Impact of Biologically and Chemically Synthesized Zinc Oxide Nanoparticles on Seed Germination and Seedlings’ Growth

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

This is a very interesting piece of research, where the authors aim at comparing the effects of zinc oxide nanoparticles, obtained by biological synthesis and chemical synthesis, on different plant parameters in seeds and seedlings of Capsicum annuum and Solanum lycopersicum. The results indicated that Zinc oxide nanoparticles obtained by green synthesis are more effective in generating bioactive compounds and activating the enzyme defense system due to being more biocompatible and they improved seed germination rate.

 

The objectives are clearly presented, although the study lucks of scientific hypotheses. As this is an experimental study, those hypotheses are to be posed and tested. Hence, the drawing of the final conclusions should be based on the hypothesis. All this needs to be addressed in the manuscript, before it can be considered for publication.

 

The reason for having washed the seeds with ethanol is to sterilize them. Please, make it clear in the manuscript.

Why where 100, 200, 300, and 400 mg L−1 of BZnONP or CZnONP the treatments used and not 50, 150, 250 and 500? Explain that end, please.

Does one petri dish with 10 seed equal one box? Is so, please, make it clearer, or even better refer to the petri dishes as petri dishes instead of boxes. Otherwise, explain the replicates in a clearer way.

Why was temperature constant during the germination texts? There is discrimination in the among of light, but not in the temperature. This needs to be explained and justified.

 

Is table 1 really needed? The very same information had already been indicated in the text.

Why was taken the fresh weight of the plumule and not the dry weight of them, as it is the most common way to measure biomass production. The fresh weight contains water that might mask the actual biomass production, hence giving little information with biological meaning.

 

It is very unclear what the real sample sizer were used in the measurement of each variable. If at 7 days several germinant were destroyed to provide their weight and length, how many seedlings remained to take the rest of the measurements (i.e. photosynthetic pigments, bioactive compounds and enzymatic activity)? It looks like the sample sizes were very reduced and clearly insufficient to achieve the conditions for an ANOVA. How did you test that data were normal and homoscedastic.

 

The quality of the figures needs to be improved.

I would suggest that the authors separate Results and Discussion in two independent sections. In the current form, it is somehow confusing to follow the relation among all the measured variables.

 

Lines 196-197: it is said that ‘The nanoparticles influence seed germination because they penetrate the seed coat and increase its permeability’ How do you know that this is the actual reason and action mechanisms of nanoparticles for the increase of germination?  Couldn’t it be that there is another reason why seed germination increases like other ways in which nanoparticles aid the seed in breaking their dormancy? This needs further discussion and explanation.

Following the above, when concentrations of nanoparticles are low, they enhance germination as they aid coat penetration, however, at high concentrations the result is phytotoxic. How do you relate these two distinct effects, one mechanical and the other chemical.  According to your discussion there is no chemical involvement in the first action of the nanoparticles at low concentrations. The same applies for the results regarding the pigment concentrations.

 

Line 215: it is said that ‘The vigor index is a measure of seedling vigor’ what is something similar to saying that The water is H₂O.  What does actually measures seed vigor? From your text, seed vigor can be increased by the nanoparticles. However, seed vigor is an inherent characteristic of seed lots. This needs to be clarified.

 

I did not understand clearly what are the mechanisms behind the increase of enzymatic activity due to the presence of nanoparticles in seeds.

Comments for author File: Comments.pdf

Comments on the Quality of English Language

Not major issues detected.

Author Response

Dear Reviewer,

We are truly thankful for your evaluation. Please refer to the attached dox document for our full response. 

 

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

 

Dear Authors, in your manuscript, the following points should be added/changed to further improve it:

 

1.       Abstract: Please add information on the size (nm) of the ZnO nano used in the research.

2.       Introduction: I have a comment on the sentence “Zinc ion reduction reactions result in nanocrystalline zinc oxide nanoparticles 45 (ZnONP).” What kind of reduction is being referred to here? Has there been a change in the oxidation state of the Zn2+ ion? We can talk about the reduction reaction when the product is obtained in the form of metallic zinc.

3.       Introduction: In your description of the state of the art review, please include the publication “DOI: 10.3390/ma13122784”. An important parameter to pay attention to is the chemical purity of the ZnO samples used for research.

4.       Materials and Methods: Did the manufacturer state the chemical purity of the ZnO produced? Is there a known method for the chemical synthesis of ZnO? If the authors are in possession of this data, please add the information to the manuscript.

5.       Conclusions: Please add information about the sizes of ZnO used in the research.

 

 

Author Response

Dear Reviewer,

Thank you! We appreciate your help in making our manuscript better. Attached please find a response to your comments.

We hope that you will find our reply satisfactory. 

Author Response File: Author Response.pdf

Reviewer 3 Report

Comments and Suggestions for Authors

The authors describe a research article entitled “Impact of biologically and chemically synthesized zinc oxide nanoparticles on seed germination and seedlings' growth”. The topic of the manuscript is interesting, and the manuscript constitutes an interesting article concerning the potential impact of the synthetic method used for generating nanoparticles on seed germination and seedlings' growth. A detailed conclusion highlighting the main results of this work. However, no perspectives are provided at the end of this work.

The work is well-written and a well-constructed introduction has been established by the authors. Sufficient spectra and figures are included in the manuscript for comprehension and clarity. Numerous figures in colors have been introduced in the manuscript, rendering the article more attractive. Interesting and convincing results are also presented in this manuscript. Overall, I think that this is a manuscript that I recommend for publication after inclusion of minor revisions.

1)      Perspectives should be added in the conclusion section.

2)      What about the impact of the particles size ? This point is not discussed. In this study, only particles around 70 nm size have been used.

 

Author Response

Dear Reviewer,

We truly appreciate your help in making our manuscript better. Attached please find our response to your comments.

We hope that you will find our reply satisfactory. 

Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

 

I genuinely appreciate and commend the authors for their dedicated efforts in enhancing the manuscript, diligently addressing nearly all the points I had raised in my previous report. However, there are several critical aspects that have been overlooked and hold significant importance within a scientific text.

The first crucial aspect pertains to the absence of clearly defined scientific hypotheses. The authors have omitted to provide a response to this essential question. In the context of an experimental study, it is imperative to include the hypotheses that are being tested. Are you conducting these experiments with specific hypotheses in mind, or is it an exploratory endeavor without predefined expectations? Given your esteemed reputation as a scientist, the latter seems unlikely.

 

To the question Why where 100, 200, 300, and 400 mg L−1 of BZnONP or CZnONP the treatments used and not 50, 150, 250 and 500? The authors state that “Our previous work used concentrations of 50, 100, 150, 200, and 250 mgL-1 for preliminary germination study. That article was focused on the synthesis of the NPs. (https://doi.org/10.3390/su15043080)” please, make this end clear in the new version of the manuscript.

 

 

I appreciate your clarification regarding the sample size. It is true that, in germination studies, each seed represents a binary response (germination or non-germination), making parametric statistical tools less suitable. However, the choice of statistical methods used in this study has not been adequately addressed by the authors. Given the nature of the data collected, the adoption of non-parametric tests would have been more appropriate. This critical issue, which has not been addressed by the authors, is a significant concern and remains a major reason for my decision to reject the manuscript.

 

I concur with the authors regarding the decision to combine the Results and Discussion sections. It is indeed an author's prerogative to do so when the outcome is a coherent piece of research. However, I must emphasize that the current manuscript lacks a comprehensive discussion that integrates individual experiment interpretations into a unified research context. The absence of specific hypotheses for testing has contributed to this lack of clarity and a sense of direction. This issue could have been resolved through a single, cohesive discussion section, followed by conclusions derived from the results, aligned with the proposed hypotheses.

 

An example of this lack of coherence is the introduction of ideas from other studies that are not adequately discussed in the current manuscript, such as the effects of nanoparticles on seed dormancy, the combined physical and chemical effects of nanoparticles on this process, and the apparent toxicity exerted by specific nanoparticle concentrations. These points are mentioned in the context of other studies but lack a substantial foundation in the current research.

 

This fundamental issue remains unresolved and impacts the manuscript's readiness for publication. It is indeed a critical factor in my decision to reject the manuscript.

Author Response

Dear Reviewer,

Thank you for your contribution to our manuscript. We have included your suggestions as listed in the attached document. 

Author Response File: Author Response.pdf

Round 3

Reviewer 1 Report

Comments and Suggestions for Authors

Thank you very much for having made the effort to improve the quality of the manuscript. At a first sight, it looked like if you had made a major review of it. However, there  changes are minor, although sufficiente.

In Table 1, please chage 'plúmula' by 'plumule'.