Cytological Characterization of vrnp 1, a Pollen-Free Male Sterile Mutant in Mung Bean (Vigna radiata)

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The manuscript is namely “Cytological characterization of vrnp 1, a pollen-free male sterile

3 mutant in mung bean (Vigna radiata)” evaluated by me. 

You can find my comments below.

-        In the abstract section, more quantitative results should be provided.

-        More information about the fertilization biology of mung bean should be included in the introduction (or in plant material secition)

-        In the Material Method section - 2.3. Observation of anther paraffin sections-; the last sentence- How was the final observation done? with a microscope? Or direct observations.

-        Results and discussion sections  The results and discussion section is quite explanatory and sufficient.

-        Fertilization biology  is the most important element of the formation of a new plant, seed and fruit. The study contains very valuable results in this regard. - Therefore, it can be published after minor changes are made.

Author Response

Comments 1: [In the abstract section, more quantitative results should be provided.]

Response 1: [Thank you for your professional review of our manuscript. We have thoughtfully considered your feedback. While we recognize the potential of the proposed experiments, our current limitations in material resources and time regrettably prevent us from including this additional experimental content in the current manuscript. We are currently conducting the localization of the mutant gene and validating the DEGs identified through transcriptome sequencing. These processes are both time-consuming and require significant effort. We intend to incorporate these results, along with the quantitative data you recommended, into our future studies. We view this as a valuable addition that we had not previously considered.]

Comments 2: [More information about the fertilization biology of mung bean should be included in the introduction (or in plant material secition)]

Response 2: [ Thank you for your professional advice. To enhance the clarity of the logic and structure of the article, we have incorporated content related to the fertilization biology into the introduction and result section, specifically between lines 45-49 and lines 227-228. Your suggestion has greatly aided us in presenting a clearer understanding of the factors contributing to the formation of the vrnp 1 pod.

Comments 3: [In the Material Method section - 2.3. Observation of anther paraffin sections-; the last sentence- How was the final observation done? with a microscope? Or direct observations.]

Response 3: [Thank you for your suggestions to enhance the rigor of the section. Finally, the sections were analyzed under an optical microscope (Nikon Eclipse E100, Japan) equipped with an imaging system (Nikon DS-U3). We concur with your viewpoint and have made corresponding revisions in lines 166-182, which has further improved the comprehensiveness of our “Observations of anther paraffin sections” part. We hope that the revisions and responses made in the revised manuscript will adequately address the previous shortcomings in our language expression.]

The attachment contains the manuscript with highlighted revisions to facilitate your clear review of the updates we have made.

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

Comments regards to the paper you can find in attached pdf document.

Comments for author File: Comments.pdf

Author Response

Comments 1: [Is this correct steps? After rinsing of the samples you stained it firstly, and than embedded into paraffin? Should it be staining of the paraffin embedded material, after cutting? By which protocol you did this? Please add it. Also, add type of the microtome add section thickness.]

Response 1: [Thank you for your identifying the errors in our manuscript. We have carefully reviewed the sections you pointed and have conducted a thorough revision and supplementation at lines 166-182 in the revised manuscript. We hope that the revisions and responses made here will adequately address the previous mistake and shortcomings in our manuscript. We also have carefully reviewed the sections you pointed and have conducted a thorough revision and supplementation at lines line 249-250 in the revised manuscript.]

Comments 2: [It should state - continued Figure 2 with E, F....., or  Figure 3 and start with A.]

Response 2: [As suggested, we have added '(Continued Figure 2 with E-G)' to Figure 3.]

Taking into account the other detailed suggestions for revisions offered via PDF annotations, we have crafted a comprehensive summary in response to the additional minor adjustments.

Textual Changes: We have revised the captions of Figures 2 to 4, including the suggested deletions of three specific sentences as recommended.

Lexical Corrections: We have made the following adjustments as per suggestions: corrected the terms 'programmed cell death' to 'PCD' and 'imaging' to 'staining', modified the label 'vrnp-1' to 'vrnp 1', and adjusted a bracket. These changes enhance the precision and clarity of our manuscript.]

The attachment contains the manuscript with highlighted revisions to facilitate your clear review of the updates we have made.

Author Response File: Author Response.pdf

Reviewer 3 Report

Comments and Suggestions for Authors

The manuscript contains the results of research with mung bean (Vigna radiata). It is an important species due to the numerous advantages it possesses. I would like to ask the Authors to indicate the possibility of implementing the obtained research results into variety breeding. What difficulties can be expected?  

The manuscript lacks conclusions.

Comments

Materials and Methods

Please attach a figure with the location of the research site.

Where was the research carried out?

In which year were they carried out?

Please attach a brief description of the mung bean variety tested

Please provide the full name of the producer of the statistical software used to analyse the data.

References

Large number of publications (68). Some of them were published more than 10 years ago and some even in the 20th century. These I propose to delete and limit to the most recent.

Author Response

Comments 1: [I would like to ask the Authors to indicate the possibility of implementing the obtained research results into variety breeding. What difficulties can be expected? ]

Response 1: [Thank you for the time and attention you've given to reviewing this manuscript. In response to your meaningful questions, I believe there is great potential in the localization of the mutation gene within the mungbean male sterility material. This could pave the way for future gene-editing endeavors aimed at developing mungbean varieties that boast enhanced traits. Such progress might substantially increase the likelihood of utilization of mungbean hybrid vigor. Moving forward, it seems we will face some challenges, particularly in establishing a more efficient mungbean genetic transformation system and in the development of gene-editing vectors with unique intellectual property rights. I look forward to the advancement of mungbean research in this field and am confident that researchers working with mungbean and other crops will rise to these challenges as we progress. Thanks for your patience and professional insights.]

Comments 2: [The manuscript lacks conclusions.]

Response 2: [We understand and respect your suggestion to add a conclusion section. And we have added a conclusion section as follows: 'In this study, the vrnp 1 mutant, characterized by a complete male sterility phenotype, was generated through EMS mutagenesis. Genetic analysis indicated that a single recessive nuclear gene was the determinant of this sterility. Furthermore, cytological and histological analyses revealed that the sterility in the vrnp 1 mutant originated post-tetrad stage due to tapetal developmental defects, delayed PCD, and microspores failing to absorb nutrients from tapetum in a timely manner. These factors led to the disruption of pollen coat deposition and failure of pollen wall maturation, ultimately culminating in premature microspore degeneration and the complete manifestation of the sterile phenotype in the vrnp 1 mutant.' in the manuscript following our discussion. We are confident that these additions will render our research findings more accessible and comprehensible. Thank you for your suggestion.]

Comments 3: [Please attach a figure with the location of the research site.Where was the research carried out?In which year were they carried out?]

Response 3: [Thanks for your inquiry. In our study, there were two research site: Gongzhuling and Hainan. The majority of the experiments were conducted in experimental fields of the Institute of Crop Germplasm Resources, Jilin Academy of Agricultural Sciences (Northeast Agricultural Research Center of China), located in Gongzhuling city. The experimental fields in Hainan were used solely for breeding purposes. The EMS treatment of Jilv 10 began in 2018, and the acquisition of the homozygous sterile mutant was achieved in 2022. Further cell biology research and analysis were conducted between 2023 and 2024. I hope this response adequately addresses your question.This part of the content has been added to the manuscript.]

Comments 4: [Please attach a brief description of the mung bean variety tested.]

Response 4: [Thank you for your interest and curiosity about our variety. The Jilv10 variety used in this research is characterized by a sturdy main stem, robust lodging resistance, yellow flowers, and an extensive root system. This upright mungbean cultivar has an average maturation period of 86 days and a hundred-seed weight of 5.1g. The seeds are rich in starch and protein content, which highlights its superior and well-rounded characteristics. After careful consideration, we have decided not to include an excessive amount of detailed physiological characteristics of Jilv 10 in the text to maintain its fluidity and readability. We sincerely hope you can understand this decision. I hope this response fully answers your question.]

Comments 5: [Please provide the full name of the producer of the statistical software used to analyse the data.]

Response 5: [In this study, the only software used for statistical data analysis is WPS Office, developed by Kingsoft Corporation.This part of the content has been added to the article at line 163.]

Comments 6: [Large number of publications (68). Some of them were published more than 10 years ago and some even in the 20th century. These I propose to delete and limit to the most recent.]

Response 6: [Thank you for suggesting that we refresh our reference list with more recent literature and consider the removal of older citations. We have taken your advice seriously and appreciate the importance of staying current in our field. Simultaneously, we believe that some historically significant papers continue to provide a solid theoretical foundation for current research. Their sustained impact and frequency of citation in academic discourse highlight their persistent relevance. Finally, following your suggestion, we have removed three older references (dating from 1968, 2000, and 2005) and retained other necessary older literature. Additionally, we have added two recent and relevant references (from 2019 and 2021) to ensure our research both respects the historical context and looks forward to future developments. We are grateful for your support of our research.]

The attachment contains the manuscript with highlighted revisions to facilitate your clear review of the updates we have made.

 

Author Response File: Author Response.pdf

Reviewer 4 Report

Comments and Suggestions for Authors

Dear Editor,

I am pleased to have reviewed the manuscript "Cytological characterization of vrnp 1, a pollen-free male sterile mutant in mung bean (Vigna radiata)." The manuscript is interesting due to its potential to enhance food production. However, despite its significance, some points still need clarification in the manuscript, mainly to ensure its reproducibility.

Abstract
No comments.

Introduction
The authors provide an introduction that contextualizes the study; however, some points should be addressed:

1. "Mung bean seems to be widely welcomed by the general populace, especially in the post-epidemic era [9]." The introduction is sometimes vague, e.g., the text described does not provide measurable information and is overly general. It could be rewritten to emphasize specific data.
2. I noticed some issues with the flow. While important topics such as the utility of mung bean and the importance of male sterility are mentioned, some arguments are not fully developed. For example, the discussion on the tapetum and pollen wall development is technical but does not clearly establish why these topics are relevant to current research in mung bean.
3. Some concepts are repeated without adding new information. For instance, the role of the tapetum and pollen walls could be condensed into a single, clearer paragraph.
4. The final statement about the study’s findings is useful but should be moved to the "Results" section or briefly summarized here as an objective.

Materials and Methods

The methodology presented is detailed but has some omissions, inconsistencies, and areas that could benefit from greater clarity or justification. Here are some points for improvement:

2.1. Induction of mutants using EMS

1. The EMS volume used is not mentioned, which could be important for reproducibility. Likewise, environmental conditions should be included.
2. It is stated that materials were cultivated and raised from 2018 to 2023, but the duration of each cultivation cycle is not specified (e.g., how many months in the field per generation?).
3. It is also unclear whether there were periods of seed storage or dormancy between cycles.
4. The light conditions (photoperiod, light intensity in lux, or type of lighting), temperature, and humidity in which the plants were cultivated are not detailed. These variables are critical, as they can influence development and trait expression.
5. The specific soil conditions or whether soil analysis was conducted before planting are not described.
6. Although “standard field management” is mentioned, specific practices are not detailed, such as irrigation (frequency, method), fertilization (type, dosage), or pest and disease management.
7. It is not indicated whether coverings or greenhouses were used or if the plants were completely exposed to natural conditions.
8. The number of seeds treated with EMS and the proportion of mutagenized seeds that germinated, survived, or exhibited sterile traits in the M2 generation are not detailed.
9. There is no mention of tests to evaluate the success of EMS treatment, such as expected mutation rates.

2.2. Genetic analysis of male sterile mutants

1. It is not explained how fertile heterozygous and sterile individuals were selected for crossings. Nor is it described whether the crossings were performed manually or if any specific technique was used to prevent undesired pollination.
2. While segregation and F2 analysis are mentioned, it is not specified whether F2 generations were grown under the same conditions as parental generations or if there were changes.
3. The total size of the F2 population analyzed (number of fertile vs. sterile plants) is not indicated.
4. It is not explained how crossing success was measured or how potential errors during pollination were controlled.

2.3. Observation of anther paraffin sections

1. The software used should include the version.
2. Criteria for selecting anthers (size, developmental stage) and the number of anthers per sample are not described. Nor is it clear whether samples came from different plants or were limited to a single individual.
3. Preliminary tests to optimize fixation time in FAA, section thickness, or staining times are not mentioned.
4. It is not indicated whether staining solutions were prepared fresh or stored, which could affect results.
5. Magnifications used and whether images were taken under standardized conditions (such as lens type or lighting parameters) are not provided.

2.4. Observation of pollen grains

1. While it is mentioned that inflorescences were collected at 9 a.m., no justification is provided for this timing, nor whether variability in pollen quality depended on climate or season.
2. It is not specified how long inflorescences were stored in 75% ethanol or whether this duration could have affected pollen quality or staining.

2.5. Observation of pollen wall

1. The number of anthers or pollen grains processed for pollen wall analysis is not mentioned, nor whether controls, such as known fertile varieties, were included.
2. It is not indicated whether samples were coated with gold or another conductive material before analysis.
3. While morphological analysis of the pollen wall is mentioned, the criteria used to classify or describe observations are not explained.

Results
No comments.

Discussion
The discussion is interesting; however, the manuscript needs improvement to strengthen it.

1. In the statement "we have developed genetically stable male-sterile progenies," there is no information on how many generations were evaluated or how genetic stability was determined.
2. There are insufficient or missing references in several sections, such as "ultimately leading to microspore degeneration," which weakens the discussion.
3. There is a lack of depth in comparisons with other studies. While genes like NEF1, ROXY1, and HKM are mentioned, an exhaustive comparison of how the vrnp 1 mutant phenotype differs from or resembles previously described mutants is absent. A detailed comparison would strengthen the study's relevance.
4. Several phrases repeat similar ideas, such as "delayed programmed cell death in the tapetal layer" and "the failure of pollen grains to absorb degradation products." Reducing these redundancies would make the discussion more concise.
5. Limitations of the study are not mentioned; that is, methodological limitations, such as potential errors in staining or biases in microscopic observations, are not discussed. Including these limitations would demonstrate greater scientific rigor.
6. The statement "the inability to thicken the pollen wall is due to the failure of pollen grains to absorb degradation products from the tapetum" does not explain why the degradation products are not absorbed. This requires more detail.
7. The mention of "RNA-seq analysis" and "Mut-map sequencing" as future steps is valuable, but it does not explain how they will be implemented or what type of data is expected. A more concrete description would be beneficial.
8. It is recommended to review the text. In some sections, the logical flow between sections is interrupted, such as in the transition from findings related to the tapetum to genetic references. Reorganizing these ideas could improve clarity.
9. Although figures 3 and 5 are repeatedly mentioned, the key results that support the conclusions are not sufficiently described. The connection between visual findings and conclusions needs to be more explicit.
10. While the mutant is mentioned as "promising for hybrid vigor," there is no discussion on how these findings could be implemented in mung bean breeding programs.

Conclusions
No comments.

References
No comments.

The manuscript addresses an interesting topic. However, the methodology and discussion sections still need improvement to strengthen the document.

 

Comments for author File: Comments.pdf

Author Response

We are deeply grateful for your thorough review and the time you have invested in providing such detailed feedback. Your insightful comments have been instrumental in refining our work. Below, we provide our detailed responses, explanations for some sections, and descriptions of the revisions.

Comments 1: [Mung bean seems to be widely welcomed by the general populace, especially in the post-epidemic era [9]." The introduction is sometimes vague, e.g., the text described does not provide measurable information and is overly general. It could be rewritten to emphasize specific data.]

Response 1: [Thank you for your thorough review of our manuscript. We have referred to the data on mung bean consumption from Allied Market Research, MarkWide Research, and the National Bureau of Statistics of China for the years 2018 to 2024. We have observed that the demand for mung beans has increased, particularly after 2022. However, we agree with your advice on maintaining the rigor of citing information, and we have revised the relevant statement. These revisions are located at lines 36-38 in the updated manuscript.]

 

Comments 2: [I noticed some issues with the flow. While important topics such as the utility of mung bean and the importance of male sterility are mentioned, some arguments are not fully developed. For example, the discussion on the tapetum and pollen wall development is technical but does not clearly establish why these topics are relevant to current research in mung bean.]

Response 2: [This study primarily explores the cytological basis of the vrnp 1 male sterility mutant, which appears to be associated with developmental defects in the tapetum and pollen wall. In the introduction, we focused on citing research papers that investigate these two structures, given their importance for fertility. As research on male sterility in mung beans is relatively limited, we have also referred to studies on model plants and other crops to provide a broader context. We hope that the revisions and responses made in the revised manuscript will adequately address the previous shortcomings in our language expression.]

 

Comments 3: [Some concepts are repeated without adding new information. For instance, the role of the tapetum and pollen walls could be condensed into a single, clearer paragraph.]

Response 3: [Thank you for your thoughtful consideration of the overall structure of the manuscript and the frequency of key terms. We have revised the text to better integrate the roles of the tapetum and pollen wall, in line with your suggestions. These revisions are located at lines 59, 62-64, 72-95, 114-127 in the updated manuscript.]

 

Comments 4: [The final statement about the study’s findings is useful but should be moved to the "Results" section or briefly summarized here as an objective.]

Response 4: [Thank you for your thoughtful input on the manuscript structure. We have followed your advice and simplified the presentation of results in the introduction section.These revisions are located at lines 144-153 in the updated manuscript.]

 

Comments 5: [The EMS volume used is not mentioned, which could be important for reproducibility. Likewise, environmental conditions should be included]

Response 5: [The EMS seed treatment involved approximately 1000 seeds per group, while the control group consisted of about 400 seeds. Given the randomness and irreproducibility of EMS mutagenesis, and the lack of a direct correlation between the mutagenesis volume and the phenotypes of the mutants in this study, we chose not to elaborate further on this detail. We sincerely hope you can understand this decision.]

 

Comments 6: [It is stated that materials were cultivated and raised from 2018 to 2023, but the duration of each cultivation cycle is not specified (e.g., how many months in the field per generation?).]

Response 6: [The growth period for our summer-sown materials spans from May to August each year, while the winter breeding in Hainan (South Breeding Program) occurs from November to January. These revisions are located at lines 167-168 in the updated manuscript.]

 

Comments 7: [It is also unclear whether there were periods of seed storage or dormancy between cycles.]

Response 7: [In our routine work, all harvested seeds are stored, dried, and labeled in the seed room without any differential treatment. In this experiment, both Jilv 10 and vrnp 1 were sown and harvested together. Across each generation, from sowing to harvesting, we have not observed any phenotypic differences attributable to seed storage. Therefore, we chose not to elaborate further on this point. We sincerely hope you can understand this decision.]

 

Comments 8: [The light conditions (photoperiod, light intensity in lux, or type of lighting), temperature, and humidity in which the plants were cultivated are not detailed. These variables are critical, as they can influence development and trait expression.]

Response 8: [Thank you for your meticulous suggestions, which helped address the insufficiencies in our statements. All materials used in this study were cultivated in open-air experimental fields. The changes related to this are reflected in line 165 of the updated manuscript. We didn’t measure daily light intensity, humidity, or temperature in the open-air environment. These values, which vary with daily climate and weather conditions, are unlikely to have a significant impact on the emergence of mutant phenotypes. Given this, we felt there was no need to highlight these aspects further. We sincerely hope you can understand this decision.]

 

Comments 9: [The specific soil conditions or whether soil analysis was conducted before planting are not described.]

Response 9: [We have carefully considered this suggestion. In this study, the experimental fields used for growing plant materials are the same fields employed for routine breeding activities. Each year, we use relatively fixed plots within our institution that maintain a stable soil environment. The plant materials in this study were cultivated on adjacent ridges without any separate treatment. Given these conditions, we did not conduct soil condition analyses or pre-planting soil assessments under normal growth conditions. Moreover, we believe that soil conditions and soil analysis data are not relevant to the cytological defects causing male sterility between the mutants (as no phenotypic changes were observed before between vrnp 1 mutants). Therefore, we chose not to elaborate further on this point. We sincerely hope you can understand this decision. ]

 

Comments 10: [Although "standard field management" is mentioned, specific practices are not detailed, such as irrigation (frequency, method), fertilization (type, dosage), or pest and disease management.]

Response 10: [Thank you for your meticulous suggestions on the Materials and Methods section. we have carefully considered these suggestions. All plantings for this study were conducted within our open-air experimental fields, where management practices included applying basal fertilizer and routine care. In cases of extreme weather, we manually watered the plots and applied pesticides specifically to areas affected by pests or diseases. However, for the two materials in our study, no additional or specialized fertilization or pesticide treatments were applied. Moreover, over the years of planting and experimentation, no phenotypic instability has been observed in these materials. Due to space constraints, we chose not to include more detailed descriptions of these practices in the manuscript. And we feel that it may not align perfectly with the overall objectives of our study. We sincerely hope you can understand this decision. ]

 

Comments 11: [It is not indicated whether coverings or greenhouses were used or if the plants were completely exposed to natural conditions.]

Response 11: [All experimental materials in this study were grown in open-air experimental fields. These revisions are located at line 165 in the updated manuscript.]

 

Comments 12: [The number of seeds treated with EMS and the proportion of mutagenized seeds that germinated, survived, or exhibited sterile traits in the M2 generation are not detailed.]

Response 12: [The experimental materials used in this study were derived from EMS-mutagenized seeds. The EMS seed treatment involved approximately 1000 seeds per group, while the control group consisted of about 400 seeds. Due to the large number of mutagenized seeds, we sowed and phenotyped the M0-M1 materials in batches. Given the phenotypic uncertainty and irreproducibility associated with EMS mutagenesis, we did not conduct germination or sowing trials for all M2 generations prior to sowing.In this paper, we would like to highlight that we obtained a single male-sterile mung bean mutant within the M2 population and promptly preserved the germplasm. While we recognize the potential interest in the phenotypic classification and data statistics of other M2 materials, we have decided not to elaborate on these aspects, as they have limited relevance to the cytological defects that are the primary focus of our study. And we feel that it may not align perfectly with the overall objectives of our study. We sincerely hope you can understand this decision.]

 

Comments 13: [There is no mention of tests to evaluate the success of EMS treatment, such as expected mutation rates.]

Response 13: [Thank you for your comments. Before we identified the male-sterile mutant discussed in this paper, we didn’t carry out sowing and phenotypic evaluation for all the EMS-mutagenized seeds. Our initial aim in conducting EMS mutagenesis was to obtain mutants with superior agronomic traits or those suitable for breeding purposes. So, after we identified the male-sterile mutant in the M2 generation through batch screening, we focused on studying the sterility mechanism of this mutant.Given the randomness of EMS mutagenesis and the irreproducibility of the traits, we also obtained other mutants with reduced fertility or increased stem hardness. Compared to their mutagenic background, Jilv 10, these materials exhibited phenotypic stability. We hope this response fully answers your question.]

 

Comments 14: [It is not explained how fertile heterozygous and sterile individuals were selected for crossings. Nor is it described whether the crossings were performed manually or if any specific technique was used to prevent undesired pollination.]

Response 14: [As noted in the text, “we proceeded to cross the progeny utilizing the heterozygous fertile and sterile plant lines as parents to breed the generations” (lines 177-178), we carried out crosses with heterozygous fertile and sterile plants as the parental lines. Fertility and sterility were determined by microscopic examination of pollen grains stained with I₂-KI. Given the strict self-pollinating nature of mung beans, hybridization was performed manually. These revisions are reflected in line 175 of the revised manuscript.]

 

Comments 15: [While segregation and F2 analysis are mentioned, it is not specified whether F2 generations were grown under the same conditions as parental generations or if there were changes.]

Response 15: [Thank you for your comment and detailed feedback. In each of our sowing and phenotypic observation experiments, the mutagenic background, Jilv 10, was sown simultaneously and in the near place with the mutant to serve as a phenotypic control. Since we aim to utilize this male-sterile mutant for future hybrid breeding in mung beans, we are particularly concerned about whether the emergence of this phenotype might affect other phenotypic traits of the mung bean. As described in the text, “no fertility restoration was observed in vrnp 1, thereby confirming the stable, pollen-free male sterility trait” (lines 230-231), and “Up to this stage, no notable developmental differences appeared to be observed between the vrnp 1 mutant and Jilv 10 plants” (lines 283-284), the mutant exhibits stable male sterility without any impact on other phenotypes beyond fertility. The revisions related to this point can be found on lines 224-225 of the revised manuscript.]

 

Comments 16: [The total size of the F2 population analyzed (number of fertile vs. sterile plants) is not indicated.]

Response 16: [Thank you for your review. As shown in Table 1 (on line 232), among 286 F2 plants, we identified 214 fertile plants and 72 sterile plants. This phenotypic segregation ratio of 3:1 is consistent with the expected pattern for a trait controlled by a recessive gene.]

 

Comments 17: [It is not explained how crossing success was measured or how potential errors during pollination were controlled.]

Response 17: [Thank you for your meticulous attention to the accurate presentation of the experimental results and for your professional suggestions. Given the strict self-pollinating nature of mung beans, hybridization must be performed manually. Flower buds that have been emasculated (such as the sterile vrnp1 mutant, which cannot produce seeds) are incapable of yielding seeds. When pollinating with the fertile Jilv 10 as the male parent, if the stigma of the hybridized flower bud swells within two days, this serves as a preliminary indication that the hybridization may have been successful. We then continue with subsequent observations and management until the pod forms and the seeds mature. The hybrid pod is harvested for further planting, and completing this process is regarded as a successful hybridization. During the pollination process, similar to artificial hybridization in other crops, potential sources of error include incomplete emasculation and selecting the female parent at a later stage, both of which can affect the success of the pollination. Since manual hybridization of mung beans is a relatively routine breeding procedure, we did not provide an extensive description here. We sincerely hope this response fully answers your question and you can understand this decision.]

 

Comments 18: [The software used should include the version.]

Response 18: [These revisions are located at line 202 in the updated manuscript.]

 

Comments 19: [Criteria for selecting anthers (size, developmental stage) and the number of anthers per sample are not described. Nor is it clear whether samples came from different plants or were limited to a single individual.]

Response 19: [In the absence of specific references for the developmental stages of mung bean anthers, we used the well-documented stages of Arabidopsis thaliana anthers as a reference and conducted a detailed comparison for each corresponding stage. The developmental patterns of mung bean anthers closely parallel those of Arabidopsis.These revisions are located at lines 202-203 in the updated manuscript.]

 

Comments 20: [Preliminary tests to optimize fixation time in FAA, section thickness, or staining times are not mentioned.]

Response 20: [FAA fixative is a common fixative in tissue biology, used for various plant tissues such as roots, stems, leaves, anthers and ovaries. The ratio of its reagent components is more critical than the duration of preservation. As shown in the text “FAA fixative solution (70% ethanol 90mL + glacial acetic acid 5mL + formalin 5mL) ” (on lines 186-187), the FAA fixative we prepared was specifically adjusted to suit the tenderness of mung bean anther tissues. The I₂-KI and acetocarmine staining methods were chosen based on preliminary tests conducted before the main experiments. After confirming the expected results through preliminary experiments with a small number of pollen grains and anther sections, we proceeded with the experimental procedures described in the “Materials and Methods” section of this paper. All experiments were strictly conducted according to the principles of single-variable experimental design. We hope our responses can address your concerns.]

 

Comments 21: [It is not indicated whether staining solutions were prepared fresh or stored, which could affect results.]

Response 21: [In this study, staining reagents such as potassium iodide (I₂-KI), acetocarmine staining solution, and Safranine O-fast Green staining solution were prepared and stored strictly according to the recommended conditions (e.g., avoiding light and using them shortly after opening). This adherence ensured the reliability and reproducibility of our experimental results. We sincerely hope this response fully address your concerns.]

 

Comments 22: [Magnifications used and whether images were taken under standardized conditions (such as lens type or lighting parameters) are not provided]

Response 22: [In addition to the scale bars shown in each image, the staining observations of pollen grains were conducted using a microscope at 40× magnification. For scanning electron microscopy (SEM), the magnification of the images we present ranges from 100-10000×, depending on the specific areas and objectives to be highlighted. Therefore, to ensure the accuracy of the images, we think, the scale bars in the figures provide a more intuitive and accurate depiction of the proportional relationships within the anther structures. We sincerely hope this response fully answers your question and you can understand this decision.]

 

Comments 23: [While it is mentioned that inflorescences were collected at 9 a.m., no justification is provided for this timing, nor whether variability in pollen quality depended on climate or season.]

Response 23: [Thank you very much for your valuable comments and suggestions. The peak flowering period for mung beans is typically in July, with flowers blooming between 8 a.m. and 11 a.m. Collecting flower samples too early in the morning may cause pollen to clump due to heavy dew, while collecting in the afternoon or later can result in closed flower buds. Both situations can impact the quality of material collection. Either situation can affect the quality of material collection. To maintain consistency in our sampling results, we collect materials around 9 a.m. We sincerely hope this response fully address your concerns.]

 

Comments 24: [It is not specified how long inflorescences were stored in 75% ethanol or whether this duration could have affected pollen quality or staining.]

Response 24: [To prevent dehydration and deformation, the inflorescences collected from the field were immediately preserved in a 75% alcohol solution and transported to the laboratory for further analysis. Since the collection, staining, and microscopic examination were performed in real-time as the experiment progressed, prolonged storage was not an issue. Additionally, we have confirmed that storing flower buds in 75% alcohol for up to two days does not affect the staining efficacy of I₂-KI or acetocarmine staining solution. All experimental materials in this study were collected and examined on the same day. We sincerely hope this explanation helps to address your concerns.]

 

Comments 25: [The number of anthers or pollen grains processed for pollen wall analysis is not mentioned, nor whether controls, such as known fertile varieties, were included.]

Response 25: [In the section on pollen wall observation methods, to maintain the logical structure of the paper, we focused solely on describing the methods used for observing the pollen wall. Addressing your concerns, the relevant results are presented in the Results section. For example, as mentioned from line 419, “we conducted scanning electron microscopy (SEM) on two distinct sets of samples” and from line 445, “The three photographs displayed the same anther at varying magnification levels”. We performed SEM on more than three sets of materials for both Jilv 10 (the mutagenic background, serving as the control) and vrnp1. We hope this explanation addresses your concerns.]

 

Comments 26: [It is not indicated whether samples were coated with gold or another conductive material before analysis.]

Response 26: [In this experiment, for the conductive treatment of samples in the scanning electron microscopy (SEM) section, we adhered the samples to a conductive carbon adhesive tape and mounted them onto the sample stage of the ion sputtering instrument. The samples were then coated with gold for approximately 30 seconds.]

Comments 27: [While morphological analysis of the pollen wall is mentioned, the criteria used to classify or describe observations are not explained.]

Response 27: [In the Methods section, we focused solely on describing the observation methods. To address your concerns, we have detailed the developmental stages of the materials used for pollen wall morphology analysis in the text, specifically in “In the imminently blooming buds” (on lines 420-421) and “upon examination of smaller buds beyond the tetrad stage” (on lines 430-431). The rationale for this analysis is that both our anther sectioning results and pollen grain observations indicate that developmental abnormalities in this male-sterile mutant occur after the tetrad stage. Additionally, the disappearance of petrified pollen grains in the anthers before flowering prompted us to conduct a more intuitive examination of “the imminently blooming buds” We hope this explanation addresses your concerns.]

 

Comments 28: [In the statement "we have developed genetically stable male-sterile progenies," there is no information on how many generations were evaluated or how genetic stability was determined.]

Response 28: [We apologize that the timeline was not clearly presented in the manuscript. Here is a clearer summary: In 2018, we conducted EMS mutagenesis and sowed a portion of the M1 mutant seeds for phenotypic observation, using control materials as a reference. In 2019, we grew the M2 generation and found a male sterile mutant. To preserve this germplasm, we immediately crossed it with Jilv 10, producing F1 hybrid pods. We then grew the F1 generation in Hainan and harvested the F2 seeds in the same year. In 2020, we planted the F2 generation and statistically analyzed the phenotypic segregation ratio. Since then, as part of our ongoing breeding efforts, we have continued to multiply seeds and observe phenotypes in both Gongzhuling and Hainan. We used microscopy and I₂-KI staining to assess pollen grain fertility and selected fertile plants from the same ridge to pollinate the sterile plants based on experimental results. By 2022, in the F6 generation, we confirmed the sterile plants as vrnp 1, based on the stable male sterile phenotype observed since the F2 generation. We hope this detailed explanation clarifies any confusion.]

 

Comments 29: [There are insufficient or missing references in several sections, such as "ultimately leading to microspore degeneration," which weakens the discussion.]

Response 29: [We apologize that we didn’t understand your question regarding this part. The reference we cited describes male sterile mutants with defective tapetal development reported by other researchers—distinct from our mutant. The phrase “ultimately leading to microspore degeneration” is a summary of those studies and does not pertain to our mutant. The unique cause of male sterility in our vrnp 1 mutant, which sets it apart from others, is precisely what makes this completely sterile mutant valuable. We sincerely hope this clarifies your concerns.]

 

Comments 30: [There is a lack of depth in comparisons with other studies. While genes like NEF1, ROXY1, and HKM are mentioned, an exhaustive comparison of how the vrnp 1mutant phenotype differs from or resembles previously described mutants is absent. A detailed comparison would strengthen the study's relevance.]

Response 30: [Upon obtaining this completely sterile male-sterile material, we focused on identifying the underlying causes of this sterility, including cytological factors (which we have already characterized) and molecular mechanisms (which are still under investigation). After presenting our experimental results in this study, we reviewed the literature on tapetal development defects and pollen wall abnormalities. The cytological defects in our study are not similar to those associated with genes such as NEF1, ROXY1, and HKM, as described in the literature. However, by comparing our findings with these related studies , we can see that our male-sterile mutant differs from the mutations of the aforementioned genes. This distinction highlights the unique value of our research. As a result, we decided not to explore these similarities and differences in greater detail. Moving forward, genes involved in tapetal development, such as NEF1, ROXY1, and HKM, will likely be important in our future genetic analyses. We hope this explanation may provides some clarification.]

 

Comments 31: [Several phrases repeat similar ideas, such as "delayed programmed cell death in the tapetal layer" and "the failure of pollen grains to absorb degradation products."Reducing these redundancies would make the discussion more concise.]

Response 31: [Thank you for your suggestion on making the discussion more concise. We have prudently made the necessary deletions, and the revised sections can be found on lines 503-505. ]

 

Comments 32: [Limitations of the study are not mentioned; that is, methodological limitations, such as potential errors in staining or biases in microscopic observations, are not discussed. Including these limitations would demonstrate greater scientific rigor.]Response 32: [Thank you for your suggestion. We have thoughtfully considered your feedback and fully recognized the importance of acknowledging the limitations of our study to ensure transparency and integrity. We have carefully considered your advice and incorporated additional text to address this. These revisions can be found on lines 534-539.]

 

Comments 33: [The statement "the inability to thicken the pollen wall is due to the failure of pollen grains to absorb degradation products from the tapetum" does not explain why the degradation products are not absorbed. This requires more detail.]

Response 33: [Thank you for your detailed question. In this part, our cytological experiments suggest that the failure of the pollen wall to thicken may be due to the inability of pollen grains to absorb the degradation products of the tapetum. We plan to explore the underlying reasons further through future molecular experiments, as a cytological perspective alone cannot fully explain the mechanisms behind a developmental defect. Based on our current findings, delayed tapetal degradation seems to prevent the release of various substances necessary for microspore maturation. This, in turn, could lead to the absence of mature pollen grains and result in male sterility. We hope this explanation helps to clarify your doubts.]

 

Comments 34: [The mention of "RNA-seq analysis" and "Mut-map sequencing" as future steps is valuable, but it does not explain how they will be implemented or what type of data is expected. A more concrete description would be beneficial.]

Response 34: [For the RNA-seq analysis, we are working to identify differentially expressed genes during key developmental stages, as well as those downstream genes of the male sterility gene. With Mut-map sequencing, we hope to use this technique to accurately point the location of the gene in vrnp 1 mutant. Validating these genes will likely have a significant impact on the planning of our future experiments. As mentioned in the text (lines 524 and 529), We have already outlined our initial thoughts and plans for this aspect of the work. We sincerely hope this explanation provides some clarity.]

 

Comments 35: [It is recommended to review the text. In some sections, the logical flow between sections is interrupted, such as in the transition from findings related to the tapetum to genetic references. Reorganizing these ideas could improve clarity.]

Response 35: [Thank you for your suggestions on improving the logical flow of the manuscript. We have incorporated your feedback and made revisions to enhance the coherence of the text. These changes can be found on lines 508-509.]

 

Comments 36: [Although figures 3 and 5 are repeatedly mentioned, the key results that support the conclusions are not sufficiently described. The connection between visual findings and conclusions needs to be more explicit.]

Response 36: [Thank you for your suggestion regarding the need to more closely link the conclusions to Figures 3 and 5. We have carefully considered your advice and appreciate your input. In the Results section, we have already provided detailed descriptions of the developmental differences between Jilv 10 and vrnp 1 at various stages of anther development (Figures 2-3), as well as the morphological differences in pollen grains within the anthers when developmental abnormalities occur (Figure 5).To keep the Conclusions section concise and avoid redundancy, we have decided not to repeat specific figure references there. Instead, we have added more descriptive language in the Results section to better link the “visual findings” with the results, as you suggested. The revised sections can be found on lines 360-361, 429-430, 432-433, 435-436. We hope these changes effectively address your concerns. Thank you again for your valuable feedback.]

 

Comments 37: [While the mutant is mentioned as "promising for hybrid vigor," there is no discussion on how these findings could be implemented in mung bean breeding programs.]

Response 37: [Thank you for your interest in our future plans. We do plan to map the male-sterility gene in mung beans to develop male-sterile lines with desirable agronomic traits. Moreover, the complete male sterility of this mutant significantly reduces the labor required for manual emasculation, which is highly beneficial for mung bean breeding programs. However, this paper is focused solely on the current causes of sterility in the mutant and the upcoming gene mapping. We have not yet implemented plans for further applications. To avoid overstating the role of the sterile mutant in this paper, we decided not to include more future plans at this stage. We hope this makes sense.]

Author Response File: Author Response.pdf

Round 2

Reviewer 4 Report

Comments and Suggestions for Authors

Dear Editor,

I have thoroughly reviewed the manuscript once again and appreciate the authors' efforts in addressing the observations I previously made. The document has significantly improved since the initial revision, and the authors have successfully incorporated my suggestions. The manuscript is clear, well-written, and of interest to the readers. Therefore, I believe it is now ready to be accepted for publication in its current form.