Influence of Light Conditions and Medium Composition on Morphophysiological Characteristics of Stevia rebaudiana Bertoni In Vitro and In Vivo

Round 1

Reviewer 1 Report

The reviewed manuscript describes the studies on the influence of some chemical (PGRs in culture media) and physical (light spectral composition) culture conditions on selected phenotypic traits, multiplication and rooting in vitro, and production of diterpene glycosides, stevioside and photosynthetic pigments in vivo of Stevia rebaudiana Bertoni.

However, in spite of the fact that Stevia rebaudiana is a crop of rapidly growing importance and that research on this species attracts great attention, which could make the subject of the research interesting from a practical point of view, the quality of the manuscript is not up to standard. 

The theoretical background presented in the “Introduction” is rather cursory and general. I would expect at least a short summary of previous studies in which similar explants and culture conditions were tested and a clear, specifically justified aim.

There are many contradictions between the sections "Material and Methods" and "Results and Discussion" which extremely impede reading and understanding the manuscript. For example:

- in spite of many analyses and measurements described in "Material and Methods", Authors presented only selected results, without their statistical significance;

- in the section “Material and Methods” Authors introduced abbreviations for different media formulations (MS 1, MS 2, MS 3). What for, I ask, if in “Results and Discussion” they used denotations like: hormone-free, Epin MS composition, and BAP?;

- the results of particular analysis are presented in a different order than they were described in the "Materials and Methods".

There is no real discussion of obtained results. Sentences given in lines 165-167, 173-175, 274-275, 316-318 are truisms and obviousnesses. Besides one paragraph (lines 207-219) the rest of the section "Results and Discussion" provides only results and fragments repeated from "Materials and Methods” or which should be included there (lines 167-172, 265-267, 290-293).

The section "Conclusion" is only a copy of the "Abstract". Moreover, it mostly repeats some truisms and a few sentences from the “Results and discussion”, while it should emphasize the uniqueness of the study, its potential application and the advantage of presented results over other similar findings published so far.

Overall conclusion: 

The presented manuscript is of insufficient quality and should be completely reworded and rewritten. Also, the language of the manuscript should be improved, because it contains a lot of grammatical errors.

Author Response

Dear Reviewer,

On behalf of ourselves and the coauthors, we thank you for your criticism of our manuscript, as well as for your valuable comments and questions. We are confident that your comments and corrections will make our manuscript better.

First, I would like to say that we have tried to significantly improve the editing of the English language and style. We hope that our article will be clearer for reviewers.

The theoretical background presented in the “Introduction” is rather cursory and general. I would expect at least a short summary of previous studies in which similar explants and culture conditions were tested and a clear, specifically justified aim. 

Response 1: We agree with your comments. We have supplemented the «Introduction» with the available research on in vitro Stevia culture and the effect of culture conditions (genetic, physiological and physical) on the accumulation of secondary metabolites (the text highlighted in green). We hope that the extended introduction leads to a clearer goal of the study. We also supplemented the list of cited literature.

There are many contradictions between the sections "Material and Methods" and "Results and Discussion" which extremely impede reading and understanding the manuscript. For example:

- in spite of many analyses and measurements described in "Material and Methods", Authors presented only selected results, without their statistical significance;

Response 2: We agree with your comments. We have made the appropriate corrections in the Materials and Methods section. In our investigation we used statistical analyses according to analysis of variance (ANOVA). Figures 6-10 show the mean values ± standard error after ANOVA test at a = 0.05.

- in the section “Material and Methods” Authors introduced abbreviations for different media formulations (MS 1, MS 2, MS 3). What for, I ask, if in “Results and Discussion” they used denotations like: hormone-free, Epin MS composition, and BAP?;

Response 3: We agree with your comments. We have made the appropriate corrections in «Materials and Methods» (147-151 lines) and "Results and Discussion" (300-318 lines) sections (the text highlighted in yellow).

- the results of particular analysis are presented in a different order than they were described in the "Materials and Methods".

Response 4: We made the appropriate corrections.

There is no real discussion of obtained results. Sentences given in lines 165-167, 173-175, 274-275, 316-318 are truisms and obviousnesses. Besides one paragraph (lines 207-219) the rest of the section "Results and Discussion" provides only results and fragments repeated from "Materials and Methods” or which should be included there (lines 167-172, 265-267, 290-293).

Response 5: We took into account your comment (the text highlighted in red).

The section "Conclusion" is only a copy of the "Abstract". Moreover, it mostly repeats some truisms and a few sentences from the “Results and discussion”, while it should emphasize the uniqueness of the study, its potential application and the advantage of presented results over other similar findings published so far.

Response 6: The section "Conclusion" is rewritten. (the text highlighted in green, 368-394 lines).

Once again, we are so grateful for your critical review and valuable comments. In addition, we also send you a WORD document with the text, taking into account the comments.

Best regards,

Marat Khaliluev

Author Response File: Author Response.docx

Reviewer 2 Report

The Authors try to present the morphophysiological changes related to the optimization of stevia cultivation. They are specifying the light spectrum and the presence of PGR in the culture medium.
The diagram of the experiment shown in Table 3 is somewhat misleading. The reader is looking for a crazy matrix of dependencies instead of 3 types of medium and four light ranges. The figure can be simplified, and only the spectra must remain; the medium is described above in quite a detail.
The first figure would use some kind of scale; it is difficult to refer to morphological changes without a centimetre scale visible.
In the figure 2 description, there are two terms, "a" and "b", which are not in the picture itself. It needs to be completed.
The third figure lacks statistical analysis, and the caption lacks information on the number of repetitions. Similarly, in the following figure, it is necessary to calculate and mark the significance of the data on the graph. And the next runway. 
The captions of all figures should be completed with the information on the number of repetitions and the statistical test used. Statistically significant results should be marked on the graph according to a uniform convention. The signatures of the chart axes should be standardized; sometimes, they start with lowercase, sometimes with a capital letter.
The addition of a graphic summary would greatly simplify the reception of the manuscript.
In summary, the manuscript is ready for publication after significant corrections.

Author Response

Dear Reviewer,

On behalf of ourselves and the coauthors, we thank you for your criticism of our manuscript, as well as for your valuable comments and questions. We are confident that your comments and corrections will make our manuscript better.

First, I would like to say that we have tried to significantly improve the editing of the English language and style. We hope that our article will be clearer for reviewers and readers.

The diagram of the experiment shown in Table 3 is somewhat misleading. The reader is looking for a crazy matrix of dependencies instead of 3 types of medium and four light ranges. The figure can be simplified, and only the spectra must remain; the medium is described above in quite a detail.

Response 1: We agree with your comments. The experimental diagram shown in Fig. 3 (Table 3) has been edited (150 line).

The first figure would use some kind of scale; it is difficult to refer to morphological changes without a centimetre scale visible.

Response 2: We agree with your comments. In Figure 4, we added a scale (ruler) to morphological differences shown in the results were obvious (229 line).

In the figure 2 description, there are two terms, "a" and "b", which are not in the picture itself. It needs to be completed.

Response 3: We agree with your comments. In fig. 5 (figure 2), we added the necessary designations, as well as in the figure caption.

The third figure lacks statistical analysis, and the caption lacks information on the number of repetitions. Similarly, in the following figure, it is necessary to calculate and mark the significance of the data on the graph. And the next runway.

Response 4: We agree with your comments. In our investigation we used statistical analyses according to analysis of variance (ANOVA). Figures 6-10 show the mean values ± standard error after ANOVA test at a = 0.05. In the captions for each figure, we added the necessary data on the number of repetitions (lines 293, 319, 330, 348, 366) (the text highlighted in red).

The captions of all figures should be completed with the information on the number of repetitions and the statistical test used. Statistically significant results should be marked on the graph according to a uniform convention. The signatures of the chart axes should be standardized; sometimes, they start with lowercase, sometimes with a capital letter.

Response 5: The captions of all figures completed with the information on the number of repetitions and the statistical test used (lines 293, 319, 330, 348, 366) (the text highlighted in red). The signatures of the chart axes standardized.

In addition, we have supplemented the "Introduction" (lines 67-84; 85-87; 93-102) and "Conclusion" (lines 369-395)sections in accordance with the comments of another reviewer. We also supplemented the list of cited literature.

Once again, we are so grateful for your critical review and valuable comments. In addition, we also send you a WORD document with the text, taking into account the comments.

Best regards,

Marat Khaliluev

Author Response File: Author Response.docx

Round 2

Reviewer 1 Report

Dear Authors,

The manuscript has been roughly corrected so I decided to give it a chance to be published, however, the text still needs a lot of improvement to fulfill the principles of scientific writing. Here I provided some general guidelines, please find also more detailed remarks in the text of the manuscript (Word file attached).

1. Please add at the very beginning of the section “Material and Methods” the subsection 2.1 entitled “Plant material” and put all information about the material (i.e. who provided plants and what was the explant source) ther.
2. In each subsection of the section “Material and Methods” concerning in vitro culture please, specify:

• what type of explants was used in particular experiment (nodes with two buds? whole microshoots? both? Unfortunately it is unclear now). If the explants were the same in all experiments, you can provide this information in the subsection “Plant material”.
• what kind of chemical and physical conditions of culture were applied (medium composition, type and colour of light, photoperiod, temperature etc.) and what was used as a control.
• how long did the treatment last?
• what kind of parameters were measured or determined after that time.

3. In each subsection of the section “Material and Methods” concerning biochemical assays please:

• specify, what kind of plant material was taken for analyses (only leaves? whole shoots? roots? It is unclear now.)
• describe shortly the equipment and procedure

4. Be precise. For example, if the subsection concerns quantitative determination of photosynthetic pigments (line 152), do not describe phenotypic assays there (lines 153-156). Adjust the titles of the subsections to their content and vice-versa. Avoid unnecessary repetitions and truisms.
5. Adjust the titles of the figures to theirs content. Figures 1-3 are not experimental designs, but just the graphs of light spectra used in particular experiments.
6. Be consequent using abbreviations of PGRs and Latin binomial names of the species. At the first use give the full name and the abbreviation, in the rest of the text - only abbreviations.
7. Still, there is lack of superscripts indicating the statistical significance of the differences between means presented on the Figures 6-10.
8. The section “Conclusion” is far better than it was previously, however, there are still some unnecessary repetitions. Also, too much details is dispensable, since they can be found in the section “Results and Discussion”.

Comments for author File: Comments.docx

Author Response

Dear Reviewer,

We thank you for reviewing of our manuscript and for your valuable comments. We are confident that your comments and corrections will make our manuscript better.

Remarks in the text of the manuscript

• At the first use, the full binomial Latin name of the species should be given. But it is accepted, that subsequently the first part (generic name) is abbreviated. Please, be consequent and correct it at the rest of the text.

Response 1: We agree with your comments. At the first use, the full binomial Latin name we used (Stevia rebaudiana Bertoni). In the following, we have used the abbreviated name (S. rebaudiana).

• Please, consider the creation of the additional subsection entitled “Plant material”

Response 2: We creation of the additional subsection entitled “Plant material” (107-112 lines).

• If all PGRs were studied both in isolation and in combination with each other, the detailed results of these studies should be available in supplementry materials

Response 3: We agree with your comments. We have edited your comment. We have made the appropriate corrections (117-122 lines).

• Blue is not a part of the red spectrum

Response 4: We have made the appropriate corrections (127-136 lines).

• This description has nothing in common with quantitative determination of photosynthetic pigments. Move it to the apropriate section / sections

Response 5: We agree with your comments. We have made the appropriate corrections and delete from section «photosynthetic pigments».

• A node with two axillary buds was an explant source or just an explant? Please specify, which part of a plant was chosen and put on the culture medium. And move this information to the section “Material and Methods”. It would be ideally to create a subsection “2.1 Plant material”, and put all information about the material there (and only there), i.e. who provided plants, what was the explant source and which parts of a plant served exactly as explants in particular experiments.

Response 6: We agree with your comments. We creation of the additional subsection entitled “Plant material” (107-112 lines). We used a node with two axillary buds as an explant source for in vitro experiments. (111-112 lines).

• Please, move it to the apropriate subsection of “the section Material and Methods”

Response 7: We have supplemented this data in the “Materials and Methods section” (130-131 lines).

• If the figures a1 and a2 illustrate the same result, one of them is unnesecarry and shold be removed. The same applies to the figures b1 and b2.

Response 8: We have changed the figures according to your comments.

Remarks in the review

1. Please add at the very beginning of the section “Material and Methods” the subsection 2.1 entitled “Plant material” and put all information about the material (i.e. who provided plants and what was the explant source) ther.

Response 1: We have significantly changed the section materials and methods, as well as made additions and changes in accordance with the comments of the reviewer (the text highlighted in yellow). We creation of the additional subsection entitled “Plant material” (107-112 lines). We have changed according to the remarks.

2. In each subsection of the section “Material and Methods” concerning in vitro culture please, specify:

• what type of explants was used in particular experiment (nodes with two buds? whole microshoots? both? Unfortunately it is unclear now). If the explants were the same in all experiments, you can provide this information in the subsection “Plant material”.

Response 2: We have changed according to the remarks (111-112 lines)

• what kind of chemical and physical conditions of culture were applied (medium composition, type and colour of light, photoperiod, temperature etc.) and what was used as a control.

Response 3: We agree with your comments. We have edited your comment. We have made the appropriate corrections:

medium composition (117-122 lines);

photoperiod, temperature etc. (123-126 lines);

type and colour of light (127-147 lines);

what was used as a control (lines 121; 130-134; 139).

• how long did the treatment last?

Response 4: We have changed according to the remarks. (lines 121-122; 127-140; 155)

• what kind of parameters were measured or determined after that time.

• Response 5: We have changed according to the remarks. (lines 121-122; 159-163)

3. In each subsection of the section “Material and Methods” concerning biochemical assays please:

• specify, what kind of plant material was taken for analyses (only leaves? whole shoots? roots? It is unclear now.)

Response 6: We have changed according to the remarks in the title of subsections (lines 173-174; 183-184).

• describe shortly the equipment and procedure

Response 7: In addition, we have significantly expanded the relevant sections for better understanding.

4. Be precise. For example, if the subsection concerns quantitative determination of photosynthetic pigments (line 152), do not describe phenotypic assays there (lines 153-156). Adjust the titles of the subsections to their content and vice-versa. Avoid unnecessary repetitions and truisms.

Response 8: We agree with your comments. We have made the appropriate corrections and delete from section «photosynthetic pigments».

5. Adjust the titles of the figures to theirs content. Figures 1-3 are not experimental designs, but just the graphs of light spectra used in particular experiments.

Response 9: We agree with your comments. We have made the appropriate corrections (lines 141, 144, 156)

6. Be consequent using abbreviations of PGRs and Latin binomial names of the species. At the first use give the full name and the abbreviation, in the rest of the text - only abbreviations.

Response 10: We agree with your comments. At the first use, the full binomial Latin name we used (Stevia rebaudiana Bertoni). In the following, we have used the abbreviated name (S. rebaudiana). The same applies to PGRs.

7. Still, there is lack of superscripts indicating the statistical significance of the differences between means presented on the Figures 6-10.

Response 11: In Figures 6-10, a symbol (asterisk) has been added for those treatments that differ significantly from the control. In addition, a legend has been added (the text highlighted in green).

8. The section “Conclusion” is far better than it was previously, however, there are still some unnecessary repetitions. Also, too much details is dispensable, since they can be found in the section “Results and Discussion”.

Response 12: The section "Conclusion" is rewritten. (the text highlighted in green, 406-420 lines).

Once again, we are so grateful for your critical review and valuable comments. In addition, we also send you a WORD document with the text, taking into account the comments.

Best regards,

Marat Khaliluev

Author Response File: Author Response.docx

Reviewer 2 Report

The authors made sufficient corrections. In my opinion, the manuscript is suitable for publication.

Author Response

Dear Reviewer,

We thank you for reviewing of our manuscript and for your valuable comments. 

Best regards,

Marat Khaliluev 

Round 3

Reviewer 1 Report

Dear Authors,

thank you for taking my comments into consideration and making all required corrections. I have some small last remarks to the text of the manuscript (please find Word file attached) but overall I can now recommend your work for publication.

Comments for author File: Comments.docx

Author Response

Dear Reviewer,

Thanks again from ourselves and all co-authors for a critical review of the manuscript and valuable comments, which significantly improved our article. All your valuable comments and corrections have been taken into account in the final version of the manuscript. 

Best regards and obligatory health in our difficult times,

Marat Khaliluev

Author Response File: Author Response.docx