Phenotypic Evaluation of Saccharum spp. Genotypes during the Plant-Cane Crop for Biomass Production in Northcentral Mississippi^†

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The topic of the article entitled “Phenotypic Evaluation of Saccharum spp. Genotypes during the Plant-Cane Crop for Biomass Production in Northcentral Mississippi” is very interesting. In this study the yield and other parameters of several energycane genotypes were evaluated. Two field experiments were conducted according to randomized complete block design, while the authors measured several parameters. In general the article is well written and are presented data that deserved to be published after minor revision.

 Comments

Abstract: This part should be revised, and some data should be presented.

Line 23: Replace “Review of literature” with Introduction.

Introduction section: This part is well written. Some minor changes should be made.

Lines 41-42: How much higher is the production of fiber of energycane compared to sugarcane? Some references should be added.

Line 42: How much greater have the energycane compared to sugarcane? Some data should be added.

Lines 72-73: The objective of this study should be revised. Also, in the material and methods it is not obvious which genotypes is new.

Material and methods: This section needs minor revision.

Lines 79-80: More information should be presented about the genotypes used in this study. It is not obvious which populations are the energycane populations. For each genotype may be presented which hybrid is each genotype (S. spontaneuum wit x S. …..)?

Lines  82-83: The authors should explain why use the genotypes Ho 02-113 and L 01-299 as control genotypes.

Tables 1 to 4: The tables should be improved. The font size of letters is greatar that that in the main text.

Lines 181-182: The font size of letters in equation 6 is greater than in the main text.

Results: This part is well written.

 

Lines 258-264: This part should be moved to material and methods.

Lines 380-310: This part should be moved to material and methods.

Lines 320-323: This sentence should be deleted since it is mentioned in the material and methods.

Discussion section: This section needs major revision. The authors should be compared for all parameters their data with data from other studies. For example, for dry matter yield the authors mainly focused in the differences between experimental years and not in the differences between the genotypes. Their data for this parameter should be compared with the results of other studies to show if the productivity is low or high compared to other genotypes. The same corrections should be made and for other parameters measured in this study.

Author Response

Abstract: This part should be revised, and some data should be presented.  You are correct and the abstract has been updated to include data.

Line 23: Replace “Review of literature” with Introduction.  Corrected

Introduction section: This part is well written. Some minor changes should be made.

Lines 41-42: How much higher is the production of fiber of energycane compared to sugarcane? Some references should be added.  This is usually defined by the industry as a reduction of sugar (with a corresponding increase in fiber).  This is reflected in the Introduction with citations (Giamalva et al. 1984; Bischoff et al., 2008).

Line 42: How much greater have the energycane compared to sugarcane? Some data should be added.  I'm not sure I understand this question.  Yield? Cold-hardiness?  We are testing both these characteristics in this manuscript.  The genotypes that were originally planted and discarded were discarded primarily because of their lack of cold hardiness.  It is impossible to compare energycane to sugarcane at 33°N latitude because sugarcane does not survive.  If you are referring to their comparative yields farther south, that is a separate manuscript.

Lines 72-73: The objective of this study should be revised. Also, in the material and methods it is not obvious which genotypes is new.  The objective has been revised to address yours and others' concerns.  However, the M&Ms (do) indicate that all the genotypes are new (except the control Ho 02-113- Table 1).

Material and methods: This section needs minor revision.

Lines 79-80: More information should be presented about the genotypes used in this study. It is not obvious which populations are the energycane populations. For each genotype may be presented which hybrid is each genotype (S. spontaneuum wit x S. …..)?  First there are no populations.  Saccharum (both sugarcane and energycane are vegetatively propagated, so all Ho designations are each a single genotype.  As stated in the Introduction, they are a cross of domestic sugarcane (whose specific epithet is under dispute) and S. spontaneum. All the genotypes used in this study are energycane genotypes.

Lines  82-83: The authors should explain why use the genotypes Ho 02-113 and L 01-299 as control genotypes.  These are the controls, energycane and sugarcane controls, respectively.  In a number of places in the M&Ms it indicates Ho 02-113 is the energycane control.  I added verbiage to indicate "L 01-299 (the sugarcane check)" did not survived.

Tables 1 to 4: The tables should be improved. The font size of letters is greatar that that in the main text.  You are correct.  Apparently when the staff converted the document to single space and changed the font, coding moved.  In spite of my best efforts to remove that coding, I could not.  I have added a second document to this submission (Figures and tables) with the figure and tables in correct and uniform formatting.

Lines 181-182: The font size of letters in equation 6 is greater than in the main text. see above

Results: This part is well written.

 

Lines 258-264: This part should be moved to material and methods.

Lines 380-310: This part should be moved to material and methods.

Lines 320-323: This sentence should be deleted since it is mentioned in the material and methods.   Here I respectfully disagree.  Those lines are presented at the beginning of each section as introductory paragraphs to remind and explain why we are discussing what we are.  I personally dislike the separation of Results and Discussion because I have to keep referring back to the M&M and Results when I read the Discussion.  This formatting makes it easier for the reader.

Discussion section: This section needs major revision. The authors should be compared for all parameters their data with data from other studies. For example, for dry matter yield the authors mainly focused in the differences between experimental years and not in the differences between the genotypes. Their data for this parameter should be compared with the results of other studies to show if the productivity is low or high compared to other genotypes. The same corrections should be made and for other parameters measured in this study.  Here, I strongly disagree and so do two of the other internal reviewers.  The objective indicates that we are searching for any of the genotypes with better performance than the energycane control (Ho 02-113).  All genotypes are compared to Ho 02-113 which is the objective.  Sections 4.5 and 4.6 make a clear assessment of the genotypes' production of ethanol, their rank and where they place in comparison to the control.  The only time citations are added to the discussion is to justify a procedural event or statement made.  The objective is to obtain information on the production of total ethanol of these new genotypes and compare it to the existing energycane variety (Ho 02-113 control).  

Reviewer 2 Report

Comments and Suggestions for Authors

The purpose of this study is novel. However, there are still some flaws in the research content and methods. The main comments are as follows:

1. It is recommended to use a map to show the planting of sugarcane in different regions.

2. What are the differences in the phenotype of energy sugarcane? Please show what sugarcane looks like. Secondly, are there any differences between different varieties in terms of plant height, leaf length, leaf width, etc.? What are the differences in photosynthesis and respiration?

3. Since the temperature and rainfall in 2020 and 2021 are different, the soil moisture content should be tested and analyzed. Secondly, it is also recommended to test and analyze soil microorganisms and nutrients including N, P, K, etc. In particular, what changes or advantages will fallow soil bring? These require data support.

4. There are still too few comparative analyses among twenty energycane genotypes. I believe the author's research goal is to select energy sugarcane that is more adaptable to low temperatures and has higher yields through two years of growth comparison. Then, the comparative analysis between all energy sugarcane genotypes should be richer. Secondly, Tables 3 and 4 show the data for 2020 and 2021, respectively, which are poor in comparison and readability.

 

5. The abstract does not fully describe the main results or findings of the study. 

Author Response

The purpose of this study is novel. However, there are still some flaws in the research content and methods. The main comments are as follows:

1. It is recommended to use a map to show the planting of sugarcane in different regions.  Every map I tried to insert was low quality.  However, I added text indicated the closest sugarcane production region's latitude and that we at 33°N latitude are 345 km north of normal production areas.
2. What are the differences in the phenotype of energy sugarcane? Please show what sugarcane looks like. Secondly, are there any differences between different varieties in terms of plant height, leaf length, leaf width, etc.? What are the differences in photosynthesis and respiration?  Since these species are fully sexually compatible there aren't difference other than those described in the Introduction.  Energycane (in this study is 50% sugarcane genetically) is generally described by the industry as being low in sugar, and correspondingly high in fiber.  That is what defines them.  Since this work is in its infancy, with no disrespect, how they look doesn't matter.  What matters is survival and productivity.
3. Since the temperature and rainfall in 2020 and 2021 are different, the soil moisture content should be tested and analyzed. Secondly, it is also recommended to test and analyze soil microorganisms and nutrients including N, P, K, etc. In particular, what changes or advantages will fallow soil bring? These require data support.  The data on soil fertility was provided.  "Nutrients were added according to soil test. Nitrogen was added at a rate of 168.1 kg N ha^-1 ."  Since this was dryland production and there wasn't irrigation available, soil moisture data is pointless.  Testing the soil for microbes is well beyond the scope of this study and is actually being pursued by and soil microbiologist in a different manuscript.
4. There are still too few comparative analyses among twenty energycane genotypes. I believe the author's research goal is to select energy sugarcane that is more adaptable to low temperatures and has higher yields through two years of growth comparison. Then, the comparative analysis between all energy sugarcane genotypes should be richer. Secondly, Tables 3 and 4 show the data for 2020 and 2021, respectively, which are poor in comparison and readability.  The objective of this research is to compare these new genotypes to the control (not necessarily to each other).  Anything that doesn't yield more ethanol than Ho 02-113 is to be discarded.  There was a single genotype that did.  Sections 4.5 and 4.6 do make comparisons among genotypes for ethanol production.  Regarding Tables 3 and 4, you are correct.  It seems during the conversion to single spacing applied by the editorial staff some coding inadvertently became included in the tables.  I was unable to remove or otherwise override that coding, so a separate second document has been uploaded with tables clearer, in appropriate font and font size and correctly formatted.

 

5. The abstract does not fully describe the main results or findings of the study.  Absolutely correct and it has been modified.

Reviewer 3 Report

Comments and Suggestions for Authors

This study evaluates the yield and productivity of 20 new energycane genotypes compared to a check variety, focusing on their ability to yield both fermentable and cellulosic ethanol. In light of climate change, the decrease in fossil fuel supplies, and the necessity to search for alternative sources, this study could be of interest.  However, the MS requires to undergo revision to be acceptable for publication. I have provided my edits and comments directly in the MS file (attached). The main points are:

·       The abstract: I think the abstract is incomplete. Please add more results, discuss briefly, and conclude, emphasizing the potential impact of your research.

·       The introduction: I think using "Introduction" is better than “Review of literature”. The authors may need to restructure the write-up for a more consolidated, coherent, and sequential introduction.

·       Materials and methods: The data analysis part is not clear to me. I could not understand if the data from the two seasons were combined and analyzed and if the interactions were studied. What was the statistical basis for determining the stability of a genotype?

·       The results: I think the authors need to reconsider how their results are presented. Some parts should be moved from the discussion to the results section, as suggested in the attached file. Please carefully check your figures in Tables 3 and 4.  I could not understand how data were pooled across years.

·       The discussion: I think your discussion should be based on the objectives of the study, i.e., how the energycane genotypes performed compared with the check under the experimental conditions. This applies to all traits studied.

·       Summary and conclusions: This is fragmented and requires restructuring to be more coherent and compelling.

 Additional comments and edits can be found in the MS file.

Comments for author File: Comments.pdf

Comments on the Quality of English Language

Author Response

• The abstract: I think the abstract is incomplete. Please add more results, discuss briefly, and conclude, emphasizing the potential impact of your research.  You are correct and the abstract has been modified to include data.
•  
• The introduction: I think using "Introduction" is better than “Review of literature”. The authors may need to restructure the write-up for a more consolidated, coherent, and sequential introduction. Yes, and it has been corrected as you suggest.
•  
• Materials and methods: The data analysis part is not clear to me. I could not understand if the data from the two seasons were combined and analyzed and if the interactions were studied. What was the statistical basis for determining the stability of a genotype?  The analysis indicated an interaction for all characteristics measured (except percentage cellulose).  As such, data must be presented by year (Tables 3 and 4).  The vast change in rank among most of the genotypes would suggest, little stability, though a few were consistently near or above Ho 02-113.  That is the objective of this study is to find out if any of the new genotypes exceed Ho 02-113 for their ability to produce ethanol.
•  
• The results: I think the authors need to reconsider how their results are presented. Some parts should be moved from the discussion to the results section, as suggested in the attached file. Please carefully check your figures in Tables 3 and 4.  I could not understand how data were pooled across years.  Data were not pooled across years (except percentage cellulose), that is why the data from 2020 is in table 3 and 2021 in table 4.  Regarding Tables 3 and 4, you are correct.  It seems during the conversion to single spacing applied by the editorial staff some coding inadvertently became included in the tables.  I was unable to remove or otherwise override that coding, so a separate second document has been uploaded with tables clearer, in appropriate font and font size and correctly formatted.
•  
• The discussion: I think your discussion should be based on the objectives of the study, i.e., how the energycane genotypes performed compared with the check under the experimental conditions. This applies to all traits studied.  

  The objective indicates that we are searching for any of the genotypes with better performance than the energycane control (Ho 02-113).  All genotypes are compared to Ho 02-113 which is the objective.  Sections 4.5 and 4.6 make a clear assessment of the genotypes' production of ethanol, their rank and where they place in comparison to the control.  The only time citations are added to the discussion is to justify a procedural event or statement made.  The objective is to obtain information on the production of total ethanol of these new genotypes and compare it to the existing energycane variety (Ho 02-113 control).  

• Summary and conclusions: This is fragmented and requires restructuring to be more coherent and compelling. - I am not sure what you mean here.  We address that only a single genotype outperforms the control for ethanol production.  I am willing to entertain additional commentary here.

 Additional comments and edits can be found in the MS file.  This file was most helpful.  As you know, once you start editing, line numbers are only a ball park guide.  Thank you for including this.

Round 2

Reviewer 2 Report

Comments and Suggestions for Authors

I think my comments have been answered fairly well. I now agree that this article should be accepted for publication.

Author Response

I think my comments have been answered fairly well. I now agree that this article should be accepted for publication.

no further comments necessary.

Reviewer 3 Report

Comments and Suggestions for Authors

Dear Authors,

Thank you for your efforts to revised your MS. I think your MS is now in a better shape. However, sometimes, I could not understand your response well. So, would you please clarify your response to the following:

1-Materials and Methods:  The data analysis part is not clear to me. I could not understand if the data from the two seasons were combined and analyzed and if the interactions were studied. What was the statistical basis for determining the stability of a genotype? Please indicate to the reader the steps you followed to analyze your data. For example, the data of each location-year was analyzed separately, OR the data of the two locations were combined and analyzed, and the genotype x location-year interaction was significant. Therefore, the results of each location-year are presented separately. Please also show which statistical method you used for mean separation and to determine the stability of a given genotype.

2- The results: I think you need to reconsider how your results are presented. Some parts should be moved from the discussion to the results section, as suggested in the MS file of the first round, for example, the correlation table (Table 5). If you think the way you present your results is appropriate, please explain.

3- The discussion: I think your discussion should be based on the objectives of the study, i.e., how the energycane genotypes performed compared with the check under the experimental conditions. This applies to all traits studied. I am not sure if the discussion is really based on how the new energycane genotypes performed compared to the check with regards to different traits studied. For instance, the check genotype (Ho 02-113) was mentioned only once in the discussion section (Juice volume). So, I still think that you need to revise your discussion.

4- Summary and conclusions: This is fragmented and requires restructuring to be more coherent and compelling. For example, please review the first paragraph. You started with general statements, then mentioned the correlation between DMY and TTEY, then moved to a different topic of Ho 02-113 was exceeded in mean DMY only by Ho 14-9213,... Don't you agree with me that you need to consolidate your conclusion in a better way?

Please check the MS file I attached during the first round of the review and try to address the points raised, agree or disagree with them, and give your reasons.

Comments on the Quality of English Language

Minor English language editing might be required

Author Response

1-Materials and Methods:  The data analysis part is not clear to me. I could not understand if the data from the two seasons were combined and analyzed and if the interactions were studied. What was the statistical basis for determining the stability of a genotype? Please indicate to the reader the steps you followed to analyze your data. For example, the data of each location-year was analyzed separately, OR the data of the two locations were combined and analyzed, and the genotype x location-year interaction was significant. Therefore, the results of each location-year are presented separately. Please also show which statistical method you used for mean separation and to determine the stability of a given genotype.  The statements you give as examples were used in the analysis description.  I understand why you didn't understand.  The description was split in two locations.  I moved the statements of mean separation (section 3.1 (the part in question) up to line 216.

2- The results: I think you need to reconsider how your results are presented. Some parts should be moved from the discussion to the results section, as suggested in the MS file of the first round, for example, the correlation table (Table 5). If you think the way you present your results is appropriate, please explain. I am having trouble with your request - not that it is wrong - the discussion (among the authors) to assess correlations and where to put it came down to, correlations are generated after all data have been produced.  Correlations are an effort to explain and thus belong in discussion. 

3- The discussion: I think your discussion should be based on the objectives of the study, i.e., how the energycane genotypes performed compared with the check under the experimental conditions. This applies to all traits studied. I am not sure if the discussion is really based on how the new energycane genotypes performed compared to the check with regards to different traits studied. For instance, the check genotype (Ho 02-113) was mentioned only once in the discussion section (Juice volume). So, I still think that you need to revise your discussion.  I find it especially troublesome when results and discussion are separate. The relative performance of various genotypes is compared to the check (Ho 02-113; the objective) appear in every section of every characteristic tested.  For DMY lines 279 and 286; for JV lines 292 and 298; for Brix lines 304 and 308; for % cellulose lien 337 and for TTEY lines 358 and 359-360.  To me, discussion is an attempt to explain why the data is "falling out" the way it is.

4- Summary and conclusions: This is fragmented and requires restructuring to be more coherent and compelling. For example, please review the first paragraph. You started with general statements, then mentioned the correlation between DMY and TTEY, then moved to a different topic of Ho 02-113 was exceeded in mean DMY only by Ho 14-9213,... Don't you agree with me that you need to consolidate your conclusion in a better way?  Review of the S&C, you are correct, that fragment has no business there.  It has been reworded.  I would like your opinion, I am willing to drop the 2nd and tird paragraph in S&C if you believe it doesn't contribute. 

Please check the MS file I attached during the first round of the review and try to address the points raised, agree or disagree with them, and give your reasons.

Author Response File: Author Response.pdf