols.io. Read more information on sharing protocols at . Additionally, PLOS ONE offers an option for publishing peer-reviewed Lab Protocol articles, which describe protocols hosted on protocols.io. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols....

We look forward to receiving your revised manuscript.

Kind regards,

Debasis Mitra

Academic Editor

PLOS One

Journal Requirements:

If the reviewer comments include a recommendation to cite specific previously published works, please review and evaluate these publications to determine whether they are relevant and should be cited. There is no requirement to cite these works unless the editor has indicated otherwise.

Please review your reference list to ensure that it is complete and correct. If you have cited papers that have been retracted, please include the rationale for doing so in the manuscript text, or remove these references and replace them with relevant current references. Any changes to the reference list should be mentioned in the rebuttal letter that accompanies your revised manuscript. If you need to cite a retracted article, indicate the article’s retracted status in the References list and also include a citation and full reference for the retraction notice.

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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

Reviewer #2: All comments have been addressed

Reviewer #4: (No Response)

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2. Is the manuscript technically sound, and do the data support the conclusions??>

Reviewer #2: Yes

Reviewer #4: Yes

**********

3. Has the statistical analysis been performed appropriately and rigorously? -->?>

Reviewer #2: N/A

Reviewer #4: Yes

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4. Have the authors made all data underlying the findings in their manuscript fully available??>

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.-->

Reviewer #2: Yes

Reviewer #4: Yes

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5. Is the manuscript presented in an intelligible fashion and written in standard English??>

Reviewer #2: Yes

Reviewer #4: Yes

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Reviewer #2: 1- The RFV unit is given in the table as a percentage. RFV is unitless.

2- It is uncommon to see both "ABC" and "abc" lettering appearing together in tables. A change may be necessary at this point.

3- The summary could be further detailed.

Reviewer #4: Based on which parameter did you select five varieties of alfa-alfa? i.e yield or production, quality, local variety, resistance? Please describe it with a sentence to the experimental design paragraph.

Did you follow a protocol to establish 10 h light for the experiment? Please insert a reference or if this procedure is yours, please indicate within the text.

Fig 1, Fig 2 and Fig 3 please make them larger in order to read data inside the graphs.

Please describe which were principal components that you have analyzed? i.e yield, quality, production

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what does this mean?). If published, this will include your full peer review and any attached files.). If published, this will include your full peer review and any attached files.). If published, this will include your full peer review and any attached files.). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our For information about this choice, including consent withdrawal, please see our For information about this choice, including consent withdrawal, please see our For information about this choice, including consent withdrawal, please see our Privacy Policy..-->

Reviewer #2: No

Reviewer #4: No

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Effects of harvest number on the yield and quality of different alfalfa varieties under hydroponic conditions

PONE-D-25-32806R2

Dear Dr. Meiying Liu,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.

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Andrey Nagdalian

Academic Editor

PLOS One

Additional Editor Comments (optional):

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

Reviewer #2: All comments have been addressed

Reviewer #4: All comments have been addressed

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2. Is the manuscript technically sound, and do the data support the conclusions??>

Reviewer #2: Yes

Reviewer #4: Yes

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3. Has the statistical analysis been performed appropriately and rigorously? -->?>

Reviewer #2: Yes

Reviewer #4: Yes

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4. Have the authors made all data underlying the findings in their manuscript fully available??>

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.-->

Reviewer #2: Yes

Reviewer #4: Yes

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5. Is the manuscript presented in an intelligible fashion and written in standard English??>

Reviewer #2: Yes

Reviewer #4: Yes

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Reviewer #2: (No Response)

Reviewer #4: Dear Authors,

I would like to thank you for revising the manuscript carefully and for responding to all the issues raised in the previous review. I have reviewed the revised manuscript along with your response to the reviewers' comments.

The authors have successfully addressed all the issues raised in the review by making all the suggested changes, which have greatly improved the quality and scientific value of the manuscript.

In my opinion, the manuscript has greatly improved and is ready for publication in its current form.

Good luck!

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Reviewer #2: No

Reviewer #4: No

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