Improvement of Tillering and Grain Yield by Application of Cytokinin Derivatives in Wheat and Barley

Round 1

Reviewer 1 Report

The authors have considerably improved the manuscript, but it is still with some major issues. The introduction needs to be improved by stating the problem and simply describing the differences between the CKd that the authors want to test and their possible impact. I agree with the authors that this is valuable information, and although treatments yield differences with the control were not outstanding, treatments consistently increased the yield over years. In that regard, it seems the authors are not using the appropriate statistical analysis. By simply plot in a 1:1 plot the treatment against its control and running a paired T-test of all comparisons across years, the authors should be able to show this consistency in the response. Different colors of the symbols could indicate the different CKd and depend on their differences they can be treated altogether or separated. A more advanced analysis would be a combined analysis of variance where the year of the experiment is also included. This is a major issue that the authors should fix before the manuscript can be published. Also, the statistic analysis is in supplementary results while the bioassay is the first result. It should be the opposite.

Minors issues:

Abstract: add a first phrase stating the problem. Also, RR-V is never defined in the abstract and never say that isopentenyl-adenin types were measured. It is confusing. Add some numbers of the percentage of increase for example.

24-27 add references to each of these sentences.

28 “complex” What does it mean in that context? This sentence could be eliminated.

30-31 “ineffective”? please provide a reference to this sentence.

37-38 provide the reference

153 delete “GPS position” leave only the numbers

155 delete “MGS”. It is not used afterward

156 the fertilizer units should be kg ha-1. Otherwise, specify.

199-205 seems material and methods rather than results

Author Response

The authors have considerably improved the manuscript, but it is still with some major issues. The introduction needs to be improved by stating the problem and simply describing the differences between the CKd that the authors want to test and their possible impact. I agree with the authors that this is valuable information, and although treatments yield differences with the control were not outstanding, treatments consistently increased the yield over years. In that regard, it seems the authors are not using the appropriate statistical analysis. By simply plot in a 1:1 plot the treatment against its control and running a paired T-test of all comparisons across years, the authors should be able to show this consistency in the response. Different colors of the symbols could indicate the different CKd and depend on their differences they can be treated altogether or separated. A more advanced analysis would be a combined analysis of variance where the year of the experiment is also included. This is a major issue that the authors should fix before the manuscript can be published. Also, the statistic analysis is in supplementary results while the bioassay is the first result. It should be the opposite.

Minors issues:

Abstract: add a first phrase stating the problem. Also, RR-V is never defined in the abstract and never say that isopentenyl-adenin types were measured. It is confusing. Add some numbers of the percentage of increase for example.

ANSWER: Thank you for your comment. We fully agree that proper data presentation is necessary to provide reliable and useful information to the readers. However, we believe that our way of providing statistical analysis is appropriate. In last version we provided Kruskal-Wallis ANOVA with post-hoc test due to the reason that dataset does not shown normal distribution. For this reason only non-parametric test should be appropriate here. Thus we would like to present datasets in current form to not mislead readers.

However, we also prepared excel file with yield data showing tests on normality (Komologorov-Smirnov, Liliefors, Shapiro-Wilk) then we plot T-Test 1:1 treatment against its control and also ANOVA (factors treatment and year) with Fisher LSD post-hoc test. This file should serve mainly for reviewer to clarify the data structure, as we believe that parametric tests are not appropriate for our datasets.

We also wanted to stress here that irrespectively of p-values our data showed consistent trends over the years as is clearly visible from supplementary data where results for particular years are provided. This should be understood as most convincing evidence that our plant hormone derivatives are effective modulators of plant growth and stimulators of yield. For these reasons we believe that movement of ANOVA tests from Supplementary file to main text is rather confusing than helping for readers. Thus we believe that current form helps better to provide understandable information.

24-27 add references to each of these sentences.

ANSWER: Thank you for suggestion. References for both sentences have been completed.

28 “complex” What does it mean in that context? This sentence could be eliminated.

ANSWER: Thank you for comment. The word “complex” is not suitable, therefore we have substituted it for “complicated”.

30-31 “ineffective”? please provide a reference to this sentence.

ANSWER: Thank you for suggestion. Reference for this resource have been completed.

37-38 provide the reference

ANSWER: Thank you for suggestion. Reference for this resource have been completed.

153 delete “GPS position” leave only the numbers

ANSWER: Thank you for suggestion. The words about “GPS position” has been deleted.

155 delete “MGS”. It is not used afterward

ANSWER: Thank you for suggestion. The abbreviation “MGS” has been deleted.

156 the fertilizer units should be kg ha-1. Otherwise, specify.

ANSWER: Thank you for suggestion. The information about dosage of fertilizers has been completed and repaired.

199-205 seems material and methods rather than results

ANSWER: Thank you for suggestion. The part of “results” about field experiments conditions were removed into the part “materials and methods”.

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments to the Authors:

The manuscript: Improvement of tillering and grain yield by application of cytokinin derivatives in wheat and barley by Koprna et al., present the detailed analysis of the effects of various cytokinin derivatives and antagonists on winter wheat and spring barley on the number of productive tillers and grain yield. I believe that RR-G, RR-O, RR-V, and RR-P are the novel compounds that are previously not tested for their effects on the number of productive tillers and grain yield for winter wheat and spring barley.   

The manuscript is well written and presented; however, I have below mentioned a few comments.

My minor concerns are as below:

First, the abstract needs clarity. Please follow the general abstract preparation guidelines and prepare the abstract—[Introduce the topic, the research question, approach, significant results, and conclusion].

Please describe, in the introduction and discussion, why this research was important, and how it was different from the previous studies about the effects of cytokinin derivatives. (Agronomic and economic importance?)  

Line 160: Please explain the deciding factor for using the dosage concentrations.

Line 160: Please check: and as foliar treatment in a dosage of 300 l per ha. I assume 300 L per ha.

Line 165: the authors stated that they used 30 plants for the morphological analysis. Please, explain: how the plants were selected; I assume it was a random selection.  

Figures 3 & 4: Please explain whether the controls were the mock treatments or non-treated plants? If they are non-treated then why not use the single control for all the treatments?

Line 248: explain iP-type metabolites or provide the full form

Line 261: Please check

Table 1: Please correct the values presented in the table, for example, 121,79 to 121.79

Author Response

Thank you for reviewer for all comments and suggestions. We hope, the answers will reply to all questions.

First, the abstract needs clarity. Please follow the general abstract preparation guidelines and prepare the abstract—[Introduce the topic, the research question, approach, significant results, and conclusion].

ANSWER: Thank you for comment. The abstract was claritied acconding to your suggestion and abstract preparation guidelines.

Please describe, in the introduction and discussion, why this research was important, and how it was different from the previous studies about the effects of cytokinin derivatives. (Agronomic and economic importance?)  

ANSWER: Thank you for comment. Importance of the study is noted in introduction – all references about importance of maintaining yield stability, tillering support and affecting this parameter by cytokinins. Previously studies were done by our team on field crops – especially wheat and barley (Koprna, R.; De Diego, N.; Dundálková, L.; Spíchal, L. Use of Cytokinins as Agrochemicals. Bioorgan. Med. Chem., 2016, 24, 484–492).

Economic importance is depended of grain price on market, but our calculations are, if the increase of yield is more than 2.5 %, it is economically repayable. We hope that the explanation of the economic benefits in the commentary will be sufficient and does not need to be added to the article.

Line 160: Please explain the deciding factor for using the dosage concentrations.

ANSWER: Thank you for comment. Used concentrations are derived from the most suitable concentration of each substances in bioassays.

Line 160: Please check: and as foliar treatment in a dosage of 300 l per ha. I assume 300 L per ha.

ANSWER: Thank you for comment. We repaired abbreviation from “l” to “L” per hectare.

Line 165: the authors stated that they used 30 plants for the morphological analysis. Please, explain: how the plants were selected; I assume it was a random selection.  

ANSWER: Thank you for question. 30 random plants (not from borders) of each variants was used for morphological assessment.

Figures 3 & 4: Please explain whether the controls were the mock treatments or non-treated plants? If they are non-treated then why not use the single control for all the treatments?

ANSWER: Thank you for question. In manuscript is filled in “All variants were compared with non-treated control in each years of testing.“ The reason, why we did not use the single control is – there are not the same years of testing each substances, therefore we compared selected years of testing for every single case with compared controls. Data from all years with data of controls are noted in supplementary file (suppl. Table 1).

Line 248: explain iP-type metabolites or provide the full form

ANSWER: Thank you for comment. The text was changed to “iP and iPR content”.

Line 261: Please check

ANSWER: Here we are not sure what should be fixed. Mentioned line stated: “The most important exception was in the content of O-glucosides, which were downregulated in the RR-P plants in comparison to the control (Table 1.)”. We checked data mentioned there and it seems to properly describing the Table 1 results: In RR-P samples only Total CK content (as is mentioned in the sentence “Although the total CK content in the plants sprayed with CK derivatives was lower than or similar to the controls…“) and O-glucosides were down-regulated while the other compounds were stimulated. We believe that this clarification will satisfy the reviewer comment.

Table 1: Please correct the values presented in the table, for example, 121,79 to 121.79

ANSWER: Thank you for suggestion. We have changed all presented values in the table to correct form.

Reviewer 3 Report

Research addressing the use of plant biostimulants on crops are extremely important because farmers need guidance in terms of the efficacy of each compound. Therefore, this study can be useful for farmers and for upcoming research in this topic. However, this study has some flaws that should be addressed:

Line 107: Why these three specific cytokinin derivatives were selected? Need more information.

Line 108: Technically, the introduction is not the most adequate section to provide details regarding the conclusion. It can mislead the readers.

Line 144: More information regarding the tobacco callus test is necessary to be mentioned. For example: What is the time frame for the CK compounds being exposed to the tobacco callus?

Line 159: Could you be more clear in terms of how many seed and foliar applications were conducted?

Line 165: 30 plants/plot? Could you be more specific?

Line 203: Technically, it is not necessary to present the graphs/figures that are not containing any statistical significance. You can try to use 0.1 > p or simply just mention the trends in the results section.

Line 264: You cannot state “improving the plant yield” because it was not statistically significant. It is better to reword it.

Line 353: CK antagonist performed better than the others CK compounds?

Author Response

Thank you for all the valuable comments and questions. We hope that all questions about the manuscript will be answered.

Line 107: Why these three specific cytokinin derivatives were selected? Need more information.

ANSWER: Thank you for your comment. The compounds were selected according their effectivity in bioassays. The results of the bioassays are in the manuscript.

Line 108: Technically, the introduction is not the most adequate section to provide details regarding the conclusion. It can mislead the readers.

ANSWER: Thank you for your suggestion. We have modified the sentence about results and removed the information about results.

Line 144: More information regarding the tobacco callus test is necessary to be mentioned. For example: What is the time frame for the CK compounds being exposed to the tobacco callus?

ANSWER: Thank you for your comment. The tobacco callus bioassays as well as other biotest presented within the manuscript are standard procedures and were well described previously in following papers that are also referenced within the manuscript. We believe that this info will satisfy you. 

For further information about bioassay: CK-dependent tobacco (Nicotiana tabacum cv. Wisconsin 38) callus derived from a 4-week-old culture was cultivated on solid MS medium (Murashige and Skoog, 1962) supplemented with sucrose (30 g l⁻¹), naphthaleneacetic acid (NAA; 1 mg l⁻¹), and the CK to be tested (in a concentration ranging from 1 nM to 100 μM). CK activities were expressed as average fresh weight (FW) of tissue per flask after 7 weeks of cultivation in darkness at 23 °C and compared in accordance with the EC₅₀ values. Five independent experiments were set up for each CK. The control stock calli were grown on the same medium containing benzyladenine (0.2 mg l⁻¹). The tested compounds were applied in concentrations of 1×10⁻⁹, 1x10⁻⁸, 1×10⁻⁷, 1×10⁻⁶, 1×10⁻⁵, and 1×10⁻⁴ M.

Line 159: Could you be more clear in terms of how many seed and foliar applications were conducted?

ANSWER: Thank you for the suggestion. For clarity there is stated in the text now “single foliar treatment”. As we follow GEP practices the seed coating is standard agronomical procedure performed only once before seed sowing.

Line 165: 30 plants/plot? Could you be more specific?

ANSWER: Thank you for suggestion. Now, description of used method of field testing and plants evaluation is completed. Plot = 10 sq meters area of plants sown according to GEP (Good Experimental Practises) – all treatments in 5 replications (in SUM at 50 sq meters). Descriptions of picked plants were done at single plants from the plots. Description of the method is also noted in lines 148 to 154.

Line 203: Technically, it is not necessary to present the graphs/figures that are not containing any statistical significance. You can try to use 0.1 > p or simply just mention the trends in the results section.

ANSWER: Thank you for your comment. We believe that the visualization of trends in plots is most important tool to help the reader understand the manuscript “message.” We also wanted to stress here that irrespectively of p-values our data showed consistent trends over the years as is clearly visible from supplementary data where results for particular years are provided. This should be understood as most convincing evidence that our plant hormone derivatives are effective modulators of plant growth and stimulators of yield. Thus we believe that current form helps better to provide understandable information.

Line 264: You cannot state “improving the plant yield” because it was not statistically significant. It is better to reword it.

ANSWER: Thank you for your comment. We believe that are data clearly showed that there are trends repeating every season (please see Supplementary files), even if those are not significant on p-value level 0.05. However, this is mainly caused in statistically insufficient number of repeats that is almost impossible to reach in standard field-plot experiments. Moreover, the validity of p-values and all frequentists statistics for biological research have doubted numerously (Mathews 2018, R. Soc Open Sci. 5:171047;  Goodman 2018, Nature; 564:7-7.). Thus we believe that current form provides reliable and understandable information.

Line 353: CK antagonist performed better than the others CK compounds?

ANSWER: Thank you for answer. No, CK derivatives increased grain yield in all experiments (wheat and barley too), while CK antagonist increased grain yield only in case of winter wheat.

Round 2

Reviewer 1 Report

Dear Authors,

I still think that the experimental data is good and deserves to be published, but it is unacceptable to not present statistical analysis in the main manuscript. Even when results are not significantly different between treatments, the results of the test used must be somehow provided to the reader in the main text. Moreover, all explanations gave to me by the authors about the data analysis must be explained in materials and methods. It is not fully explained there.

Also, I did not receive any excel file and have not seen yet any 1:1 plot with the T-test in which yield from the control treatment is on the x-axis and yield from the three CKd are on the y-axis. With different symbols from each CKd.

From the agronomic point of view, the bioassay is less relevant than the statistical yield differences between treatments. Therefore, the bioassay (that also lacks statistical analysis) should be in the supplementary material and the yield analysis should be summarized in the main text.

There are also some minor issues such as:

Abstract: name the three compounds tested and abbreviations otherwise nobody knows what RR-V means there.  

Line 273 "applied substances" that are very unspecific.

Line 294-295: If the compounds were already tested in bioassays then there is no need to present them in the main text and first in results. Place the bioassays in the supplementary materials and add references to the materials and methods.

Author Response

Authors want to thank expert reviewer for the valuable comments, we believe that improved version of our manuscript satisfy your remarks and help the readers to better understand presented manuscript. Statistics have been changed according to suggestions and recommendations of reviewer in manuscript and supplementary file too. All replies are as “Reviewer_1_Answers_file.pdf”, or changed in enclosed manuscript.

Yours sincerely

Radoslav Koprna

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

Modern agriculture tries to improve crop yield employing a plethora of approaches. One of these approaches is to apply chemicals that enhance growth or improve yield. The cereals wheat and barley are two main crops grown worldwide. In this manuscript, Koprna et al show the effects of applying cytokinins (a plant phytohormone) derivatives to grain yield and tillering. They complement their experiment with a cytokinins quantification after these treatments and showing that these CK analogues perturb the endogenous homeostasis. 

Although the manuscript is in general well written and follow a logic reasoning, the fact that none of the phenotypic parameters measured was significantly changed resulted decisive for this reviewer to reject it for publication in agronomy. Let me add some more specific aspects that require improvements for future submissions to more specialized journals or this one after a structure reconsideration:

• General comments:
• The size of the different sections is not well balanced. The introduction and discussion are very long and provides, although correct, unnecessary information about, for example, the history of CKs. Reconsider re-write them more concisely.
• The references are not sorted following the journal rules. The first cited paper must-have reference number 1 and so on. 
• Style improvements:
• The explanation about how the CK derivatives work in lines 119-132 could be much more clear with a little diagram of (1) the structural differences with the natural compound and (2) the action mode
• Figure 1 is done with the default output style of M Excel. Consider modify it in more professional software.
• Data presented in Table 1 could be more visual just transforming these data into charts.
• Supplemental Figure 1: Although colour code is the same, the order (Control, left; treatment, right) is not the same in all the charts.
• Supplemental Figures 2 and 3: the style of the panel letters is different. Homogenise the style of all the figures.
• Minor points:
• The very first sentence of the introduction ends with a “(REF)”. Include the appropriate reference. I would ask for the same after the sentence starting at line 31. 
• Line 34: I would substitute “energy” for “resources”.
• Line 264: there is an unnecessary blue line.
• Lines 327 and 331: sometimes authors use the italics for “O-glucosides” and sometimes they do not. 
• Line 266. Which test was used for this statistical analysis?

Reviewer 2 Report

This manuscript has a great amount of data to aim the effect of derivates of cytokinin on barley and wheat yield. However, the experiments lack experimental design (or at least it is not presented by the authors), statistical analysis, and clarity on their presentation. I could not follow the manuscript well and it is not clear on what statistical basis the authors conclude about the results. This manuscript needs a lot of work.

#3 Title said in cereals which is too broad. I would narrow it down to wheat and barley.

#10 The CK is not defined. I see CKd and CKa but not CK.

#27 What’s ”REF”? it is never used in the text after this first sentence.

#111-113 this sentence should be in the results and not in the introduction.

#172 What does BBCH  21-25 means? Use worldwide recognize crop stages.

#173 What was the area harvested?

#182 Statistical analysis?

#183 In the result section, the yields should be presented first, and then the rest of the results.

Figure 1 is rudimentary and must be improved. Significant differences should be marked.

Figure 2: the numbers are small and the significant differences are not marked.

Table 1: Which analysis did the authors use?