In Vitro Assessment of Kurdish Rice Genotypes in Response to PEG-Induced Drought Stress

Round 1

Reviewer 1 Report

Title: Which response are the Kurdish rice genotypes in vitro assessed for?

Abstract:

It is confusing when mixing SSR and PEG assays results

Introduction

Line 47: It should be stated that authors refers to genetic distances among Iraqi rice cultivars. Why to cite a reference to a Vicia faba work? [4]Tahir, N.A. Identification of genetic variation in some faba bean (Vicia faba L.) genotypes grown in Iraq 555 estimated with RAPD and SDS-PAGE of seed proteins, Indian J. Biotechnol. 2015, 14, 351-356.

Line 50: why so general explanation? How large is the rice cultivation area in this region? What about rice yields in this area? How limited is water supply? Is there any estimate of actual yield losses due to irrigation constraints?

Line 70: drought tolerance and inorganic elements changes are not supported by these references.

Line 86: There are not in vitro procedures being evaluated in this work, since there is only one in vitro procedure. What is the utility of rice drought tolerant callus? This callus was not embryogenic? It did not regenerate plants? Is this the actual objective of this work? I mean, the objective was to obtain new drought-tolerant plant material derived from Kurdish cultivars? Because this is not stated at the title of the manuscript. From this not very clear title, I thought it would be to test the ability of kernels from Kurdish rice cultivars to produce callus in PEG-supplemented medium.

Or maybe authors are testing if in vitro kernel-derived callus growth in PEG-supplemented medium correlated to plant drought tolerance? Why there is not any information about previous knowledge of the level of drought stress tolerance of the cultivars used in this study? Why there is not information about previous studies about the correlation between rice in planta drought stress tolerance and the capability of kernel-derived callus to grow in PEG-supplemented medium? In vitro screening of rice cultivars for salt and drought stress has been widely reported, these references would support the actual objective of this work, whatever it would be.

Material and methods

Line 94: how many rice cultivars are used by Kurdish growers? Only six? Why to study these six? Are these cultivars drought tolerant? Are this genotypes cultivated only in this area? Kurdish growers use only local landraces?

Line 103: Wheat SSR markers?

Line 127: What for?

Results

Line 201: genetic differences among these 6 cultivars would be more useful if we knew how representative of Kurdish rice cultivars they are.

Line 206: the existence of a uniform spectrum of a marker means that one marker was monomorphic for the six cultivars?

Line 214: the objective was to determine drought stress tolerance of Kurdish rice cultivars by an in vitro assay?

Line 233: these findings would not indicate also a different ability of kernels from different cultivars to produce callus in control conditions too? I mean that ANOVA showed significant differences among cultivars, PEG treatments and also for the interaction between both factors (Table 4)? From results shown in this table, I would say that PEG treatments did not affect callus induction in some cultivars but reduced callus growth in all of them, although not at the same rate. Perhaps showing these results in three figures would help to understand this. And perhaps if authors would explain using these figures the percentage of reduction observed for each case, it would not be necessary to calculate any drought tolerance index and to elaborate new figures with the same data. This is not helping to understand their results.

Line 236: which are the most tested parameters? Is any parameter tested more than others?

Line 263: The figure doesn’t show results from unstressed and stressed plants.

Line 264: Blue dot.

Figure 1: mean separation cannot be correct for CWT. And in Line 219, authors stated that there was not a significant decrease for this trait when medium was supplemented with PEG.

Line 237: cell dimension parameters have been described with callus induction and growth parameters in the previous paragraph.

Line 269: Effectiveness in callus morphology?

Line 301: callus growth performance? These traits are related to callus morphology.

Discussion

Line 454: What?

Line 474: Because these 6 are the only existing Kurdish rice genotypes?

Line 480: phenotypic resemblance means similar performance?

Line 512: What is the meaning of encompassed less of the most? Are more essential than… what?

Authors should describe precisely, from the title up to the conclusions, that they are evaluating drought tolerance of rice callus, or the in vitro response under drought stress of the six genotypes for callus production. The drought tolerance of a genotype can be different depending on the trait that is being evaluated, therefore it is necessary to be more specific.

Line 530: Are the authors inferring that two of the studied cultivars would show in planta or much better, in field drought tolerance? Why? Is there any correlation for rice between kernel-derived callus ability to grow in PEG supplemented medium and plant drought tolerance? Have they studied this relationship? ... If not, have they described the references that support this inference? … I am afraid they didn’t.

Author Response

Authors' responses

Manuscript number: applsci-836212

Paper title: In vitro Assessment of Kurdish Rice Genotypes in Response to PEG-Induced Drought Stress

Authors: Didar Rahim, Petr Kalousek, Nawroz Tahir, Tomáš Vyhnánek, Petr Tarkowski, Vaclav Trojan, Dana Abdulkhaleq, Ahmad Hama Ameen and Ladislav Havel

Dear Editor and Reviewer

The authors would like to thank the area editor and the reviewers for their precious time and invaluable comments. We have carefully addressed all the comments. The constructive comments/ suggestions by the reviewer are appreciated. We have now completely revised the manuscript. The manuscript has been corrected by an expert of language and highlighted by trach change method. The corresponding changes and refinements made in the revised paper are summarized in our response below. The actual comments of the reviewer are in BOLDED RC, and the author's responses are italicized AC.

Reviewer-1

RC1. Line 47: It should be stated that authors refer to genetic distances among Iraqi rice cultivars. Why cite a reference to a Vicia faba work? [4] Tahir, N.A. Identification of genetic variation in some faba bean (Vicia faba L.), genotypes were grown in Iraq 555 estimated with RAPD and SDS-PAGE of seed proteins, Indian J. Biotechnol. 2015, 14, 351-356.

AC/ This reference (Tahir, N.A) has been deleted and replaced by this reference:

Khush, G.S.; Toenniessen, G.H.; International Rice Research Institute; C.A.B. International. Rice Biotechnology; C.A.B. International, 1991, ISBN 9780851987125.

RC2. Line 50: why so general explanation? How large is the rice cultivation area in this region? What about rice yields in this area? How limited is water supply? Is there any estimate of actual yield losses due to irrigation constraints?

AC/ In Kurdistan, the cultivated area and production of rice are 2000 ha and 14.49 ton, respectively, in 2018. The cultivation and growing season of rice in Kurdistan starts in June and ends in October. During this period, the production of rice depends on the water resources coming from the lake of Dukan and Darbanikhan presenting in northern Iraq. There is the loss of yield because the level of availability of water in two lakes depends on the amount of water come from Iran and this pay is trying to build some lakes in the border regions to conserve the water and to minimize the water going to Iraqi Kurdistan.

RC3. Line 70: drought tolerance and inorganic elements changes are not supported by these references.

AC/ the two references have been deleted and replaced by these references:

Salehi-Lisar, S.Y.; Bakhshayeshan-Agdam, H. Drought Stress in Plants: Causes, Consequences, and Tolerance. In Drought stress tolerance in plants. Vol 1, Physiology and biochemistry; Hossain, M.A., Wani, S.H., Bhattacharjee, S., Burritt, D.J., Tran, L.-S.P., Eds.; Springer: Switzerland, 2016; pp 1–16, ISBN 978-3-319-28899-4.

Kumar, S.; Sachdeva, S.; Bhat, K.V.; Vats, S. Plant Responses to Drought Stress: Physiological, Biochemical and Molecular Basis. In Biotic and abiotic stress tolerance in plants; Vats, S., Ed.; Springer: Singapore, 2018; pp 1–25, ISBN 978-981-10-9029-5.

RC4. Line 86: There are not in vitro procedures being evaluated in this work since there is only one in vitro procedure. What is the utility of rice drought-tolerant callus? This callus was not embryogenic? It did not regenerate plants? Is this the actual objective of this work? I mean, the objective was to obtain new drought-tolerant plant material derived from Kurdish cultivars? Because this is not stated at the title of the manuscript. From this not very clear title, I thought it would be to test the ability of kernels from Kurdish rice cultivars to produce callus in PEG-supplemented medium

AC/ The utility of rice drought-tolerant callus is to improve selection efficiency and accuracy in terms of drought tolerance in the plant because the condition of growth of callus via tissue culture is controlled completely and is not affected by the environmental factors. So, the selection of plant under field conditions is tedious due to low heritability, the time required and interaction between environmental factors and genotypes. The produced callus is non-embryogenic. This callus can not regenerate the plants. The main objective of this work is to test or screen the rice genotype responses to drought-induced by PEG via tissue culture method. This section of aim has been modified in the revised manuscript. In vitro, kernel-derived callus growth in PEG-supplemented medium may be correlated to plant drought tolerance, but it should be tested because it depends on plant species. Stress tolerance may thus be considered as a developmentally regulated, stage-specific phenomenon such that tolerance at one stage of plant development may not be linked to tolerance at other developmental stages, as already reported by:

Muscolo, A.; Junker, A.; Klukas, C.; Weigelt-Fischer, K.; Riewe, D.; Altmann, T. Phenotypic and metabolic responses to drought and salinity of four contrasting lentil accessions. J. Exp. Bot. 2015, 66, 5467–5480, doi:10.1093/jxb/erv208.

Sadat Noori, S.A.; McNeilly T. Assessment of variability in salt tolerance based on seedling growth in Triticum durum Desf. Genetic Resources and Crop Evolution 2000, 47, 285–291, doi:10.1023/A:1008749312148.

RC5. Line 94: how many rice cultivars are used by Kurdish growers? Only six? Why study these six? Are these cultivars drought tolerant? Are these genotypes cultivated only in this area? Do Kurdish growers use only local landraces?

AC/ In Kurdistan only six genotypes are available. The farmers use only local genotypes and the six genotypes are the most cultivated genotypes in this area. There is not any study about the tolerance or sensitivity of rice genotypes in this area. Our study is the first work.

RC6/ Line 103: Wheat SSR markers?

AC/ The word wheat has been deleted and replaced by rice

RC7/ Line 127: What for?

AC/ To determine the impact of PEG (Induced drought) on the size of the callus

RC8/ Line 201: genetic differences among these 6 cultivars would be more useful if we knew how representative of Kurdish rice cultivars they are.

AC/ The recognizing of genetic distance among rice genotypes is the key to find some markers related to drought tolerance. So, it is important to determine the genetic profile because the rice genotypes are tested under controlled condition, so the genetic makeup plays the main factor to determine the drought-tolerant.

RC9/ Line 206: the existence of a uniform spectrum of a marker means that one marker was monomorphic for the six cultivars?

AC/ This line is corrected in the revised manuscript. One SSR marker (RM526) reflected the existence of genotypes uniform, in other words, this marker showed the monomorphism band in all genotypes and can not differentiate all genotypes.

RC10/ Line 214: the objective was to determine drought stress tolerance of Kurdish rice cultivars by an in vitro assay?

AC/ Yes, this study aims to screen the rice genotypes under drought condition using PEG treatment through tissue culture method.

RC11/ Line 233: these findings would not indicate also a different ability of kernels from different cultivars to produce callus in control conditions too? I mean that ANOVA showed significant differences among cultivars, PEG treatments and also for the interaction between both factors (Table 4)? From the results shown in this table, I would say that PEG treatments did not affect callus induction in some cultivars but reduced callus growth in all of them, although not at the same rate. Perhaps showing these results in three figures would help to understand this. And perhaps if authors would explain using these figures the percentage of reduction observed for each case, it would not be necessary to calculate any drought tolerance index and to elaborate new figures with the same data. This is not helping to understand their results.

AC/ The results of the drought tolerance index have been deleted in the revised manuscript

RC12/ Line 236: which are the most tested parameters? This is any parameter tested more than others?

AC/ This section means that the increase of PEG concentrations caused the reduction in the most callus growth parameters like CFW, CDW, CL and CW. All parameters are tested in the same way. These parameters are added to the revised manuscript.

RC13/ Line 263: The figure doesn’t show results from unstressed and stressed plants.

AC/ The figure shows results from normal and stress conditions. The figure reveals the differentiation between plants of the optimum group and stress groups. This section has been modified in the revised manuscript.

RC14/ Line 264: Blue dot.

AC/ The word (Bleu) has been corrected (blue) in the revised manuscript

RC15/ Figure 1: mean separation cannot be correct for CWT. And in Line 219, authors stated that there was not a significant decrease for this trait when the medium was supplemented with PEG.

AC/ CI, CFW, CDW, CL, and CW traits showed significant reduction compared to the control treatment and these characteristics gradually decreased from minimal drought stress (T0.50) to severe drought stress (T1.50), while CWT showed a significant increase compared to control group and this characteristic gradually increased from minimal drought stress (T0.50) to severe drought stress (T1.50).

RC16/ Line 237: cell dimension parameters have been described with callus induction and growth parameters in the previous paragraph.

AC/ This section has been modified and corrected in the revised manuscript.

RC17/ Line 269: Effectiveness in callus morphology?

AC/ This section has been modified and corrected for: the effect of PEG on the callus morphology.

RC17/ Line 301: callus growth performance? These traits are related to callus morphology.

AC/ Figure 4 has been deleted in the revised manuscript because you requested the deletion of the results related to the drought tolerance index (see comment RC11).

RC18/ Line 454: What?

AC/ This section has been modified to: The morphological and physiological traits variation between genotypes are relatively unexplored resources that can not only provide crucial data on the capacity and performance of various crops under different climatic conditions but are also an invaluable genetic resource that could be used to improve yields. So, the morphological and physiological traits can be used in the evaluation of drought tolerance in the rice genotypes.

RC19/ Line 474: Because these 6 are the only existing Kurdish rice genotypes?

AC/ No, because the two primers have high PIC values. Increasing in PIC value refers to an increase in the showing of the variation among genotypes.

RC20/ Line 480: phenotypic resemblance means similar performance?

AC/ Yes, phenotypic resemblance means similar performance in terms of callus growth.

RC21/ Line 512: What is the meaning of encompassed less of the most? Are more essential than… what?

AC/ It means that both genotypes (Choman and Shawre) contain a small amount of, the most mineral elements and the results showed' that the mineral elements parameters are less important in the drought tolerance genotypes compared to biochemical characters. This section has been modified in the revised manuscript.

RC22/ Line 530: Are the authors inferring that two of the studied cultivars would show in planta or much better, in field drought tolerance? Why? Is there any correlation for rice between kernel-derived callus ability to grow in PEG supplemented medium and plant drought tolerance? Have they studied this relationship? ... If not, have they described the references that support this inference?

AC/ We did not study the tolerance response of two genotypes under field condition, but it is our project for the future and still, we did not do it because before starting any project, it should be tested the genotypes in the control condition (in vitro). In the control condition, we can screen the genotypes properly because in this case, only the genetic makeup plays a role in the response to drought stress. So, we, start with this project and we will test the plant in the field as a future project. Stress tolerance may thus be considered as a developmentally regulated, stage-specific phenomenon such that tolerance at one stage of plant development may not be linked to tolerance at other developmental stages, as already reported by:  

Muscolo, A.; Junker, A.; Klukas, C.; Weigelt-Fischer, K.; Riewe, D.; Altmann, T. Phenotypic and metabolic responses to drought and salinity of four contrasting lentil accessions. J. Exp. Bot. 2015, 66, 5467–5480, doi:10.1093/jxb/erv208.

Sadat Noori, S.A.; McNeilly T. Assessment of variability in salt tolerance based on seedling growth in Triticum durum Desf. Genetic Resources and Crop Evolution 2000, 47, 285–291, doi:10.1023/A:1008749312148.

Concerning the correlation between kernel-derived callus ability to grow in PEG supplemented medium and plant drought tolerance, it depends on the plant species and so, it should be tested the plants in the control condition (in vitro) and in the filed for determining this correlation.

Best regards

Author Response File: Author Response.docx

Reviewer 2 Report

The reviewed manuscript investigates several genotypes of rice on biochemical, physiological and molecular marker levels to find which of the genotypes respond better to a simulated drought stress treatment using PEG.

The article is written very clearly, all methods are sound and well explained. Nowadays, people begin to wonder whether crop breeding in the past may not be adequate for arid future in the world and whether some of the breeding will have to be done from scratch (see e.g. Jatayev et al, 2020). Knowledge of biochemical and other markers and their presence in specific genotypes is an important piece of information in this respect and could be useful in attempts to identify and select drought resistant genes for future crops. So the presented research is highly relevant.

Although the specific combination of measurements agenotypes used here seems to be highly original, not all similar works done in the past are acknowledged and cited. In fact, the Introduction is limited to what are mostly policy and trend statements and references to similar research are only introduced in the Discussion section. Some Figures are not as informative as they could be.

As a result I recommend publication of the presented research, however with a suggestion of minor changes:

1) Moving the more general and historic parts of discussion of similar work into the Introduction, to make the reader more aware early on of the context of the presented research. A work that could be added to cited references is Joshi et al., 2011 https://www.researchgate.net/publication/236018717_In_vitro_screening_of_rice_genotypes_for_drought_tolerance_using_polyethylene_glycol

because these authors also studied rice plants at the level of tissue culture, or Islam et al 2018 https://www.researchgate.net/publication/326541620_Evaluation_of_Rice_Oryza_sativa_L_Genotypes_for_Drought_Tolerance_at_Germination_and_Early_Seedling_Stage because this is a recent work with similar motivation. The existence of such works also led me to decrease the "originality" mark in this review.

2) It is difficult to follow the content of Figures 8 and 9, because the treatments shown in panels A-D are not described in captions, only shown in specific labels inside the Figure. I suggest the author expand the captions to better describe what is shown in different panels.

3) Overall, I miss a summarizing visualization that would represent the main relationship between genotype and phenotype, integrating the partial relationships evident in various tables and figures throughout the paper. Figure 11 partly foots the bill, however it separates the observed relationships into individual PEG treatments. If there is a way for the authors to show, perhaps by some multidimensional technique, such as PCA or multiple regression a relationship between markers and drought resistance that would hold accross treatments and show the differences among varieties, this would add to the informativeness and attractiveness of the manuscript. If space is limiting, perhaps some of the tables, or even 1-2 figures could be moved to supplementary online-only data (if journal supports these) to make space for the suggested figure.

Otherwise, the manuscript is very well written and I see no need to change anything else.

Author Response

Authors' responses

Manuscript number: applsci-836212

Paper title: In vitro Assessment of Kurdish Rice Genotypes in Response to PEG-Induced Drought Stress

Authors: Didar Rahim, Petr Kalousek, Nawroz Tahir, Tomáš Vyhnánek, Petr Tarkowski, Vaclav Trojan, Dana Abdulkhaleq, Ahmad Hama Ameen and Ladislav Havel

Dear Editor and Reviewer

The authors would like to thank the area editor and the reviewers for their precious time and invaluable comments. We have carefully addressed all the comments. The constructive comments/ suggestions by the reviewer are appreciated. We have now completely revised the manuscript. The manuscript has been corrected by an expert of language and highlighted by trach change method. The corresponding changes and refinements made in the revised paper are summarized in our response below. The actual comments of the reviewer are in BOLDED RC, and the author's responses are italicized AC.

Reviewer-2

General note: some figures have been deleted in the revised manuscript because the first reviewer requested it.

RC1) Moving the more general and historic parts of discussion of similar work into the Introduction, to make the reader more aware early on of the context of the presented research. A work that could be added to cited references is Joshi et al., 2011 https://www.researchgate.net/publication/236018717_In_vitro_screening_of_rice_genotypes_for_drought_tolerance_using_polyethylene_glycol because these authors also studied rice plants at the level of tissue culture, or Islam et al 2018 https://www.researchgate.net/publication/326541620_Evaluation_of_Rice_Oryza_sativa_L_Genotypes_for_Drought_Tolerance_at_Germination_and_Early_Seedling_Stage because this is a recent work with similar motivation. The existence of such works also led me to decrease the "originality" mark in this review.

AC/ The more general and historic parts of discussion have been moved to the introduction and the two mentioned references have been added to the revised manuscript.

RC2) It is difficult to follow the content of Figures 8 and 9 because the treatments shown in panels A-D are not described in captions, only shown in specific labels inside the Figure. I suggest the author expand the captions to better describe what is shown in different panels.

AC/ The caption of the figure is expanded and detailed in the revised manuscript.

RC3) Overall, I miss a summarizing visualization that would represent the main relationship between genotype and phenotype, integrating the partial relationships evident in various tables and figures throughout the paper. Figure 11 partly foots the bill, however, it separates the observed relationships into individual PEG treatments. If there is a way for the authors to show, perhaps by some multidimensional technique, such as PCA or multiple regression a relationship between markers and drought resistance that would hold across treatments and show the differences among varieties, this would add to the informativeness and attractiveness of the manuscript. If space is limiting, perhaps some of the tables, or even 1-2 figures could be moved to supplementary online-only data (if the journal supports these) to make space for the suggested figure.

AC/ A figure of PCA plot (Figure 5) representing the normal and stress conditions has been inserted to the revised manuscript.

Best regards

Author Response File: Author Response.docx

Round 2

Reviewer 1 Report

I am affraid but there are still incongruences between the objective of the research, as stated in the manuscript, and the response of the authors:

In Page 2: To screen in vitro selection for the establishment of drought-tolerant genotypes means that authors plan to obtain drought-tolerant genotypes by selection performed in vitro, while the actual objective is to perform in vitro tests to characterize the drought-tolerance of six existing rice genotypes. Or screening rice genotypes for in vitro induced drought tolerance, but not establishing, since the rice genotypes have not been established by the authors.

And yes, kernel-derived callus growth in PEG-supplemented medium may be correlated to plant drought tolerance,… and may not. This should be discussed at the introduction and taken into account for the conclusions, since the authors have not included any previous information about in field drought tolerance of these cultivars, neither tested the response of plants. Therefore we can’t know if the PEG-derived in vitro results of calli are indicating the performance of these genotypes under drought conditions at the plant level. The conclusions “In the short term, the use of callus traits is… for quick screening of resistant or sensitive rice genotypes” and “the reliable predictor of drought tolerance” are not sustained by the experimental work developed, since we have no evidence of correlation with drought tolerance of rice plants.

If further experiments are going to be performed to asses this, then explain it at the end of the manuscript.

Why not to mention at the manuscript that these six are the most cultivated cultivars in Kurdistan?

To determine the impact of PEG (Induced drought) on the size of the callus … Sorry for my unclear question. I understood why the authors performed these assays… What I wanted to bring out is that this objective is not mentioned at the introduction of the manuscript. Authors are evaluating the ability of these six rice cultivars to produce callus in PEG-supplemented medium, and characterizing this callus: cell size, proline and mineral contents, and enzymatic activities. These are the concrete objectives of the work, and I think this should be described instead of using less specific terms such as in vitro selection or response to PEG-induced stress.

This section means that the increase of PEG concentrations caused the reduction in the most callus growth parameters like CFW, CDW, CL and CW. I guess authors wanted to say that addition of PEG to medium significantly reduced the observed results for most of the callus growth parameters.

Figure 3 caption is still not correct, since it is not self-explanatory. The figure did not show results from plants: biochemical traits have been determined in kernel derived calli from six rice cultivars with a number of replications for each. This information must be included. Figure 4 caption is also not self-explanatory.

RC19/ Line 474: Because these 6 are the only existing Kurdish rice genotypes? AC/ No, because the two primers have high PIC values. Increasing in PIC value refers to an increase in the showing of the variation among genotypes. The sentence “two SSR markers can be used for identification of Kurdish rice genotypes” means all the Kurdish rice genotypes, but you studied only six, and there are others, therefore the correct sentence would be “can be used for identification of the six studied Kurdish genotypes”

The Stress Tolerance Index in Table 6 has not been previously defined in this version.

Author Response

Authors' responses

Manuscript number: applsci-836212

Paper title: In vitro Assessment of Kurdish Rice Genotypes in Response to PEG-Induced Drought Stress

Authors: Didar Rahim, Petr Kalousek, Nawroz Tahir, Tomáš Vyhnánek, Petr Tarkowski, Vaclav Trojan, Dana Abdulkhaleq, Ahmad Hama Ameen and Ladislav Havel

Dear Reviewer

The authors would like to thank the area editor and the reviewers for their precious time and invaluable comments. We have carefully addressed all the comments. The constructive comments/ suggestions by the reviewer are appreciated. We have now completely revised the manuscript. The manuscript has been corrected by an expert of the language of MDPI in the first round (see the attached Certificate-19746). The correction and modification have been highlighted (in yellow color) in the revised version. The corresponding changes and refinements made in the revised paper are summarized in our response below. The actual comments of the reviewer are in BOLDED RC, and the author's responses are italicized AC.

Reviewer-1

General point: This manuscript has been edited by English Editing expert of MDPI (Please see the attached certificate of English Editing- Certificate-19746)

RC1. In Page 2: To screen in vitro selection for the establishment of drought-tolerant genotypes means that authors plan to obtain drought-tolerant genotypes by selection performed in vitro, while the actual objective is to perform in vitro tests to characterize the drought-tolerance of six existing rice genotypes. Or screening rice genotypes for in vitro induced drought tolerance, but not establishing, since the rice genotypes have not been established by the authors.

AC/ The objective of this research is to evaluate and characterize the ability of six local rice genotypes to produce callus in PEG-supplemented medium. This section is corrected in the revised manuscript.

RC2. Yes, kernel-derived callus growth in PEG-supplemented medium may be correlated to plant drought tolerance,… and may not. This should be discussed at the introduction and taken into account for the conclusions since the authors have not included any previous information about in field drought tolerance of these cultivars, neither tested the response of plants. Therefore we can’t know if the PEG-derived in vitro results of calli are indicating the performance of these genotypes under drought conditions at the plant level.

AC/ This section is modified in the revised manuscript in this way: In the short term, the use of callus characters is useful for rapid screening of resistant or sensitive rice genotypes, and the reliable indicator of drought tolerance is not supported by the experimental work, as there is no evidence of correlation with the drought tolerance of rice plants. As a future project, Choman and Shawre should be tested in field condition to find a correlation between the responses of both genotypes under in vitro and field conditions.

RC3. If further experiments are going to be performed to asses this, then explain it at the end of the manuscript.

AC/ This section has been added to the conclusions: As a future project, Choman and Shawre should be tested in field condition to find a correlation between the responses of both genotypes under in vitro and field conditions.

RC4. Why not to mention at the manuscript that these six are the most cultivated cultivars in Kurdistan?

AC/ This line has (these six genotypes are the most cultivated cultivars in Kurdistan) been added in the Materials and Methods in the revised version.

RC5. What I wanted to bring out is that this objective is not mentioned at the introduction of the manuscript. Authors are evaluating the ability of these six rice cultivars to produce callus in PEG-supplemented medium, and characterizing this callus: cell size, proline and mineral contents, and enzymatic activities. These are the concrete objectives of the work, and I think this should be described instead of using less specific terms such as in vitro selection or response to PEG-induced stress.

AC/ Yes, the main objective in this study is to evaluate the ability of these six rice genotypes to produce callus in PEG-supplemented medium and the aim has been modified in the revised version.

RC6. This section means that the increase of PEG concentrations caused the reduction in the most callus growth parameters like CFW, CDW, CL and CW. I guess authors wanted to say that addition of PEG to medium significantly reduced the observed results for most of the callus growth parameters.

AC/ Yes, we want to say that the addition of PEG to medium significantly reduced the observed results for most of the callus growth parameters. This line has been modified in the revise version.

RC7. Figure 3 caption is still not correct, since it is not self-explanatory. The figure did not show results from plants: biochemical traits have been determined in kernel derived calli from six rice cultivars with a number of replications for each. This information must be included. Figure 4 caption is also not self-explanatory.

AC/ the caption of figures 1, 3 and 4 has been corrected and expanded in the revised version.

RC8. because the two primers have high PIC values. Increasing in PIC value refers to an increase in the showing of the variation among genotypes. The sentence “two SSR markers can be used for identification of Kurdish rice genotypes” means all the Kurdish rice genotypes, but you studied only six, and there are others, therefore the correct sentence would be “can be used for identification of the six studied Kurdish genotypes”

AC/ Yes, the two SSR primers can be used for identification of the six studied genotypes and the PIC value depends on the genotype and its number. This section has been modified in the revised version.

RC9. The Stress Tolerance Index in Table 6 has not been previously defined in this version

AC/ This index has been defined in the statistical data analysis in this way: the ranking method was used to identify the best genotypes, using various measured traits. Using this method, the stress tolerance index (STI) and the average number of ranks (ASR) were used as a criterion for choosing the best genotypes for all characteristics. In this method, each trait's best performance recorded the lowest rank; thus, the best genotypes were identified with the highest STI and lowest ASR values.

Best regards

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

I attached a review file for this manuscript.

Comments for author File: Comments.pdf

Author Response

Authors' responses

Manuscript number: applsci-782071

Paper title: In vitro Assessment of Kurdish Rice Genotypes in Response to PEG-Induced Drought Stress

Authors: Didar Rahim, Petr Kalousek, Nawroz Tahir, Tomáš Vyhnánek, Petr Tarkowski, Vaclav Trojan, Dana Abdulkhaleq, Ahmad Hama Ameen and Ladislav Havel

Dear Editor and Reviewer

The authors would like to thank the area editor and the reviewers for their precious time and invaluable comments. We have carefully addressed all the comments. The constructive comments/ suggestions by the reviewer are really appreciated. We have now completely revised the manuscript All corrections are highlighted by the red line. The manuscript has been corrected by an expert of language. The corresponding changes and refinements made in the revised paper are summarized in our response below. The actual comments of the reviewer are in BOLDED RC, and the author's responses are italicized AC.

Reviewer-1

Specific Comments:

RC1. What did the dendrogram by 11 SSR markers contribute for the discussion of this study? I think the dendrogram has been deleted in this study, because you did not a well-known tolerant or susceptive rice genotype. And although you concluded that six rice genotypes were divided into three distinct clusters, the divided clusters could not account for your conclusion (line 416~418).

AC/ The dendrogram has been deleted

RC2. Although in the abstract (line 25~28), you introduced that Drought stress has had a significant impact on callus growth and morphology parameters comprising callus induction (CI), callus fresh weight (CFW), callus dry weight (CDW), relative water content (RWC), callus length (CL), callu width (CW) and callus wall thickness (CWT) and in results, it has various and rich findings about callus growth and morphology parameters, you did not assess response (tolerant or susceptive) of each rice genotype.

AC/ The detail about the callus growth and morphology has been added in abstract and results and discussion

RC3. You concluded that the extent of relative water content, antioxidant enzyme activities of CAT and accumulation of K, Mg and Chai in White Bazyan and Gwll Swr genotypes showed their rate of tolerance under drought stress (line 414~416), but you have introduced (line 63~70) and discussed (line 377~393) that enzyme activity and proline content were various enzymatic and nonenzymatic antioxidant defense systems.

AC/ The structure of conclusion has been modified

General Comment: English mistakes

AC/ English editing and correction of grammatical mistakes have been done in the revised manuscript.

Best regards

Corresponding author

Author Response File: Author Response.docx

Reviewer 2 Report

I can see that the authors have made some efforts to address previous concerns in relation to this manuscript. There are, however, many minor and a few major errors throughout the paper that need to be addressed. I must stress that the whole paper needs to be looked at and revised not just the points written here.

The major concern is that the results from this work are not clearly and accurately presented. This was an original concern of mine and I see the authors have made efforts in this direction, but it is still unclear. I now understand what is been presented, but there are still major issues.

• The unproven level of accuracy is a big issue. I do not believe that 2 decimal places is appropriate, can this be justified?
• There are missing values and it is unclear why this is
• The units are still not formatted correctly
• There are extra letters after some values - why?
• Linking the letters to meanings would make this clearer a, b, c? what do the letters related too?

The other major issue is that there are ALOT of formatting inconsistencies that should be thoroughly corrected eg page 3 line 117-127 'x %' vs 'x%', page 3 line 123 and page 6 line 231-236 'µm' instead of 'µM', etc etc. This needs to be fully revised.

Other issues -

the abstract is far too long, it should be concise and too the point. Progress was made here, but it has gone backwards.

page 2 line 54 'our lands' is not appropriate for publication. I am pleased to see that the rational for this work has more completely been described. Careful use of language is important, this work should not be politicised.

Related to the major issue - values etc page 7 line 243 are presented to a unproven level of accuracy. These need to be reported to 2 or 3 significant figures at a maximum unless this can be justified.

There are several English errors throughout the paper - eg Page 7 line 241-241 – ‘was noticed to have the’

Author Response

Authors' responses

Manuscript number: applsci-782071

Paper title: In vitro Assessment of Kurdish Rice Genotypes in Response to PEG-Induced Drought Stress

Authors: Didar Rahim, Petr Kalousek, Nawroz Tahir, Tomáš Vyhnánek, Petr Tarkowski, Vaclav Trojan, Dana Abdulkhaleq, Ahmad Hama Ameen and Ladislav Havel

Dear Editor and Reviewer

The authors would like to thank the area editor and the reviewers for their precious time and invaluable comments. We have carefully addressed all the comments. The constructive comments/ suggestions by the reviewer are really appreciated. We have now completely revised the manuscript All corrections are highlighted by the red line. The manuscript has been corrected by an expert of language. The corresponding changes and refinements made in the revised paper are summarized in our response below. The actual comments of the reviewer are in BOLDED RC, and the author's responses are italicized AC.

Reviewer-2

Specific Points:

RC1. The unproven level of accuracy is a big issue. I do not believe that 2 decimal places is appropriate, can this be justified?

AC/ The decimal values have been corrected in the revised manuscript.

RC2. There are missing values and it is unclear why this is

AC/ The missing value has been corrected in the revised manuscript

RC3. The units are still not formatted correctly

AC/ The units have been corrected in the revised manuscript.

RC4. There are extra letters after some values - why?

AC/ The extra letters have been corrected in the revised manuscript.

RC5. Linking the letters to meanings would make this clearer a, b, c? what do the letters relate too?

AC/ the means within a column are not connected by the same letter are significantly different.

For example

The difference in means between White Bazyan, and Red Bazyan is significant because the means is connected by two different letters (a for White Bazyan and c for Red Bazyan).

The difference between Choma mean and White Bazyan mean is not significant because the two means have a common letter (a). The same explication applies for Gwll Swr and Kalar because of the means of Gwll Swr and Kalar connected by the same letter (bc). So, a common letter between two means indicates the non-significant difference.

In addition, the number of letters (a, b, c, d or a, b, c, d, e, f……etc) depends on the degree of significance between means within a column

• If the significance is high between means, the column takes a greater number of letters (a, b, c, d, e, f).
• If the level of significance is moderate between means, the column takes a moderate number of letters (a, b, c, d).
• If the level of significance is low between means, the column takes a few numbers of letters (a, b).

RC6. The other major issue is that there are ALOT of formatting inconsistencies that should be thoroughly corrected eg page 3 line 117-127 'x %' vs 'x%', page 3 line 123

AC/ All errors have been corrected in the revised manuscript.

RC7. Page 6 line 231-236 'µm' instead of 'µM', etc etc. This needs to be fully revised

AC/ Concerning Page 6 line 231-236, µm unit (micrometre unit), it is a metric unit and usually, it writes as µm because micrometre (µm) is the unit of measurement of dimension (Length, width and thickness). So, the writing µM is not correct. We always capitalize the M for the concentration like Molar or Molarity. In addition, (nm-nanometre) is the unit of wavelength so it writes in minuscule form.

RC8. the abstract is far too long, it should be concise and too the point. Progress was made here, but it has gone backwards.

AC/ The abstract has been improved

RC9. Page 2 line 54 'our lands' is not appropriate for publication. I am pleased to see that the rational for this work has more completely been described. Careful use of language is important, this work should not be politicised.

AC/ English Corrections have been made in the revised manuscript

RC10. Related to the major issue - values etc page 7 line 243 are presented to a unproven level of accuracy. These need to be reported to 2 or 3 significant figures at a maximum unless this can be justified.

AC/ The section has been modified in the revised manuscript

RC11. There are several English errors throughout the paper - eg Page 7 line 241-241 – ‘was noticed to have the’

AC/ English Corrections have been made in the revised manuscript

General Comment: English mistakes

AC/ English editing and correction of grammatical mistakes have been done in the revised manuscript.

Best regards

Corresponding author

Author Response File: Author Response.docx

Round 2

Reviewer 1 Report

I attached a reviewing document file for this study.

Comments for author File: Comments.pdf

Reviewer 2 Report

I think this work could have been done with more traditional methods; but after a number or round and a full re-submission this paper is now ready for publication. A final read through is needed, but this can be done at the proof stage. Thank you for your efforts in addressing my concerns.