Characterization of Sugarcane Germplasm for Physiological and Agronomic Traits Associated with Drought Tolerance Across Various Soil Types

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

There are some suggested revisions as follows:

 

1. Keywords

It is suggested to add the keywords "soil type", "genotype", and "physiological characteristic", and delete the keywords "NDVI", "SPAD", and "HGR".

 

2. Line 48 on page 2

It is suggested that "yield and adaptation" be revised to "adaptation and yield", because the yield should be regarded as the result.

 

3. Lines 58-59 on page 2

Although it is generally believed that the SPAD value is closely related to the chlorophyll content in leaves, for some plants, the relationship between the two is not a simple linear one. Therefore, it is suggested that the author specify the relationship between the two in sugarcane (for example, a strong positive correlation).

 

4. Introduction

It is suggested to add a paragraph to explain the necessity of conducting experiments in different types of soil in this study.

 

5. Line 80 on page 2

It seems that "In sandy loam soil" was omitted before "The minimum daily air temperature ranged from 9.4 to 29.4°C". It is suggested to take supplements.

 

6. Figure 7

From the perspective of facilitating readers' reading, it is suggested that the depth of the color be revised to be positively correlated with the magnitude of the correlation.

 

7. Lines 192 on page 8

Suggest italicizing the letter “p”.

 

     8. Conclusion:

This section contains too much content. It is recommended to simplify it.

Author Response

Comments and Suggestions for Authors

There are some suggested revisions as follows: 

1. Keywords

It is suggested to add the keywords "soil type", "genotype", and "physiological characteristic", and delete the keywords "NDVI", "SPAD", and "HGR".  

                Response: We would like to thank you for your valuable suggestions. We have replaced the keywords for better, "soil type", "genotype", and "physiological characteristic" [Page 1: Line 31-32].

 

1. Line 48 on page 2

It is suggested that "yield and adaptation" be revised to "adaptation and yield", because the yield should be regarded as the result.  

                Response: Thank you for pointing out this issue. We have replaced “adaptation and yield” with “yield and adaptation” to reflect the result [Page 2: Line 49].

 

1. Lines 58-59 on page 2

Although it is generally believed that the SPAD value is closely related to the chlorophyll content in leaves, for some plants, the relationship between the two is not a simple linear one. Therefore, it is suggested that the author specify the relationship between the two in sugarcane (for example, a strong positive correlation)

                Response: We sincerely appreciate your valuable comments and suggestions. We have added more details about the relationship between SPAD and chlorophyll content in sugarcane. “SCMR in sugarcane has a significant positive correlation with the chlorophyll content in the leaves under drought conditions” [Page 2: Line 59-60].

4. Introduction

It is suggested to add a paragraph to explain the necessity of conducting experiments in different types of soil in this study. 

                Response: We have added a paragraph to explain the necessity of conducting experiments in different types of soil “Conducting experiments in different soil types is crucial for assessing sugarcane genotype stability under drought. Soil properties influence water availability and plant stress responses. Sandy soil loses moisture more quickly than loam, worsening drought conditions [21].  Studies by [22] showed that soil type alters physiological and agronomic responses. Thus, including multiple soil types improves genotype evaluation and broadens applicability across environments [Page 2: Line 74-79].

5. Line 80 on page 2

It seems that "In sandy loam soil" was omitted before "The minimum daily air temperature ranged from 9.4 to 29.4°C". It is suggested to take supplements. 

                Response: Thank you for pointing this out. We have added “In sandy loam soil” before "The minimum daily air temperature ranged from 9.4 to 29.4°C" [Page 2: Line 88].

 

6. Figure 7

From the perspective of facilitating readers' reading, it is suggested that the depth of the color be revised to be positively correlated with the magnitude of the correlation. 

        Response:  We sincerely appreciate your valuable comments and suggestions. We have revised the color depth in Figure 7 to be positively correlated with the magnitude of the correlation.

 

7. Lines 192 on page 8

Suggest italicizing the letter “p”.  

Response: We would like to thank you for your valuable suggestions. We have replaced the italicizing the letter “p” in the detail. “p < 0.05 and p < 0.01” [Page 9: Line 333].

 

8. Conclusion:

This section contains too much content. It is recommended to simplify it.

Response: We have revised and simplified the content to improve readability and focus on the key points. Redundant explanations and overly detailed descriptions have been removed to ensure the section is more concise and aligned with the journal’s expectations. The revised version can be found on Page 12: Line 518-520. “In loam soil, MPT13-118, MPT07-1, and Q47 belonged to cluster 1, while F153 and CP75-330 were in cluster 2. MPT07-1 not only excelled in yield but also in drought tolerance, whereas K88-92 struggled under drought conditions” 

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

The paper needs improvement to meet the publication criteria imposed by Stresses journal
The author has results without responding to certain data collection methods analyzed.
Here are some of the suggestions that the author should take into account to improve the presentation of the obtained results      (I have attached the pdf paper, where I have placed all the suggestions in the track change):

• The analysis of climatic indicators is not well presented and is not by the experimental data
  he analysis of climatic indicators is not well presented and is not in accordance with the experimental data
  1. The experiment was carried out during November-January so you must provide climatic data from this period
  2. The experiment was carried out over 3 experimental years. Here, you must have monthly averages for each experimental year
  3. To highlight the amounts of precipitation, it is important to highlight the total monthly precipitation over the entire experimental period (3 years) to highlight the moments (maybe even months) where there was a lack of precipitation or small amounts that coincide with certain important vegetation phases for the studied species!
  
  
• All these evaluator suggestions and others are placed in trackchange in the attached document.

• There are no clear data regarding the two types of soil, soil texture (Clay and humus content, Degree of compaction, and soil structure to know exactly where the CO is for the two types of soil.

  Also, the amounts of water used for irrigation for the two types of soil and the equipment for monitoring the soil moisture status during the experimental period are not mentioned.

   

   

  
  

Comments for author File: Comments.pdf

Author Response

Comments and Suggestions for Authors

The paper needs improvement to meet the publication criteria imposed by Stresses journal

Response: We appreciate your observation regarding the need for improvement to meet the publication standards of Stresses journal. In response, we have carefully revised the manuscript to enhance its clarity, completeness, and scientific rigor. We believe that the revised version now aligns more closely with the journal’s requirements and expectations.

The author has results without responding to certain data collection methods analyzed.

Response: We acknowledge the oversight regarding the explanation of certain data collection methods that were used in the analysis. In the revised manuscript, we have now clearly described the methodologies employed for data collection, ensuring that all analyzed results are adequately supported by corresponding methodological details. We believe this clarification enhances the transparency and reliability of the study.

Here are some of the suggestions that the author should take into account to improve the presentation of the obtained results (I have attached the pdf paper, where I have placed all the suggestions in the track change):

1. The analysis of climatic indicators is not well presented and is not by the experimental data

Response: We respectfully believe that the analysis of climatic indicators in the previous version was clearly presented. The climatic data, including detailed information such as rainfall and maximum–minimum temperatures, were aligned with the experimental period. Numbers 1 to 365 in the figure represent the first and last days of the experimental period [Page 2-3 , Lines 88-95]. “In sandy loam soil the minimum daily air temperature ranged from 9.4 to 29.4°C, and the maximum temperature ranged from 19.3 to 40.8 °C (Figure 1a). The total rainfall during the drought stress period was 185.5 mm, while the cumulative rainfall after the drought stress period was 1,302.91 mm during the growing season in sandy loam soil (Figure 1a). In loam soil, the minimum daily air temperature ranged from 10.0 to 29.4 °C and the maximum temperature ranged from 18.9 to 40.0 °C. The total rainfall during the drought stress period was 276 mm in loam soil. In contrast, the cumulative rainfall during the growing season in loam soil after the drought stress period was 950.5 mm (Figure 1b).

2. The experiment was carried out during November-January so you must provide climatic data from this period

Response: We sincerely apologize for the confusion regarding the experimental timeline and conditions. The experiment was indeed conducted from November to January, but it was carried out across two different soil types with slightly different planting schedules.

Specifically:

• In the sandy loam soil, sugarcane was planted and grown from November 2020 to November 2021.
• In the loam soil, planting took place from January 2021 to January 2022.
• Numbers 1 to 365 in the figure represent the first and last days of the experimental period.

We have now revised the manuscript to clearly present this information in the Materials and Methods section [Page 11, Lines 434-437], to avoid any further misunderstanding. We appreciate the reviewer’s attention to this detail, which helped us improve the clarity of our study.

3. The experiment was carried out over 3 experimental years. Here, you must have monthly averages for each experimental year.

Response: We apologize for the confusion. The experiment was conducted under two different soil conditions. In the sandy loam soil, sugarcane was planted and grown from November 2020 to November 2021 and planting in the loam soil was conducted from January 2021 to January 2022 [Page 11, Line 434-437].

 

4. To highlight the amounts of precipitation, it is important to highlight the total monthly precipitation over the entire experimental period (3 years) to highlight the moments (maybe even months) where there was a lack of precipitation or small amounts that coincide with certain important vegetation phases for the studied species!

Response: The experiment was conducted under two different soil conditions. In the sandy loam soil, sugarcane was planted and grown from November 2020 to November 2021 and planting in the loam soil was conducted from January 2021 to January 2022 [Page, Lines]. In response to your suggestion, we have now amounts of precipitation from planted to tillering stage (drought period). “The total rainfall during the drought stress period was 185.5 mm, while the cumulative rainfall after the drought stress period was 1,302.91 mm during the growing season in sandy loam soil (Figure 1a). In loam soil, the minimum daily air temperature ranged from 10.0 to 29.4 °C and the maximum temperature ranged from 18.9 to 40.0 °C. The total rainfall during the drought stress period was 276 mm in loam soil. [Page 2-3, Line 89-95].

5. All these evaluator suggestions and others are placed in track change in the attached document.

Response: Thank you for your valuable suggestions. All your comments and suggestions, as well as additional modifications, have been carefully addressed and are shown using track changes in the revised manuscript, which is attached with this response. We hope these revisions meet your expectations and clarify the points you raised.

 

6. There are no clear data regarding the two types of soil, soil texture (Clay and humus content, Degree of compaction, and soil structure to know exactly where the CO is for the two types of soil.

Response: The soil samples were systematically collected from two experimental plots at depths of 15 cm and 30 cm to assess soil texture composition. The sandy loam with a composition of 70.86% sand, 23.47% silt, and 5.67% clay. In contrast, the loam soil contained 42.14% sand, 33.54% silt, and 24.32% clay [Page 11, Line 452-454]. We apologize for not measuring the soil compaction level.

 

7. Also, the amounts of water used for irrigation for the two types of soil and the equipment for monitoring the soil moisture status during the experimental period are not mentioned.

Response: We have now included detailed information regarding the amounts of water used for irrigation for the two soil types. “Water was supplied under the control condition (CT) through 3 irrigation events, with a total cumulative amount of 266.67 mm, which was sufficient to meet the water requirements of sugarcane. For drought conditions (DS), water was withholding water from 0 to 4 months after planting and then re-watering during 2 irrigation events after 4 to 12 months. In sandy loam soil, the cumulative water amount under CT was 1932.86 mm, while under DS it was 1666.19 mm. In loam soil, the cumulative water amount under CT was 1670.95 mm, and under DS it was 1404.28 mm [Page11, Line 441-447].” Additionally, we have described the equipment and methods used to monitor soil moisture status throughout the experimental period. We have now added “The soil moisture content (SMC) was measured 3, 6, and 9 months after planting using the auger gravimetric method at 0-45 cm depths in the soil. Soil samples were weighed and oven-dried at 105 °C for 48 hours. The soil moisture content was calculated through the following equation [Page12, Line 463-466]”: 

 

Author Response File: Author Response.pdf

Reviewer 3 Report

Comments and Suggestions for Authors

This is a nice work but need some improvements before publication. Please, find my specific comments in the PDF attached. 

Comments for author File: Comments.pdf

Author Response

Comments and Suggestions for Authors

This is a nice work but need some improvements before publication. Please, find my specific comments in the PDF attached. 

Response: We appreciate your recognition of the quality of our work and acknowledge that certain improvements are necessary before publication. We have carefully reviewed all your specific comments provided in the attached PDF and have made corresponding revisions to enhance the manuscript. We believe these changes have strengthened the clarity and scientific rigor of the study.

 

1. I suggest the authors remove this part and make the title shorter.

Response: Thank you for your valuable feedback. We believe that the presented content is both accessible for further literature review and highly relevant to readers interested in the classification of parental lines based on agronomic and physiological traits under drought conditions, especially considering soil variability. Therefore, we respectfully suggest retaining this section to support the clarity and relevance of the study. In addition, other reviewers agreed with the chosen research topic.

 

2. Please, just mention the geographic region from where those genotypes were taken.

            Response: We have added supplements about Geographic origins of the genotypes. [Page 11, Line 448]

 

Table S1 List of the 120 sugarcane genotypes accessions used in the study to characterize sugarcane germplasm for physiological and agronomic traits associated with drought tolerance across various soil types.

Variety/Clone/Code	Institution/Country
3-2-023L	Department of Agriculture, Thailand
B41721, B4744, B76718	Barbados
Biotec2, Biotec3, Biotec4, Biotec5	National Science and Technology Development Agency (NSTDA) and Kasetsart University, Thailand
BO14, BO24	Barbados Agricultural Management Company (BAMC)
CAC57-23	U P. College of Agri., Philippines
CN1	Department of Agriculture in Chainat, Thailand
Co1148, Co1287, Co290, Co331	Sugarcane Breeding Institute, Coimbatore India
CP110, CP45-150, CP63-306, CP72-120, CP75-330	United States
D158	British Guyana
DB7160	Guyana and Barbadose
Ebene1/37	Mauritius
Eyos	Colombia
F170, F148, F152, F153, F140, F174, F177	Taiwan
Fiji105	Sugar Research Institute of Fiji (SRIF)
GALOA	Fiji Sugar Corporation (FSC) with the Sugar Research Institute of Fiji (SRIF)
GT29	Sugarcane Research Institute of Guangxi Academy of Agricultural Sciences
IAC49-131, IAC51-205, IAC52-156	Institute Agronomico de Campinas, Brazil
IRK67-1	Unknown
K84-200, K88-92, K92-80, K95-84, K93-219	Kanchanaburi, Office of the Cane and Sugar Board (OCSB), Thailand
KK3	Khon Kaen Field Crops Research Center, Thailand, Thailand
KKU99-01, KKU99-02, KKU99-03, KKU99-06	Khon Kaen University, Thailand
Kps01-12, Kps01-25	Sugarcane and Sugar Research and Development Center, Kam Paeng San Campus, Kasetsart University, Thailand
LF48-8077, LF76-2300, LF79-2964	Fiji
LK92-11	Office of the Cane and Sugar Board, Thailand
M124/59, M13/58	Mauritius
Mackay	Australia
Mossman	Australia
MP1, MP2, MP3, MPT02-458, MPT03-166, MPT04-169, MPT04-55, MT05-187, PT06-171, MPT06-26, MPT06-362, MPT06-367, MPT07-1, MPT07-152, MPT07-71, MPT08-191, MPT08-3, MPT08-50, MPT09-118, MPT09-296, MPT12-1782, MPT13-118, MPT14-1-468, MPT14-1-892, MPT14-1-902, MPT14-5-216, MPT14-6-63, MPT99-1444	Mitrphol Innovation and Research Center, Thailand
My55-14	Cuba
N50-211	South Africa
NCo310, NCo382	India
Phil66-07	Philipines (SRA)
Pindar	Australia
PL310
PR3067	Insular Experiment Station, Rio Piedras, Puerto Rico
PSA63	Philippines Sugar Association, Philippines
Q100, Q115, Q117, Q146, Q208, Q47, Q77, Q81, Q83	Australia
R397	Reunion
ROC1, ROC22, ROC7	Materials imported from Taiwan, China
SP50	Suphan Buri FCRC, DOA, Thailand
TBy20-2248, TBy26-1255	Kasetsart University, Thailand
TUC725, TUC74-6	Argentina
UT1, UT12, UT3, UT5, UT6, UT8	Suphanburi Field Crops Research Center, Department of Agriculture (DOA) Thailand
YASAWA	Sugar Research Institute of Fiji (SRIF)

 

 

3. Soil Moisture Content and Meteorological Conditions subsection would fit better in the site description in materials and methods as these are not results from the study.

Response: Thank you for your valuable suggestion. We have included it in the Results section because the information provides verification of the actual water received under the two irrigation treatments during the experimental period.

 

4. Please increase font size in the axis, it is hard to read and understand as it is right now.

Response: We have revised the figures by increasing the font size on both the X and Y axes to enhance readability and ensure that the graphical presentation is clearer and easier to interpret.

 

5. Discussion is too short and vague. Very descriptive. Lacking a solid and critical discussion with current literature. cites only a very few studies.

                Response: We sincerely appreciate your valuable comment regarding the Discussion section. In response, we have revised and expanded the Discussion to address “The maintenance of growth enhances the plant is capacity to adapt to water deficit stress by modulating nutrient allocation and photosynthetic activity. Moreover, con-tinued growth facilitates the sustained productivity of the crop under drought condi-tions [11,28]. Drought stress adversely affects key physiological indices in plants, in-cluding Fv/Fm, SCMR, and pigment content, all of which tend to decrease under such conditions. These indicators provide valuable insights into plant health and stress re-sponses, supporting the effective development of drought-tolerant cultivars [29].The HHH group employs three acclimation strategies to sustain high cane yields. This group maintained SCMR and NDVI during the water stress period (Figure 5a, b, e, and f and Table 1). Additionally, all sugarcane cultivars exhibited no significant variation in Fv/Fm. (Figure 5c, d). The morpho-physiological traits of sugarcane genotypes deter-mine phenotypic variability in their responses to recovery and drought treatments throughout the growth phase [30,31,32]. Drought stress during the early growth stage significantly affected almost all growth parameters [28]. The results indicated that growth-related traits (HGR) and photosynthesis-related traits (SCMR and NDVI) were affected under early drought stress conditions. This is consistent with the findings of Dinh et al. [33], who reported that drought stress significantly reduced plant height and SPAD Chlorophyll Meter Reading (SCMR) in sugarcane under drought stress at the early growth stage.” [Page 9-10, Line 344-361] and “Drought stress is a major limiting factor for sugarcane productivity. The identification of reliable physiological traits is therefore crucial for breeding programs aimed at en-hancing drought tolerance. This study found that Height Growth Rate (HGR), Nor-malized Difference Vegetation Index (NDVI), and SPAD Chlorophyll Meter Reading (SCMR) are effective non-destructive indicators for evaluating sugarcane genotypes under water-deficient conditions [48]. These traits allow for rapid assessment and are particularly suitable for large-scale germplasm screening. Their application enables precise selection of drought-tolerant genotypes and supports the development and advancement of breeding populations with improved adaptability to drought-prone environments.” [Page 11, Line 422-430]

 

5.1 Besides that, the authors could make a better contextualization and discussion with climate change and the increasing on extreme weather events, including longer drought periods.

            Response: We have added sentence “The large agricultural regions, particularly in tropical and subtropical zones, are expected to experience increased variability in rainfall patterns and prolonged dry pe-riods [24,25], which pose significant challenges to limit sugarcane growth and productivity [11,26,27] [Page 9, Line 339-342].”

 

5.2 Additionally, I miss a bit more on the quality of sugarcane. Do you have data on total sugars? pol? juice purity? etc (no need to add in this article, but it would be interesting)... always remember that at the end of the day the sugarcane will be processed and these parameters are as important as yield.

                Response: The authors acknowledge that quality-related parameters of sugarcane—such as total sugars, pol, and juice purity—are essential and provide valuable supplementary insights for evaluating sugarcane performance alongside yield. However, we focus of the present study was on physiological and agronomic traits associated with drought tolerance across different soil types (to characterize and selection parents in sugarcane breeding for drought tolerance programs). Nonetheless, we recognize the importance of these quality traits and intend to incorporate them in future research to allow for a more comprehensive evaluation of sugarcane genotypes under drought stress conditions.

 

 

 

6. For each location, please add:

6.1 Geographical coordinates;

Response: We have added geographical coordinates follow: In the sandy loam soil (16.311328, 102.191785), sugarcane was planted and grown from November 2020 to November 2021 and planting in the loam soil (16.465202, 102.111236) was conducted from January 2021 to January 2022” [Page 11, Line 434-437].

 

6.2 Type of climate (Köppen?);

Response: We have added type of climate in the sentence “The experiment was conducted under a field trial in a tropical climate zone [Page 11, Line 434].

 

 

 

6.3 Soil characterization: soil type based on an international recognized classification system + at least clay, sand and silt % (physical) + at least pH (chemical). Also add a brief description of the soil preparation.

Response: We have added “The soil samples were systematically collected from two experimental plots at depths of 15 cm and 30 cm to assess soil texture composition. The sandy loam with a composition of 70.86% sand, 23.47% silt, and 5.67% clay. In contrast, the loam soil contained 42.14% sand, 33.54% silt, and 24.32% clay” [Page 11, Line 451-454].

 

7. Please, double-check these abbreviations: you are using H for all.

Response: We apologize for any confusion caused. We have removed the mentioned text and replaced it with the following: The subplot included 120 sugarcane genotypes evaluated during the formative stage at two field locations. “These genotypes exhibited varying degrees of drought tolerance, as evidenced by differences in cane yield and drought tolerance index under drought conditions” [Page 11, Line 449-450].

 

8. Based on previous study? on This study? Please, make it clear.

Response: We apologize for any confusion caused. We have removed the mentioned text and replaced it with the following: The subplot included 120 sugarcane genotypes evaluated during the formative stage at two field locations. These genotypes exhibited varying degrees of drought tolerance, as evidenced by differences in cane yield and drought tolerance index under drought conditions in this study conditions [Page 11, Line 447-450].

Author Response File: Author Response.pdf

Reviewer 4 Report

Comments and Suggestions for Authors

The study is devoted to the current topic - resistance of sugar cane to drought. In general, the study gives a good impression, it is logical, well built. The figures and tables meet the requirements. The volume of experimental material is sufficient. 

Comments

1. In the study is poorly formulated scientific novelty of this work - please explain what has already been done in this topic and what is the novelty of your study.
2. In our view, the Discussion could be extended by considering the physiological significance of the studied indicators - growth, Fv/Fm, content of pigments. What is an increase or decrease in these indicators under stress? What is their physiological essence? The volume of Discussion, in our opinion, is small. We recommend expanding it.
3. Indicate in which biological and statistical repetitions your experiments were carried out? How many plants did you measure the height?

Author Response

Comments and Suggestions for Authors

The study is devoted to the current topic - resistance of sugar cane to drought. In general, the study gives a good impression, it is logical, well built. The figures and tables meet the requirements. The volume of experimental material is sufficient. 

Comments

1. In the study is poorly formulated scientific novelty of this work - please explain what has already been done in this topic and what is the novelty of your study.

Response: We agree that clarifying the scientific novelty is crucial for enhancing the strength of our manuscript. Previous studies on Determination of Morpho-Physiological Traits for Assessing Drought Tolerance in Sugarcane have primarily focused on general physiological responses or germplasm screening under greenhouse conditions. However, only limited work has been done under field conditions, especially different soil types, to comprehensively evaluate genotype performance under water deficit and recovery phases.

The novelty of our study lies in:

1. Conducting experiments across different soil types.
2. Using integrated physiological, agronomic, and Normalized Difference Vegetation Index (NDVI) indicators to assess sugarcane performance.
3. Identifying specific genotype clusters that consistently exhibit drought tolerance across different soil types.

 

2. In our view, the Discussion could be extended by considering the physiological significance of the studied indicators - growth, Fv/Fm, content of pigments. What is an increase or decrease in these indicators under stress? What is their physiological essence? The volume of Discussion, in our opinion, is small. We recommend expanding it.

Response: We sincerely thank the reviewer for this insightful comment. We agree that a deeper discussion on the physiological significance of the measured indicators (growth, Fv/Fm, and pigment content) under stress conditions will strengthen the manuscript. We have revised and expanded the Discussion to address “The maintenance of growth enhances the plant is capacity to adapt to water deficit stress by modulating nutrient allocation and photosynthetic activity. Moreover, con-tinued growth facilitates the sustained productivity of the crop under drought condi-tions [11,28]. Drought stress adversely affects key physiological indices in plants, in-cluding Fv/Fm, SCMR, and pigment content, all of which tend to decrease under such conditions. These indicators provide valuable insights into plant health and stress re-sponses, supporting the effective development of drought-tolerant cultivars [29].The HHH group employs three acclimation strategies to sustain high cane yields. This group maintained SCMR and NDVI during the water stress period (Figure 5a, b, e, and f and Table 1). Additionally, all sugarcane cultivars exhibited no significant variation in Fv/Fm. (Figure 5c, d). The morpho-physiological traits of sugarcane genotypes deter-mine phenotypic variability in their responses to recovery and drought treatments throughout the growth phase [30,31,32]. Drought stress during the early growth stage significantly affected almost all growth parameters [28]. The results indicated that growth-related traits (HGR) and photosynthesis-related traits (SCMR and NDVI) were affected under early drought stress conditions. This is consistent with the findings of Dinh et al. [33], who reported that drought stress significantly reduced plant height and SPAD Chlorophyll Meter Reading (SCMR) in sugarcane under drought stress at the early growth stage.” [Page 9-10, Line 344-361] and “Drought stress is a major limiting factor for sugarcane productivity. The identification of reliable physiological traits is therefore crucial for breeding programs aimed at en-hancing drought tolerance. This study found that Height Growth Rate (HGR), Nor-malized Difference Vegetation Index (NDVI), and SPAD Chlorophyll Meter Reading (SCMR) are effective non-destructive indicators for evaluating sugarcane genotypes under water-deficient conditions [48]. These traits allow for rapid assessment and are particularly suitable for large-scale germplasm screening. Their application enables precise selection of drought-tolerant genotypes and supports the development and advancement of breeding populations with improved adaptability to drought-prone environments.” [Page 11, Line 422-430]

 

3. Indicate in which biological and statistical repetitions your experiments were carried out? How many plants did you measure the height?

            Response: We thank the reviewer for the helpful question. The experiment was conducted using two replications per treatment in a split-plot design, which served as statistical repetitions. For biological repetitions, plant height was measured on three individual plants per plot. These three measurements were then averaged to represent the value for each plot. “Growth and physiological traits in this study were measured on three individual plants per plot. These three measurements were then averaged to represent the value for each plot” [Page 12, Line 470-472].

Author Response File: Author Response.pdf

Reviewer 5 Report

Comments and Suggestions for Authors

This manuscript studies drought tolerance in sugarcane genotypes. This is an important topic, and the authors propose approaches to selecting genotypes that are most tolerant to water stress. 
However, the manuscript is poorly written and illogical, critical measurements are missing, and the experimental design and clustering principles are not clear.

1. It is not entirely clear how the experiment was designed, how the genotypes were initially selected, and how the soil type was chosen.

2. This work is devoted to drought, and no indicator evaluates the water status (tissue hydration, RWC, osmotic or water potentials of the cell contents), which is mandatory for studies of this kind.

3. Lines 105-109 Here individual genotypes are named and a reference to the figure is given, but they are not indicated in any way in the figure.

4. 118-123 It remains unclear how out of 120 genotypes there remained 36 + 15 genotypes. Also, where is the general list of genotypes? What criteria were used to make the selection?

5. The principles of grouping require a more detailed description. Designations and descriptions do not coincide in the results (118-123) and in the materials and methods (lines 278-282).

6. It is completely unclear at what stage the clustering was carried out. If based on the results to which Figures 4 and 5 belong, then why do we first see clusters, and then in Section 2.3 it is said how they were identified? It is very difficult to understand this. It is necessary to organize the narrative, clearly display what followed what and why.

7. Physiological indicators and yield were assessed at different stages. In this regard, it is poorly understood whether the yield was looked at for already formed groups or was an indicator for identifying clusters.

8. The legends do not always match the descriptions of the figures. It is difficult to understand what Q1, Q3 and IQR are in figures 4 and 5.

Due to the shortcomings of the work, as well as the fact that the manuscript is poorly organized and unclear, I propose to reject it from publication.

Author Response

Comments and Suggestions for Authors

This manuscript studies drought tolerance in sugarcane genotypes. This is an important topic, and the authors propose approaches to selecting genotypes that are most tolerant to water stress. However, the manuscript is poorly written and illogical, critical measurements are missing, and the experimental design and clustering principles are not clear.

1. It is not entirely clear how the experiment was designed, how the genotypes were initially selected, and how the soil type was chosen.

Response: We thank the reviewer for pointing out this important concern. We have now revised the Materials and Methods section to clearly describe the experimental design, genotype selection criteria, and soil type determination. “The experiment was conducted under a field trial in a tropical climate zone. In the sandy loam soil (16.311328, 102.191785), sugarcane was planted and grown from No-vember 2020 to November 2021 and planting in the loam soil (16.465202, 102.111236) was conducted from January 2021 to January 2022 at the Mitr Phol Innovation and Research Center, Chaiyaphum 36110, Thailand. The experiment was set up as a split-plot arrangement, with a randomized complete block design and two replications. The main plot consisted of two water applications: non-water-stress (CT) and drought conditions (DS) at the early growth stage. Water was supplied under the control condition (CT) through 3 irrigation events, with a total cumulative amount of 266.67 mm, which was sufficient to meet the water requirements of sugarcane. For drought conditions (DS), water was withholding water from 0 to 4 months after planting and then re-watering during 2 irrigation events after 4 to 12 months. In sandy loam soil, the cumulative water amount under CT was 1932.86 mm, while under DS it was 1666.19 mm. In loam soil, the cumulative water amount under CT was 1670.95 mm, and under DS it was 1404.28 mm.The subplot included 120 sugarcane genotypes (Table s1.) evaluated during the formative stage at two field locations. These genotypes exhibited varying degrees of drought tolerance, as evidenced by differences in cane yield and drought tolerance index under drought conditions. The soil samples were systematically collected from two experimental plots at depths of 15 cm and 30 cm to assess soil texture composition. The sandy loam with a composition of 70.86% sand, 23.47% silt, and 5.67% clay. In contrast, the loam soil contained 42.14% sand, 33.54% silt, and 24.32% clay”[Page 11, Line 434-454]. This experiment aimed to characterize and identify drought tolerance in parental lines for use in a breeding program.

Specifically:

• The experiment was designed as a split-plot design with two replications, where the main plots consisted of two water regimes (non-stress and drought stress), and the sub-plots consisted of 120 sugarcane genotypes.
• The genotypes were initially selected based on their contrasting responses to water availability, including previously known drought-tolerant and susceptible lines, as well as commercial varieties commonly cultivated in the region. We selected genetically diverse germplasm and representative soil types from this region.
• The soil types at both field locations were selected to represent two dominant agricultural soils of the region: sandy loam and loam “The sandy loam with a composition of 70.86% sand, 23.47% silt, and 5.67% clay. In con-trast, the loam soil contained 42.14% sand, 33.54% silt, and 24.32% clay”. These clarifications have been added to the manuscript on [Page 11, Line 452-454]. The use of different soil types can lead to variability in drought intensity, potentially affecting the drought response mechanisms of various sugarcane genotypes.

 

2. This work is devoted to drought, and no indicator evaluates the water status (tissue hydration, RWC, osmotic or water potentials of the cell contents), which is mandatory for studies of this kind.

Response: We appreciate the reviewer’s suggestions. Measuring RWC was limited by the large number of samples (480 samples), which could compromise data accuracy if conducted on such a scale. However, we monitored soil water status through soil moisture content, which was consistent with the imposed water regimes. In the present study, we primarily focused on physiological and morphological responses such as plant height, pigment content, and chlorophyll fluorescence (Fv/Fm) as indirect indicators of plant stress under drought conditions. However, we agree that direct measurements of plant water status—such as tissue hydration, relative water content (RWC), or water/osmotic potentials—are crucial for a comprehensive understanding of drought stress response.

 

3. Lines 105-109 Here individual genotypes are named and a reference to the figure is given, but they are not indicated in any way in the figure.

            Response: Now we have revised the figure by adding appropriate labels or annotations to clearly indicate the specific genotypes referred to in the text. This modification aims to improve clarity and ensure consistency between figure 3 and the manuscript content [Page 4, figure 3].

 

4. 118-123 It remains unclear how out of 120 genotypes there remained 36 + 15 genotypes. Also, where is the general list of genotypes? What criteria were used to make the selection?

            Response: We appreciate the reviewer’s helpful suggestion. However, 120 genotypes derived from genetically diverse germplasms (as listed in Table S1) were evaluated under early-stage drought conditions. The objective was to identify drought-tolerant genotypes based on physiological and agronomic traits, which could be recommended as parental lines in sugarcane breeding programs aimed at improving drought tolerance.

            To clarify, based on comprehensive analysis, including drought tolerance indices and yield performance, we selected 36 genotypes showing superior tolerance and 15 genotypes with contrasting responses for more detailed study. “In this study, 120 sugarcane genotypes were selected to represent the following groups: high cane yield in irrigated conditions, high cane yield in rainfed conditions, and high drought tolerance index (HHH); and high cane yield in irrigated conditions, low cane yield in rainfed conditions, and low drought tolerance index (HLL). These groups were categorized based on significant differences in cane yield under non-water stress and drought conditions, with the DTI of cane yield under drought stressed conditions. The HHH group consisted of 14 genotypes in sandy loam soil and 6 genotypes in loam soil. In HLL group comprises 13 genotypes in sandy loam soil and 9 genotypes in loam soil. (Figure 3a, 3b)” [Page 5, Line 172-179].

            For the selection criteria, clustering method and statistical analysis, we have explained in the Materials and Methods section. “Data were compiled using Microsoft Excel 365. The experimental data were sub-jected to an analysis of variance (ANOVA). Significant differences between means were assessed using the least significant difference (LSD) test at the 0.05 probability level in Statistix 10 software. The Pearson correlation coefficient between attributes was calcu-lated using the R statistical program, version 4.3.2. Cluster sugarcane by calculating the standard deviation of traits, grouping genotypes with cane yield under both water conditions with DTI (use means and standard deviation), and using statistical methods like k-means clustering for analysis [14,46,47], employing the UPGMA algorithm in GENESIS software version 1.8.1. [Page 13, Line 502-506].”

 

5. The principles of grouping require a more detailed description. Designations and descriptions do not coincide in the results (118-123) and in the materials and methods (lines 278-282).

            Response: Now we have revise sentence “In this study, 120 sugarcane genotypes were selected to represent the following groups: high cane yield in irrigated conditions, high cane yield in rainfed conditions, and high drought tolerance index (HHH); and high cane yield in irrigated conditions, low cane yield in rainfed conditions, and low drought tolerance index (HLL). These groups were categorized based on significant differences in cane yield under non-water stress and drought conditions, with the DTI of cane yield under drought stressed conditions. The HHH group consisted of 18 genotypes in sandy loam soil and 6 genotypes in loam soil. In HLL group comprises 18 genotypes in sandy loam soil and 9 genotypes in loam soil. (Figure 3a, 3b) [Page 5, Line 172-179]. We have removed “In this study, we selected sugarcane genotypes representing the following groups: high cane yield in irrigated conditions (H), high cane yield in rainfed conditions (H), and high drought tolerance index (H); and high cane yield in irrigated conditions, low cane yield in rainfed conditions, and low drought tolerance index (HLL). These groups were cat-egorized based on significant differences in cane yield under non-stressed conditions and the DTI of cane yield under drought-stressed conditions  

            In Materials and Methods, we added “Cluster sugarcane by calculating the standard deviation of traits, grouping genotypes with cane yield under both water conditions with DTI (use means and standard devia-tion), and using statistical methods like k-means clustering for analysis [14,46,47], em-ploying the UPGMA algorithm in GENESIS software version 1.8.1.” [Page 13, Line 502-506].

 

6. It is completely unclear at what stage the clustering was carried out. If based on the results to which Figures 4 and 5 belong, then why do we first see clusters, and then in Section 2.3 it is said how they were identified? It is very difficult to understand this. It is necessary to organize the narrative, clearly display what followed what and why.

            Response: We would like to clarify that the clustering was conducted based on physiological responses under drought conditions, as illustrated in Figure 3. This figure represents the step at which genotypes were grouped. Figures 4 and 5 were subsequently used to demonstrate that the identified groups differed significantly in all measured traits, thereby supporting the validity of the grouping. Section 2.3 provides an extended analysis of physiological parameters within each group [figures 6]. This was done to further characterize the distinct drought response patterns and to strengthen the interpretation of group-specific physiological behavior under drought stress.

 

7. Physiological indicators and yield were assessed at different stages. In this regard, it is poorly understood whether the yield was looked at for already formed groups or was an indicator for identifying clusters.

            Response: We sincerely appreciate the reviewer’s thoughtful comment. We fully understand the concern regarding the timing and function of yield assessment in relation to the clustering procedure. Based on findings from numerous previous studies, the assessment of physiological traits during both drought stress and recovery phases provides a reliable evaluation of plant responses indicative of drought tolerance capacity. Which often influences yield performance at the harvest stage. However, such studies are often conducted with a relatively limited number of genotypes. Therefore, in this study, we evaluated drought-related physiological traits across a large and genetically diverse germplasm collection to validate their utility in identifying parental lines for sugarcane breeding programs aimed at improving drought tolerance.

                To provide clarification, yield data were employed as one of the primary parameters for identifying genotype clusters. The clustering analysis was conducted based on yield performance under both non-water-stress (CT) and drought-stress (DS) conditions, with the drought tolerance index. Following the establishment of these clusters, physiological parameters were subsequently assessed within each group to further characterize their differential responses to drought stress.

 

8. The legends do not always match the descriptions of the figures. It is difficult to understand what Q1, Q3 and IQR are in figures 4 and 5.

            Response: To avoid potential confusion, we have removed some redundant or unclear descriptions from the figure legends. We believe that this change improves the clarity and readability of the figures without compromising the essential information. We removed details of “Figure 4. SPAD chlorophyll meter reading (SCMR) in sandy loam soil (a) and loam soil (b), maximum quantum efficiency of photosystem II photochemistry the (Fv/Fm) in sandy loam soil (c) and loam soil (d), and normalized difference vegetation index (NDVI) in sandy loam soil (e) and loam soil (f) of cluster 1 and 2 during drought and recovery periods. Different letters on the boxes indicate significant differences based on the least significant difference (LSD) test at p < 0.05. The box’s horizontal line represents the median. The lower and upper limits of the box, as well as the lower and upper whiskers, represent Q1 (25th percentile), Q3 (75th percentile), (Q1−1.5IQR), and (Q3 + 1.5IQR), respectively. IQR, interquartile range. [Page 6, Line 239-241].

Figure 5. Height growth rate (HGR) in sandy loam soil (a) and loam soil (b), leaf rolling (LR) score in sandy loam soil (c) and loam soil (d) of clusters 1 and 2 during drought and recovery periods. Different letters on the boxes indicate significant differences based on the least significant dif-ference (LSD) test at p < 0.05. The box’s horizontal line represents the median. The lower and upper limits of the box, as well as the lower and upper whiskers, represent Q1 (25th percentile), Q3 (75th percentile), (Q1−1.5IQR), and (Q3 + 1.5IQR), respectively. IQR-interquartile range”[Page 7, Line 261-263].

 

9. Due to the shortcomings of the work, as well as the fact that the manuscript is poorly organized and unclear, I propose to reject it from publication.

            Response: We sincerely appreciate the time and effort the reviewer has taken to evaluate our manuscript and provide valuable feedback. We acknowledge the concerns regarding the organization and clarity of the manuscript as well as the identified shortcomings. We are committed to thoroughly revising the manuscript to address these issues, including improving the structure, clarifying ambiguous sections, and strengthening the scientific content. We believe that with these improvements, the manuscript will meet the publication standards. We kindly ask the reviewer to consider our revised version upon resubmission and hope for the opportunity to demonstrate the value of our work.

 

Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

The manuscript has been well revised, but there is still one problem. In line 76 of page 2, the reference cited by the author does not seem to mention "loam soils", but rather "organic soils". The original text in this cited literature is: ‘sugarcane growing on mineral (sand) soils where plants are more often exposed to unfavorable conditions as compared with plants on organic soils.’. Therefore, it is suggested that the author modify "loam soils" here to "organic soils". At the same time, using "faster" here to represent the differences among various soils doesn't seem very appropriate, as it doesn't indicate the consistency of other conditions. Therefore, it is suggested to change "faster" to "easier".

Author Response

Comments and Suggestions for Authors

The manuscript has been well revised, but there is still one problem. In line 76 of page 2, the reference cited by the author does not seem to mention "loam soils", but rather "organic soils". The original text in this cited literature is: ‘sugarcane growing on mineral (sand) soils where plants are more often exposed to unfavorable conditions as compared with plants on organic soils.’. Therefore, it is suggested that the author modify "loam soils" here to "organic soils". At the same time, using "faster" here to represent the differences among various soils doesn't seem very appropriate, as it doesn't indicate the consistency of other conditions. Therefore, it is suggested to change "faster" to "easier".  

Response: We sincerely thank the reviewer for careful reading and constructive comments. As suggested, we have corrected “loam soils” to “organic soils” in line 76 of page 2 to be consistent with the original text of the cited literature.

                  In addition, we agree with the reviewer’s observation that the use of the word “faster” is not appropriate in this context. Therefore, we have revised it to “easier” in line 76 of page 2 to better reflect the differences among soil types without implying inconsistency of other conditions.

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

Dear author,

You have not completed your paper with the necessary information suggested to be included in the Materials and Methods section.

Information is still missing, such as the equipment for monitoring soil moisture status, the graph with the moisture status during the analyzed period in all experimental variants, etc.

Without this information, as well as that mentioned in the "Tracking changes" section, the paper cannot be published.

Comments for author File: Comments.pdf

Author Response

Comments and Suggestions for Authors

              You have not completed your paper with the necessary information suggested to be included in the Materials and Methods section. Information is still missing, such as the equipment for monitoring soil moisture status, the graph with the moisture status during the analyzed period in all experimental variants, etc. Without this information, as well as that mentioned in the "Tracking changes" section, the paper cannot be published.

              Response: We sincerely thank the reviewer for highlighting this issue. In response, we have carefully revised the Materials and Methods section to include the previously missing information. Specifically, we have added the following:

• Details of the equipment used to monitor soil moisture – Soil water content was measured using the auger gravimetric method at 3, 6, and 9 months after planting, covering the entire experimental period. [Page 12, Line 469]
• Graphical representation of soil moisture status – Soil moisture (±SE) at 3, 6, and 9 months after planting is presented for all experimental variants and both soil types. [Figure 2]
• Additional clarifications as suggested in the “Tracking changes” section – We have ensured that all recommended details are now clearly included.

                   We believe that these revisions fully address the reviewer’s concerns and provide a complete and transparent description of the experimental procedures.

1. some suggestions were not responded to in the material and method, you were asked to mention the quantities of water used for irrigation for the two types of soil, throughout the entire vegetative period. It is necessary to enter in the form of a graph the quantities of water but also the soil moisture status for each irrigation variant, even the control (only water from precipitation) throughout the vegetative period). Also, in the material and method, enter the equipment through which you monitored the soil moisture for each irrigation variant)

Response: We sincerely thank the reviewer for the valuable comments and suggestions. In response, we have revised the Materials and Methods section to include the missing information as follows:

1. Quantities of water used for irrigation: we have now included the quantities of water used for irrigation for both types of soil throughout the entire vegetative period. [Page 11, Lines 446-452]
2. Graphical representation of water quantities and soil moisture status: we have now included a figure showing soil moisture status [Figure 2] and a figure showing rainfall amounts [Figure 1], along with details of irrigation for each variant, including the control (rainwater only), throughout the vegetative period [Page 11, Lines 446-452]. The content is sufficient and comprehensively covers the experimental work. In addition, other reviewers agreed with the chosen research topic.
3. Details of the soil moisture monitoring equipment – Information on the equipment used to monitor soil moisture for each irrigation variant has been added. [Page 12, Lines 468-471]

We hope that these additions adequately address the reviewer’s concerns and improve the clarity and completeness of the manuscript.

 

2. For figure 1a also place details (November 1, 2020-November 30, 2021)

Response: Thank you for your suggestion. The period (November 1, 2020 - November 1, 2021) and (January 1, 2021 - January,1 2022) have now been clearly added to the caption of Figure 1a to clarify the timeframe of the data presented. [Page 3, Lines 130-131]

 

3. For figure 1 a-climatic data recorded in the experimental plot - sandy-loamy soil b-climatic data for the experiment with loamy soil variety

Response: We sincerely appreciate your valuable feedback. We believe that the presented information effectively facilitates the understanding of Figure 1. Therefore, we respectfully propose to retain this section as originally presented. Furthermore, other reviewers have concurred with the explanation provided for Figure 1. [Page 3, Lines 129-131]

 

4. A lot of data on soil texture was introduced (clay and humus content, degree of compaction, soil structure to know exactly where the CO2 is for the two types of soil). Very important data, thank you to the author.

Response: We sincerely thank the reviewer for acknowledging the importance of the soil texture data presented in our study. The authors acknowledge that soil-related parameters, such as clay and humus content, degree of compaction, and soil structure (to determine precisely the distribution of CO₂ in the two soil types), are important and provide valuable supplementary information for evaluating sugarcane performance. However, the focus of the present study was on physiological and agronomic traits associated with drought tolerance across different soil types (to characterize and select parental lines for drought-tolerant sugarcane breeding programs). Nevertheless, we recognize the importance of these soil composition traits and plan to incorporate their analysis in future research to allow for a more comprehensive evaluation of sugarcane genotypes under drought stress conditions.

 

5. Also, the amounts of water used for irrigation for the two types of soil were mentioned

Response: We have now included detailed information regarding the amounts of water used for irrigation for the two soil types. “Water was supplied under the control condition (CT) through 3 irrigation events, with a total cumulative amount of 266.67 mm, which was sufficient to meet the water re-quirements of sugarcane. For drought conditions (DS), water was withholding water from 0 to 4 months after planting and then re-watering during 2 irrigation events after 4 to 12 months. In sandy loam soil, the cumulative water amount under CT was 1932.86 mm, while under DS it was 1666.19 mm. In loam soil, the cumulative water amount under CT was 1670.95 mm, and under DS it was 1404.28 mm. [Page 11, Lines 446-452].”

 

6. I suggested to the author to introduce data on equipment for monitoring the soil moisture status during the experimental period.

Response: We sincerely thank the reviewer for this valuable suggestion. In response, we have now added information regarding the equipment used to monitor soil moisture status during the experimental period. The soil moisture was measured using the auger gravimetric method, and these details have been included in the Materials and Methods section in Page 12, Line 469. “The soil moisture content (SMC) was measured 3, 6, and 9 months after planting using the auger gravimetric method at 0-45 cm depths in the soil. Soil samples were weighed and oven-dried at 105 °C for 48 hours. The soil moisture content was calculated through the following equation:”

Soil moisture content (%) = [(wet weight - dry weight)/ dry weight)] x 100

 

7. Mention the soil moisture status during the mentioned period as well as the soil moisture monitoring equipment during the experimental period.

Response: We sincerely thank the reviewer for the valuable comment. We have now included the soil moisture status at 3, 6, and 9 months after planting for both soil types, covering the entire experimental period, in Figure 2. Details of the equipment used to monitor soil moisture for each irrigation variant have also been added on Page 12, Lines 468–471, providing a clear overview of soil moisture dynamics throughout the vegetative period for both soil types.

 

8. This question was not answered. In order to see the moisture status on the irrigation variants, it is necessary to enter it into the graph that provides clear information over the entire vegetation period (in both experiments - the two soil types).

Response: We sincerely thank the reviewer for the valuable comment. We have now included the soil moisture status (±SE) at 3, 6, and 9 months after planting for both soil types, covering the entire experimental period, in Figure 2.

 

 

Author Response File: Author Response.pdf

Reviewer 5 Report

Comments and Suggestions for Authors

The authors have significantly revised the manuscript, and I believe it can be published in this form.

Author Response

Comments and Suggestions for Authors

The authors have significantly revised the manuscript, and I believe it can be published in this form.

 

Response:

The quality of our work has been considerably improved as a result of these thoughtful critiques, which have guaranteed that it meets the journal's publication standards. We are grateful for the reviewer's guidance in improving our manuscript.

Round 3

Reviewer 2 Report

Comments and Suggestions for Authors

Dear author,
Your article has been greatly improved after responding to most of the reviewers' suggestions!
It makes a scientific contribution regarding the evaluation of physiological and agronomic traits of 120 sugarcane genotypes grown under early water stress conditions, on two different soil types.