Association of Physiological Responses and Root Distribution Patterns of Ratooning Ability and Yield of the Second Ratoon Cane in Sugarcane Elite Clones
Round 1
Reviewer 1 Report
The study was conducted to understand the association of physiological responses and root distribution patterns on the yield of the second ratoon cane. I think this study is useful as there is not enough information available on the effect of drought on the physiological traits and grain yield in sugarcane. However, this study will be better after considering some of the comments provided below.
Major comments:
Introduction section: Please elaborate on the rationale of your study
Materials and Methods: How many measurements were taken for the SPAD and LAI? Please explain the procedure of LAI measurements?
What statistical tools were used in the analysis? Did you measure the correlation based on the adjusted means? If not, please use the adjusted means for the correlation analysis.
Results and Discussion:
Could you please show us the ANOVA table?
I would strongly suggest you to write a separate discussion section rather than combining it with the results. Some the results are hard to follow and have some repetitive information from the introduction section.
Minor suggestion:
Line 11: Need to describe the rationale statement before beginning the objective in the abstract
Please add the year of an experiment conducted in the tables and figures.
Author Response
Response to reviewer
The manuscript is revised according to the comments and suggestions. The authors very much appreciate the commitments and suggestions to improve the manuscript.
The manuscript is also carefully checked for possible errors and revised. Where the revision is made, the blue highlight is indicated.
Response to reviewer 1
Introduction section: Please elaborate on the rationale of your study
: The rationale of the project is included in the Introduction section. (line: 62-64)
Although the responses to drought for physiological traits, root traits and germination have been studied in sugarcane, most studies were conducted in rhizoboxes or field condition with few genotypes and the relationships of these characters and cane yield of ratoon crops have not been sufficiently investigated.
Materials and Methods: How many measurements were taken for the SPAD and LAI? Please explain the procedure of LAI measurements?
The measurements were undertaken from six leaves of six stem, and, therefore, there were 18 measurements for each plot. (line 131-132)
The authors did not reported leaf area index (LAI) in the manuscript. However, the authors assumed that the reviewer is asking about Specific leaf area (SLA) which was presented in the manuscript, and, therefore, the authors explain the procedure for measuring specific leaf area.
Specific leaf area was measured using an LI-3100C Area Meter, LI-COR, Inc. Lincoln, Nebraska, USA. Six second leaves from six stalks in each plot were used for measuring specific leaf area. The leaf samples were first measured for leaf area and the leaf samples were oven-dried at 80°C for 72 hours, or until the weights were constant, and the dry weights measured. SLA was calculated as the ratio between leaf area (cm2) and leaf dry weight (g). (line 125-129)
What statistical tools were used in the analysis? Did you measure the correlation based on the adjusted means? If not, please use the adjusted means for the correlation analysis.
: The correlation was based on means for the correlation analysis.
Results and Discussion:
Could you please show us the ANOVA table?
The ANOVA table is Table 2,3, 4 and 5.
I would strongly suggest you to write a separate discussion section rather than combining it with the results. Some the results are hard to follow and have some repetitive information from the introduction section
The results and discussion are in the separate sections.
Minor suggestion:
Line 11: Need to describe the rationale statement before beginning the objective in the abstract
Poor ratooning ability of sugarcane can limit crop productivity and profitability of sugarcane growers. (line: 11-12).
Please add the year of an experiment conducted in the tables and figures.
The year is
added in the tables 2, 3, 4, 5 and 6 are included in the Results and
Discussion, and the old Tables are moved to supplementary section.
Author Response File: 
Author Response.docx
Reviewer 2 Report
This article about the association of physiological traits with sugarcane ratooning ability and yield is an important piece of work. Sugarcane ratooning ability is a major issue worldwide which has a major effect on the crop profitability.
The authors conducted an important and thorough experimental work where a lot of good quality data have been collected and therefore I don't think anymore experimental work is required.
With such good quality data the authors could clearly improve the quality of their analyses and therefore the quality of the manuscript. I would suggest to the author to have a fresh look on their data and to re-analyse them.
Please find below some comments about the manuscript:
Intro
L52 to L55. Rephrase, it is a bit confusing.
Material and methods section
Section 2.1:
- How the plants were harvested manually, with a commercial harvester or with a small mechanical harvester. This is important to mention since depending on the harvesting method, stool will be ripped off the ground.
- Do you haves any information about the soil type (facultative)?
- For each data point, how many cores were collected for the soil moisture content? There is no error bar or any measure of the variability on Fig. 1 graphs. What was the core size in this experience?
- There are issue with the way the equations appear in the pdf version of the article. Maybe an issue when converting from word to pdf.
Section 2.2.4:
Why did you combined the 0-20 cm and the 20-40 cm cores? The root length density in these two types of cores varies drastically.
Results and discussion
- L151: Table S1 mot S2
- Table S1: It is inherent that there is variability in yield between varieties so it would be better to compare yield decline between PC, 1R and 2R for each varieties rather than comparing differences in yield between varieties for PC, 1R and 2R.
- Table S1: Where are the details information (for each variety) about the germination percentage of the 1R.
- L158-159: Compare to what? To plant crop? Please explain I don’t see where 46.83 comes from.
- L166: Is it first ratoon?
- When germination was calculated and if a mechanical harvester was used, did you calculate the number of missing stool after harvest?
- Fig2b and 2; Once again explain where you’ve got these figures from?
- Fig2: While overall relationship is ok. I would rather see the correlation for each individual varieties. This will avoid misinterpretation of the correlation (Simpson's paradox).
Section 3.2
- This whole section needs to be rewritten or reformat. It is complicated to see what the point outside of describing every single results is.
- Root length density is in cm/cm3 not cm.
- According to fig 4 and 5 as well as the literature, I don’t think you can combine the 0-500px and 20-1000px for your analysis. It will results in a very high variance, while the 40-100 cm combination will have a low variance. Therefore you can’t do an ANOVA.
- L.234:UT12 is part of the varieties in 4a and 5a: varieties with high RLD in the top layer. You then say that UT12 a high RLD at depth. which is what?
Section 3.3
- P11 L37-39: Not clear, please rephrase.
- Why do you start to present SCMR result in this section while most of them are discuss in the next section.
Section 3.6
While it is true that cane yield is a complex trait and therefore it is hard to correlate it with a single trait, it looks that RLD is not correlated with cane yield, neither the percentage. I would be nice to see a graph like the one of Fig. 2 to assess the strength of the correlation visually.
Best regards
Author Response
Response to reviewers
The manuscript is revised according to the comments and suggestions. The authors very much appreciate the commitments and suggestions to improve the manuscript.
The manuscript is also carefully checked for possible errors and revised. Where the revision is made, the blue highlight is indicated.
Response to reviewer 2
Introduction section
L52 to L55. Rephrase, it is a bit confusing.
The paragraph is rewritten. (line: 53-59).
Root functions as anchor to support plant in the soil and takes up soil water and nutrient for plant growth and yield. After the canes are harvested, the ratoon crop can develop a new root system from old active roots within three days. New roots also emerge from the basal nodes of young shoots for several weeks after new shoots are developed but, at seven weeks after harvest, new roots rarely develop from old roots that have been active at the time of harvest [4]. Drought reduces root growth in upper soil layers, and root growth also shifts toward the lower soil layers to maintain water uptake [5].
Material and methods section
Section 2.1:
How the plants were harvested manually, with a commercial harvester or with a small mechanical harvester. This is important to mention since depending on the harvesting method, stool will be ripped off the ground.
The crops were harvested manually by cutting the stalks at ground level and discarding the tops. (line 162-163).
Do you haves any information about the soil type (facultative)?
The soil type at the experimental site is classified as Yasothorn soil series, which is characterized by sandy soil (84.93% sand, 10.0% silt and 5.07% clay). The soil was slightly acidic (pH=5.78), low in organic matter (0.54%), nitrogen deficient (0.03%), low in phosphorus (23.78 ppm) and sufficient in potassium (54.46 ppm). (line 82-85).
For each data point, how many cores were collected for the soil moisture content? There is no error bar or any measure of the variability on Fig. 1 graphs. What was the core size in this experience?
The error bar was included in all data points in Fig. 1.
There are issue with the way the equations appear in the pdf version of the article. Maybe an issue when converting from word to pdf.
The equations appear of the study is material and methods section (line 119 and 142).
Section 2.2.4:
Why did you combined the 0-20 cm and the 20-40 cm cores? The root length density in these two types of cores varies drastically.
The soil at 0-20 and 20-40 cm is in the tillage soil layer and the soil below 40 cm was lower than tillage layer.
Results and discussion
L151: Table S1 not S2
S2 to Table 2
Table S1: It is inherent that there is variability in yield between varieties so it would be better to compare yield decline between PC, 1R and 2R for each varieties rather than comparing differences in yield between varieties for PC, 1R and 2R.
The authors calculated yield reduction (%) (Table S1). :
Yield reductions in comparison to the planted crop were 22.26% and 36.73% for the first ratoon crop and second ratoon crop, respectively.
Table S1: Where are the details information (for each variety) about the germination percentage of the 1R.
Germination data of 1R are included in Tables S1.
L158-159: Compare to what? To plant crop? Please explain I don’t see where 46.83 comes from.
The data are included in Table S2.
L166: Is it first ratoon?
In this study, germination of new plants from harvested plants of the planted and first ratoon crops (line 187-188)
When germination was calculated and if a mechanical harvester was used, did you calculate the number of missing stool after harvest?
The crops were harvest manually.
Fig2b and 2; Once again explain where you’ve got these figures from?
The data for yield reduction are included in Table S1, and yield reduction was calculated as follows;
The yield data of plant crop (PC), the 1st ratoon crop (1R) and the 2nd ratoon crop (2R) and germination percentage of the second ratoon crop in Tables S1 in the supplementary section.
: The yield reduction data was calculated as
Yield reduction of 1st ratoon (%) = [cane yield of plant crop/cane yield of the first ratoon crop] × 100
Yield reduction of 2nd ratoon (%) = [cane yield of plant crop/cane yield of the second ratoon crop] × 100
Fig2: While overall relationship is ok. I would rather see the correlation for each individual varieties. This will avoid misinterpretation of the correlation (Simpson's paradox).
The correlations for the individual varieties are presented in Fig. 2.
Section 3.2
This whole section needs to be rewritten or reformat. It is complicated to see what the point outside of describing every single results is.
The paragraph is rewritten. (line 213-247)
Root length density is in cm/cm3 not cm.
cm to cm/cm3
According to fig 4 and 5 as well as the literature, I don’t think you can combine the 0-500px and 20-1000px for your analysis. It will results in a very high variance, while the 40-100 cm combination will have a low variance. Therefore you can’t do an ANOVA.
The data for root length density in fig 4 and 5 were not statistically analyzed, and the data were calculated for five soil layers at 20 cm intervals. The purpose is to visualize the distribution of root length density in different soil layers before the soil layers were combined into top soil and sub soil.
L.234:UT12 is part of the varieties in 4a and 5a: varieties with high RLD in the top layer. You then say that UT12 a high RLD at depth. which is what?
UT12 had high root length densities at the soil depth of 40-100 cm. (line 227)
Section 3.3
P11 L37-39: Not clear, please rephrase.
The paragraph is rewritten. (line 284-287)
KKU99-06 had high root length densities at 0-40 and 40-100 cm under at 90 DAH and high root length densities on this genotypes persisted until harvest (270 DAH). The results demonstrated high root length density and good root distribution under drought supported germination.
Why do you start to present SCMR result in this section while most of them are discuss in the next section.
SCMR is moved to discussion section.
Section 3.6
While it is true that cane yield is a complex trait and therefore it is hard to correlate it with a single trait, it looks that RLD is not correlated with cane yield, neither the percentage. I would be nice to see a graph like the one of Fig. 2 to assess the strength of the correlation visually.
Because the correlation coefficient between root length density and cane yield was low, the authors did not present the correlation in graph like that of Fig. 2.
Author Response File: Author Response.docx
Round 2
Reviewer 1 Report
The manuscript has been improved after the considering suggestions by reviewers. However, it requires minor revisions before it is considered for publication.
Minor suggestions:
Authors should clearly state what statistical software/tools were used for the analyses.
Please re-check grammar/typos.
Thank you.
Author Response
Response to Reviewer 1 Comments
The manuscript is revised according to the comments and suggestions of reviewer 1 (minor suggestions). The authors very much appreciate the commitments and suggestions to improve the manuscript.
The manuscript is also carefully checked for possible errors and revised. Where the revision is made, the blue highlight is indicated.
Point 1: Authors should clearly state what statistical software/tools were used for the analyses.
Response 1: The statistical software is included in the Materials and methods section. (Line: 163)
“All statistical analyses were conducted using the software package Statistix 10.”
Point 2: Please re-check grammar/typos.
Response 2: The manuscript is also carefully checked for possible errors and revised.
Author Response File: 
Author Response.pdf
This manuscript is a resubmission of an earlier submission. The following is a list of the peer review reports and author responses from that submission.
Round 1
Reviewer 1 Report
This study has collected some potentially interesting data from a second ratoon crop of sugarcane. However, the contribution of the work and paper is very marginal in terms of providing a deeper understanding of the factors affecting variation in yield in sugarcane. There were many aspects of the presentation and data analysis which I suggest need to be addressed before the paper should be published.
1. The hypotheses for the research were not clear. Also, is was not clear at the end of how the results would be used in the future – either for guiding further research, or breeding programs. The authors do not present their views on this issue other than say that selection of genotypes with high germination should improve ratooning ability. However, the correlation between germination and yield was quite low and not significant. A breeder would make much more effective progress by just selecting for yield directly – it is not explained why better gains would be made by using germination (or any other traits). Overall, I think the authors need to put deeper thinking into the issue of how their results could assist in breeding or other applications, because at this stage it is not convincing.
2. There is too many tables of detailed data, which is not of value to a reader. It would be better to just give summary statistics briefly in a table for all traits (overall mean, statistical significance of genetic effects) and then show relationships between traits in Figures (like Figure 3) or as a correlation matrix. The detailed matrices of genotype x trait data could be provided in supplementary tables.
3. Information on harvesting time for P, 1R, and 2R is needed. Also, I suggest yield data on P and 1R should be included if it is available.
4. The writing needs to be improved a lot throughout. There are many places where there is repetition and where the text does not flow very logically. For example, on lines 147-157 (in results section) there is material there is material that should be in the introduction, and on lines 204 – 211 (in results section) there is text that is just repeating material that should be in the materials and methods. There are many more cases of examples like these. Also, the English needs to be improved throughout.
5. Some relationships between measured traits and yield were indicated in Tables 5 and 6 which could be interesting. However, it would be important to know if these relationships arose through correlated genetic effects or correlated environmental effects within the trial. Ideally genetic correlations between traits should be determined, rather than phenotypic correlations. (Phenotypic correlations arise due to joint effect of genetic and within-experiment environmental effects – eg. see Falconer and Mackay (1995). An example of genetic correlations and methods is given by Basnayake et al. (2015).
6. The study is limited in that it was only done at one site, with one rainfed environment treatment (ie. does not include irrigated versus rainfed for comparisons). Also, roots are only sampled to 2500px – but it is known that sugarcane roots go as deep as 3m, possibly even more in some soils in dry conditions. While these limitations are not “fatal” they do weaken the study.
Basnayake J, Jackson P, Inman-Bamber N, Lakshmanan P (2015) Sugarcane for water-limited environments. Variation in stomatal conductance and its genetic correlation with crop productivity. J Exp Bot 66:3945-3958
Falconer D, Mackay T (1995) I ntroduction to Q uantitative Genetics. Longman 19:1
Reviewer 2 Report
The authors examined the distribution of sugar cane root system from various directions and investigated the ratoon cane yield and it seems to deserve publication of this journal. However, There are some parts to be corrected.
1. Make abbreviations for RWC, SCMR, SLA, RLD, DAH etc.
2. Create a Table of Genotype in Materials and Methods and specify the characteristics and the time of release of the genotype.
3. There is no description of the method or time for measuring stomatal conductance.
4. The figures are too small and unclear. Again, make it bigger.
5. Expand the Genotype column in the Tables and reduce the number of rows.
6. The literature is duplicated. Please confirm.
7. Read the past printed matter and create the manuscript.
Reviewer 3 Report
The filed experiment in the current study was conducted for only one year and I doubt the reliability and repeatability of the study. In order to be considered for the reputed Journal like this, authors must conduct an experiment for at least one more year or multiple locations. By doing so it will be intersting to a larger audience and will have an impact.
