Effects of Selenate Application on Growth, Nutrient Bioaccumulation, and Bioactive Compounds in Broccoli (Brassica oleracea var. italica L.)

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

Thank you for inviting me to assess the merits of the manuscript for publication in Horticulturae.

The aim of this manuscript is to evaluate selenium (Se) supply on productivity, Se accumulation, agronomic biofortification, broccoli quality and Se bioavailability after Se treatment and cooking.

All sections of the manuscript are very well written. A well-founded introduction, clear objectives, correct, accurate and detailed methodology, clearly presented results and a well-written discussion.

There are a few small notes I made in the text. My biggest question is why the authors didn't carry out a polynomial regression study. However, this is not a reason to reject the paper for publication.

The conclusions are in line with the results of the study.

Comments for author File: Comments.pdf

Author Response

We thank the reviewers for all your suggestions and corrections. We have agreed to include most of the changes proposed by the reviewers to improve the manuscript and make it more readable. We hope that it will now be acceptable for publication in Horticulturae. Corrected manuscript is attached with and without changes indicated by using the track changes mode in MS Word.

Thank you for inviting me to assess the merits of the manuscript for publication in Horticulturae.

The aim of this manuscript is to evaluate selenium (Se) supply on productivity, Se accumulation, agronomic biofortification, broccoli quality and Se bioavailability after Se treatment and cooking.

All sections of the manuscript are very well written. A well-founded introduction, clear objectives, correct, accurate and detailed methodology, clearly presented results and a well-written discussion.

There are a few small notes I made in the text. My biggest question is why the authors didn't carry out a polynomial regression study. However, this is not a reason to reject the paper for publication.

Line 24 unnecessary because you put Brassica oleracea var. italica

The keyword has been deleted

Line 47 What does mean?

Thank you for pointing this out. We have clarified that EFSA is European Food Safety Authority

Line 60 var.italica

This has been addressed.

Line 146 Broccoli leaves are waxy. Was an adhesive spreader (surfactant) used? If so, which one?

To ensure the effectiveness of treatment, a commercial surfactant was used. (0.1% v/v Tween 20; Sigma Aldrich, San Luis, MO, US). A reference to this effect has been introduced in the materials and methods section.

 

Line 213 why did not carry out polynomial regression study? (0 0 0,05 0 0,1 and 0,15)

We answer better this question in the following points.

Line 237 If polynomial regression study is applied, probably, a significant fit should be found for four of the five characteristics evaluated.

When polynomial regression was applied to the five variables in the table, only a significant fit was found for the Stem+Leaves height (y = 28.333 - 35.111x + 1433.333x2  - 7155.556x3; adjusted R-squared = 0.5424933; p-value = 0.02582170). Since this variable already showed significant differences in the ANOVA, we consider better not to include the polynomial analysis.

Line 299 Why was not a significant equation fitted? The results observed in the florets indicate the possibility of studying higher doses.

The reason for not including the polynomial regression analysis in the study, particularly in this case, is that we achieved our goal of sufficiently increasing the Se content in the florets with the minimum dose of sodium selenate (0.05-Se). Therefore, we considered that higher dosages of Se would not be necessary.

The conclusions are in line with the results of the study.

Reviewer 2 Report

Comments and Suggestions for Authors

In the current manuscript, the authors investigated the efficacy of varied Se foliar applications on broccoli. The manuscript is well written, but I have some minor comments:

1. Since broccoli is a Se-hyperaccumulator, it is essential to calculate how much Se (per treatment) will be afforded when people consume 100 g FW. This also will lead to knowing how much Se-enriched broccoli covers the Se daily requirements dose μg Se/day.

2. Lines 44-445 please cite: Abdalla, M.A.; Wick, J.E.; Famuyide, I.M.; McGaw, L.J.; Mühling, K.H. Selenium enrichment of green and red lettuce and the induction of radical scavenging potential. Horticulturae 2021, 7, 488.

Author Response

We thank the reviewers for all your suggestions and corrections. We have agreed to include most of the changes proposed by the reviewers to improve the manuscript and make it more readable. We hope that it will now be acceptable for publication in Horticulturae. Corrected manuscript is attached with and without changes indicated by using the track changes mode in MS Word.

In the current manuscript, the authors investigated the efficacy of varied Se foliar applications on broccoli. The manuscript is well written, but I have some minor comments:

 

1. Since broccoli is a Se-hyperaccumulator, it is essential to calculate how much Se (per treatment) will be afforded when people consume 100 g FW. This also will lead to knowing how much Se-enriched broccoli covers the Se daily requirements dose μg Se/day.

Thank you for pointing this out. This aspect was already introduced in the Discussion for the lowest Se treatment. The results for each Se treatment have now been calculated and explained, and the discussion has been expanded to include additional context.

2. Lines 44-445 please cite: Abdalla, M.A.; Wick, J.E.; Famuyide, I.M.; McGaw, L.J.; Mühling, K.H. Selenium enrichment of green and red lettuce and the induction of radical scavenging potential. Horticulturae 2021, 7, 488.

Thank you for the suggestion. We have added the reference in the Discussion.

Reviewer 3 Report

Comments and Suggestions for Authors

Summary

The study is very interesting and addresses current needs in the agricultural sector. Biofortification is an extremely relevant topic, especially in the context of modern agriculture, which must address the nutritional deficiencies of the population. The comparison between fresh and boiled material is particularly intriguing. However, there are some issues that need to be resolved. The use of only one accession (albeit very stable) is not sufficient to support the authors' hypothesis. The discussions are well-founded. The work will be accepted after the resolution of the following questions.

Title

Title requires the use the capitol letter for each world.

Abstract

Lines 17-19: “Se dose (0.05-Se) was sufficient to notably increase Se content, being enough to potentially improve human Se status” How did you deduce this? Did you calculate it, or is this conclusion based on bibliographical information? If it is based only on literature, I suggest adding “as previously documented/outlined.”

Introduction

Lines 32-39: Synthetize. Despite this paragraph is quite interesting, the enzymes that incorporate Se is not the target of Your study. Within this context, I suggest to reduce the size of this paragraph.

Line 46-47: Why You only mention Spain (and not for example Europe)? Is okay for me, but this information must be contextualized. 

Line 60: If You specify “broccoli” You must specify the varietas i.e. “italica”.

Line 83 and others: According to MDPI reference management guidelines, it is preferable not to specify the author followed by "et al." Instead, you should phrase it as "previously documented/outlined." I mentioned a similar point in the abstract, but for different reasons.

Lines 99-104: I suggest You to define better the aim of Your study, because lines 99-101 are similar to 101-104.

I highly recommend You to add some references related to the foliar, innovative treatments in broccoli (10.17660/ActaHortic.2023.1365.10; https://doi.org/10.3389/fnut.2023.1057084)

Materials and Methods

Subsection 2.1.

When describing the experimental conditions, you must specify which instruments (sensors) were used for measuring temperature and humidity. Did you use a data logger (if yes, specify the model), or did you retrieve the data from an online public database (if yes, specify the database)? Additionally, it would be better to specify the daily temperatures (maximum, minimum, mean) with, for instance, a line plot rather than only providing the average value from November to March.

Subsection 2.3.

Which seeds were used? Specify the cultivar (hybrid, landrace, breeding line) and the type of crop (apical dominance, sprouting). Who provided the seeds? You must thoroughly detail the plant material.

Line 146: Provide additional details related to the spraying procedures. Did you use an electric backpack sprayer, a manual sprayer, or another instrument? Specify the instrument and, if possible, the model. How did you ensure that only 15 ml was sprayed?

Results

Table 1. Why did You only provide the LSD for only one trait (Stem+Leaves height). In Line 241 there are two dots.

Table 3. There are several parameters without LSD.

I highly recommend providing a principal component analysis (PCA) to display the distribution of the genotype in relation to the different Se concentrations for the analysed traits. This will help to understand which parameters vary significantly as a result of the different Se doses. You can provide also two PCA plots, one for the raw florets and the other for the boiled ones.

Discussion

Why did you analyse only one cultivar? How do you know that the descriptors you analyzed were not genotype-related factors? This is a limitation of your study, and you must specify it. Brassica crops generally display extremely wide variability. In your study, the standard deviation did not reach high values. This needs to be discussed because, I suppose, you used commercial material, which is generally uniform. You must address this in your discussion.

As outlined by several authors, Se application mitigates various types of abiotic stress and increases yield. I suggest commenting on this aspect in the context of your work (in one paragraph of about 3-4 lines). In fact, your results showed an increase in florets weight as a consequence of Se application.

Comments for author File: Comments.pdf

Comments on the Quality of English Language

Author Response

We thank the reviewers for all your suggestions and corrections. We have agreed to include most of the changes proposed by the reviewers to improve the manuscript and make it more readable. We hope that it will now be acceptable for publication in Horticulturae. Corrected manuscript is attached with and without changes indicated by using the track changes mode in MS Word.

Summary

The study is very interesting and addresses current needs in the agricultural sector. Biofortification is an extremely relevant topic, especially in the context of modern agriculture, which must address the nutritional deficiencies of the population. The comparison between fresh and boiled material is particularly intriguing. However, there are some issues that need to be resolved. The use of only one accession (albeit very stable) is not sufficient to support the authors' hypothesis. The discussions are well-founded. The work will be accepted after the resolution of the following questions.

Title

Title requires the use the capitol letter for each world.

Thank you for pointing it out. This has been changed

Abstract

Lines 17-19: “Se dose (0.05-Se) was sufficient to notably increase Se content, being enough to potentially improve human Se status” How did you deduce this? Did you calculate it, or is this conclusion based on bibliographical information? If it is based only on literature, I suggest adding “as previously documented/outlined.”

Thank you for pointing this out. To make that statement, we calculated the Se content of a portion of 100 g of our boiled broccoli florets, considering an average of 10% of dry matter, and compared it with the recommended amount of Se per day. We agree with the reviewer that this must be better addressed in the abstract and we have changed it to make it clearer.

Introduction

Lines 32-39: Synthetize. Despite this paragraph is quite interesting, the enzymes that incorporate Se is not the target of Your study. Within this context, I suggest to reduce the size of this paragraph.

We have only left the initial part of the paragraph, which introduces the importance of selenium as a key part of amino acids and the references to two of the most important enxymes related, GPx and TrxR, since they are related to antioxidant activities, which are related to our study.

Line 46-47: Why You only mention Spain (and not for example Europe)? Is okay for me, but this information must be contextualized. 

Thank you for pointing this out. We have changed this to make a broader statement regarding global situation and we have maintained the Spanish reference as an example.

Line 60: If You specify “broccoli” You must specify the varietas i.e. “italica”.

This has been addressed.

Line 83 and others: According to MDPI reference management guidelines, it is preferable not to specify the author followed by "et al." Instead, you should phrase it as "previously documented/outlined." I mentioned a similar point in the abstract, but for different reasons.

Thank you for pointing this out. This has been addressed throughout the manuscript.

Lines 99-104: I suggest You to define better the aim of Your study, because lines 99-101 are similar to 101-104.

We have reformulated the second part of our aim in order to make the distinction clearer.

I highly recommend You to add some references related to the foliar, innovative treatments in broccoli (10.17660/ActaHortic.2023.1365.10; https://doi.org/10.3389/fnut.2023.1057084)

We have added the references highlighting the importance of foliar applications on broccoli.

Materials and Methods

Subsection 2.1.

When describing the experimental conditions, you must specify which instruments (sensors) were used for measuring temperature and humidity. Did you use a data logger (if yes, specify the model), or did you retrieve the data from an online public database (if yes, specify the database)? Additionally, it would be better to specify the daily temperatures (maximum, minimum, mean) with, for instance, a line plot rather than only providing the average value from November to March.

We have added the information regarding the datalogger used for the duration of the experiment. Additionally, we have added a climograph representing the weekly average of maximum minimum and relative humidity within the greenhouse.

Subsection 2.3.

Which seeds were used? Specify the cultivar (hybrid, landrace, breeding line) and the type of crop (apical dominance, sprouting). Who provided the seeds? You must thoroughly detail the plant material.

The seeds used in the experiment were from a commercial variety known as was Green Top, and commercialized by Takii Seeds (Almería, Spain). It is a precocious variety, with a dense apical production, that has been traditionally used in Spain for the last 20 years. This information has been added to the manuscript

 

Line 146: Provide additional details related to the spraying procedures. Did you use an electric backpack sprayer, a manual sprayer, or another instrument? Specify the instrument and, if possible, the model. How did you ensure that only 15 ml was sprayed?

The broccoli plants were individually treated with a 30 ml manual hand sprayer, containing the 15 ml used in every repetition. This way, we could minimize losses, ensure that there were no cross-contamination, that soil did not receive any amount of sodium selenate and that each plant received the correct dosage of the nutrient solution. We have clarified this point in the Materials and Methods section.

 

Results

Table 1. Why did You only provide the LSD for only one trait (Stem+Leaves height). In Line 241 there are two dots.

Table 3. There are several parameters without LSD.

In both tables, the letters have been added to make it clearer that there are no significant differences.

I highly recommend providing a principal component analysis (PCA) to display the distribution of the genotype in relation to the different Se concentrations for the analysed traits. This will help to understand which parameters vary significantly as a result of the different Se doses. You can provide also two PCA plots, one for the raw florets and the other for the boiled ones.

Thank you for pointing this out. We have conducted an evaluation of the PCA results for our dataset. The principal dimensions in the PCA explain less than 50% of the total variation, indicating that a significant portion of the data's variability is not captured by the first few principal components. Given this, we believe the PCA plot, for this particular experiment, may not provide substantial additional insights beyond those already offered by the ANOVA included in the manuscript.

 

Discussion

Why did you analyse only one cultivar? How do you know that the descriptors you analyzed were not genotype-related factors? This is a limitation of your study, and you must specify it. Brassica crops generally display extremely wide variability. In your study, the standard deviation did not reach high values. This needs to be discussed because, I suppose, you used commercial material, which is generally uniform. You must address this in your discussion.

Thank you for pointing this out. We used one cultivar in order to evaluate the effect of the different foliar treatments of sodium selenate on the parameters analyzed and being able to ponder the Se dosages. We agree with the reviewer that, once we have made a first but important approximation to this point, the next experiments should be oriented to evaluate the different responses of a wider range of broccoli varieties and cultivars. We have added this limitation to the discussion, also as an opportunity for the future.

 

As outlined by several authors, Se application mitigates various types of abiotic stress and increases yield. I suggest commenting on this aspect in the context of your work (in one paragraph of about 3-4 lines). In fact, your results showed an increase in florets weight as a consequence of Se application.

This potential of Se to improve yield and mitigate the negative effects of abiotic stresses is addressed in the second paragraph of the discussion as we acknowledged the effect of the biofortification on floret weight, although this increase was not significant in our case. We have highlighted this point and included additional references.

Round 2

Reviewer 3 Report

Comments and Suggestions for Authors

The authors greatly replied to all the comments, provide a significanlty improved version of the manuscript.

Comments on the Quality of English Language

The authors greatly replied to all the comments, provide a significanlty improved version of the manuscript.

Author Response

Thank you very much for your words and your corrections. We are pleased that the article is now to your liking.