Nanoextract of Zataria multiflora Boiss. Enhances Salt Stress Tolerance in Hydroponically Grown Ocimum basilicum L. var. Genovese
Round 1
Reviewer 1 Report
Comments and Suggestions for AuthorsThe manuscript titled "Effect of Zataria multiflora Boiss. extract and nano-extract on inducing salt stress tolerance of Ocimum basilicum L. var. Genovese under floating culture" presents a well-structured and methodologically sound study evaluating the role of Z. multiflora extract and its nano-formulation in enhancing basil’s tolerance to salinity stress. The work integrates physiological, biochemical, and essential oil composition analyses in a floating hydroponic system, which is both timely and relevant in the context of sustainable horticulture under abiotic stress. The manuscript is rich in data, and the experimental design appears strong. However, several issues related to clarity, figure presentation, and manuscript structure should be addressed to improve the manuscript’s overall scientific value and readability.
Comments
- The introduction is overly long and includes several loosely connected ideas. The authors should restructure it to first define the problem (salinity stress), then introduce the significance of basil as a crop, followed by the rationale for using Z. multiflora and its nano-formulation. Several cited studies are described in too much detail without clearly linking them to the current study’s hypothesis.
- Lines 174–184: The objective of the study is suppressed at the end of the introduction. It should be clearly stated earlier, in a separate paragraph, to highlight the novelty and specific aims of the research.
- Lines 194–210: The experiment design is stated, but critical details such as total number of treatments (4 salinity × 3 extract = 12), randomization procedure, and plot layout should be clarified. Also, define whether each tray represented one replicate or multiple plants per replicate were averaged.
- Figure 1 showing the TEM image of nano-extract lacks a scale bar and particle size distribution data. The characterization of the nano-extract should be expanded to include average particle size and zeta potential to validate its “nano” designation.
- Line 336 onward: The Results section lacks a concise overview or transitional paragraph. It would improve readability if each subsection began with a brief summary statement outlining key trends before presenting detailed data.
- Table 1: The unit for K and Na content is given as mg g-1 DW, but the legend does not clarify whether values are leaf-specific or averaged across tissues. Also, the significance markers (*) should be defined more clearly, and ensure column headers are not split awkwardly (Different size text in column headers).
- Lines 494–517: The antioxidant activity (DPPH and FRAP) data are well explained, but the mechanism linking extract application to enhanced activity is not discussed. The authors should include a brief discussion in the Results or Discussion section linking phenolic/flavonoid accumulation to antioxidant function.
- Lines 524–575: The essential oil composition section is informative but difficult to follow. Table 3 is too dense and contains many compounds irrelevant to the discussion. Consider condensing this table to include only the major components and moving the full dataset to supplementary material. Also can’t read all the values in file, the values are going out of the page.
- Lines 584–598: The PCA results are presented with minimal interpretation. Figure 6 is visually helpful, but the description of which traits load heavily on PC1 vs PC2 is vague. Authors should explain how these components relate to treatment differences more clearly.
- Throughout the manuscript: The nano-extract is referred to without sufficient mechanistic discussion. Authors should expand the discussion on how the nano-formulation enhances uptake, bioavailability, or physiological responses compared to the bulk extract, referencing relevant nanotechnology literature.
The manuscript is generally understandable but requires substantial English language editing. Several sentences are overly long, lack grammatical agreement, or use awkward phrasing (e.g., “the highest level of O. basilicum L.var. Genovese sodium was observed…”). Technical terms are often repeated redundantly, and there is inconsistent use of tense. Specific issues include article usage (e.g., “a essential oil” should be “an essential oil”), verb form errors, and run-on sentences. A professional native-English editing service is recommended to enhance clarity, conciseness, and scientific tone throughout the manuscript.
Author Response
Reviewer 1:
Thank you for your comments and suggestions. We are very grateful for your reflections. Your comments were helpful to further improve clarity of the manuscript. We have revised the paper according to your comments which try to meet your approval.
- The introduction is overly long and includes several loosely connected ideas. The authors should restructure it to first define the problem (salinity stress), then introduce the significance of basil as a crop, followed by the rationale for using Z. multiflora and its nano-formulation. Several cited studies are described in too much detail without clearly linking them to the current study’s hypothesis.
Answer: According to the opinion of the respected reviewer, the introduction was rewritten and the desired corrections were made.
- Lines 174–184: The objective of the study is suppressed at the end of the introduction. It should be clearly stated earlier, in a separate paragraph, to highlight the novelty and specific aims of the research.
Answer: According to the respected reviewer, the purpose of the article was added to the last section of the introduction and highlighted in red.
- Lines 194–210: The experiment design is stated, but critical details such as total number of treatments (4 salinity × 3 extract = 12), randomization procedure, and plot layout should be clarified. Also, define whether each tray represented one replicate or multiple plants per replicate were averaged.
Answer: According to the reviewer's opinion, the number of treatments, tray arrangement, and repetitions were added in red in the second paragraph of Materials and Methods.
- Figure 1 showing the TEM image of nano-extract lacks a scale bar and particle size distribution data. The characterization of the nano-extract should be expanded to include average particle size and zeta potential to validate its “nano” designation.
Answer: Information regarding the size of nano-extract particles and scale bar has been added below Figure 1 with red color according to the reviewer's opinion.
- Line 336 onward: The Results section lacks a concise overview or transitional paragraph. It would improve readability if each subsection began with a brief summary statement outlining key trends before presenting detailed data.
Answer: In the results section, as per the reviewer's recommendation, a short summary statement outlining key trends was added before presenting the data details and highlighted in red.
- Table 1: The unit for K and Na content is given as mg g-1 DW, but the legend does not clarify whether values are leaf-specific or averaged across tissues. Also, the significance markers (*) should be defined more clearly, and ensure column headers are not split awkwardly (Different size text in column headers).
Answer: Potassium and sodium were measured in dried basil leaves as mentioned in Materials and Methods Section 2.3.6, Other corrections related to the text of the table columns were made. The significant markers has been corrected in below of table, where * indicates significance at the 5% probability level and ** indicates significance at the 1% probability level.
- Lines 494–517: The antioxidant activity (DPPH and FRAP) data are well explained, but the mechanism linking extract application to enhanced activity is not discussed. The authors should include a brief discussion in the Results or Discussion section linking phenolic/flavonoid accumulation to antioxidant function.
Answer: According to the reviewer comment, possible mechanisms of how the extract affects stress resistance has been added to the discussion section.
- Lines 524–575: The essential oil composition section is informative but difficult to follow. Table 3 is too dense and contains many compounds irrelevant to the discussion. Consider condensing this table to include only the major components and moving the full dataset to supplementary material. Also can’t read all the values in file, the values are going out of the page.
Answer: While thanking the esteemed reviewer for his opinion, given the importance of some of these compounds in inducing resistance to salinity and some of them in cosmetic, health and medical applications, they should usually be fully stated in essential oil studies. Also, the table in the original file is placed in landscape format and the data is quite clear. Perhaps the failure to display all the compounds is due to a problem with the esteemed referee's laptop.
- Lines 584–598: The PCA results are presented with minimal interpretation. Figure 6 is visually helpful, but the description of which traits load heavily on PC1 vs PC2 is vague. Authors should explain how these components relate to treatment differences more clearly.
Answer: Corrections were made according to the opinion of the respected referee and added to the result section.
- Throughout the manuscript: The nano-extract is referred to without sufficient mechanistic discussion. Authors should expand the discussion on how the nano-formulation enhances uptake, bioavailability, or physiological responses compared to the bulk extract, referencing relevant nanotechnology literature.
Answer: According to the reviewer comment, possible mechanisms of how the extract affects physiological responses and stress resistance has been added to the discussion section.
Comments on the Quality of English Language
The manuscript is generally understandable but requires substantial English language editing. Several sentences are overly long, lack grammatical agreement, or use awkward phrasing (e.g., “the highest level of O. basilicum L.var. Genovese sodium was observed…”). Technical terms are often repeated redundantly, and there is inconsistent use of tense. Specific issues include article usage (e.g., “a essential oil” should be “an essential oil”), verb form errors, and run-on sentences. A professional native-English editing service is recommended to enhance clarity, conciseness, and scientific tone throughout the manuscript.
Answer: The article was re-read and items were revised as necessary.
Author Response File: Author Response.pdf
Reviewer 2 Report
Comments and Suggestions for AuthorsThis study innovatively applies plant-derived nano-extracts to salt stress management in floating cultivation systems. While rich in data and rationally designed, it should strengthen the in-depth exploration of mechanisms, quantitative data presentation, and structural and logical refinement.
Title:
The title does not reflect the core findings (such as the synergistic effect of nano-extracts). It is recommended to revise it to: "Nano-extract of Zataria multiflora Boiss. enhances salt stress tolerance in hydroponically grown Ocimum basilicum L. var. Genovese".
Abstract:
(1) Delete vague descriptions similar to "In this study, the effect of nano-extract on the content of essential oil compounds was evident (Line 28)".
(2) Information stacking leads to an unclear focus (lines 18-32), and key data are missing. It is recommended to clarify the relative advantages of nano-extracts using percentages or multiples.
(3) The use of only "highest efficiency" (line 35) is too general. Specific data comparisons should be supplemented, such as the percentage by which nano-extracts improve efficiency compared to ordinary extracts.
Introduction:
(1) The mechanism of action of Z. multiflora extract is insufficiently elaborated (e.g., how it regulates antioxidant enzymes?).
(2) The theoretical basis for nano-processing is not clarified.
(3) Information such as the impact of salt stress (Lines 59-63, Line 120-122, Line 108) and the performance of basil in soilless cultivation (Line 103-107, Line 108) is repeated in different positions. Integrate it into the most relevant thematic paragraphs to avoid redundancy.
(4) Use transition words/sentences to clarify the connections between paragraphs and enhance the fluency of logical cohesion.
(5) Research objectives and hypotheses are buried in background descriptions. The core proposition such as "This study will explore the effect of Z. multiflora extract on basil under salt stress in a floating system" should be clearly summarized.
Materials and Methods:
(1) The chapters are divided too finely. It is recommended to divide them into the following chapters:
2.1 Plant Materials, Experimental Design, and Cultivation System (merge the current 2.1 and 2.2)
2.2 Preparation of Extracts and Nanoemulsions (original 2.3)
2.3 Sampling Methods and Determination Indicators (merge original 2.4 to 2.11 and group them logically)
2.4 Essential Oil Extraction and GC-MS Analysis
2.5 Statistical Analysis (original 2.14, correct the numbering and supplement details)
(2) Other details: There is a contradiction between "harvested after 60 days" in Line 206-207 and "90 days after planting" in Line 231.
Results:
(1) The description of parameters is too verbose. It is recommended to uniformly describe parameters with the same change rules.
(2) It is recommended to add significance analysis tests in Figure 2, 3, and 4.
Discussion:
The depth of the mechanism is insufficient: it does not thoroughly explain why nano-extracts are superior to traditional extracts.
Conclusion:
The potential for technical transformation is not clarified. It is recommended to supplement application suggestions (e.g., "Under 50–100 mM salt stress, foliar spraying of 0.5% Z. multiflora nano-extract three times can maximize yield increase").
Author Response
Reviewer 2
Comments and Suggestions for Authors
This study innovatively applies plant-derived nano-extracts to salt stress management in floating cultivation systems. While rich in data and rationally designed, it should strengthen the in-depth exploration of mechanisms, quantitative data presentation, and structural and logical refinement.
Answer: While thanking the respected reviewer for his/her comments, by applying the respected reviewer's comments and expressing possible mechanisms, the technical level of the article has been added, and at the study level (despite financial limitations in the research), the results provide convincing evidence of the effectiveness of nano-extract treatments in improving basil growth under salt stress conditions.
Title:
The title does not reflect the core findings (such as the synergistic effect of nano-extracts). It is recommended to revise it to: "Nano-extract of Zataria multiflora Boiss. enhances salt stress tolerance in hydroponically grown Ocimum basilicum L. var. Genovese".
Answer: The title of the article was changed according to the opinion of the respected reviewer.
Abstract:
- Delete vague descriptions similar to "In this study, the effect of nano-extract on the content of essential oil compounds was evident (Line 28)".
Answer: The above sentence was deleted according to the opinion of the respected reviewer.
- Information stacking leads to an unclear focus (lines 18-32), and key data are missing. It is recommended to clarify the relative advantages of nano-extracts using percentages or multiples.
Answer: The abstract was rewritten according to the reviewer 's opinion, and the results were expressed as percentages or multiples.
- The use of only "highest efficiency" (line 35) is too general. Specific data comparisons should be supplemented, such as the percentage by which nano-extracts improve efficiency compared to ordinary extracts.
Answer: The abstract was rewritten according to the reviewer 's opinion, and the results were expressed as percentages or multiples.
Introduction:
- The mechanism of action of Z. multiflora extract is insufficiently elaborated (e.g., how it regulates antioxidant enzymes?).
Answer: According to the reviewer's opinion, the mechanism of action of Z. multiflora extract on secondary metabolites and antioxidant enzymes was added to the introduction.
- The theoretical basis for nano-processing is not clarified.
Answer: Corrections have been added to the introduction according to the opinion of the respected reviewer.
- Information such as the impact of salt stress (Lines 59-63, Line 120-122, Line 108) and the performance of basil in soilless cultivation (Line 103-107, Line 108) is repeated in different positions. Integrate it into the most relevant thematic paragraphs to avoid redundancy.
Answer:The introduction was completely rewritten again and the mentioned changes have been added.
- Use transition words/sentences to clarify the connections between paragraphs and enhance the fluency of logical cohesion.
Answer:The introduction was completely rewritten again and the mentioned changes have been added.
- Research objectives and hypotheses are buried in background descriptions. The core proposition such as "This study will explore the effect of Z. multiflora extract on basil under salt stress in a floating system" should be clearly summarized.
Answer: Given that this question was also one of the questions asked by reviewer 1, these changes were added to the text.
Materials and Methods:
(1) The chapters are divided too finely. It is recommended to divide them into the following chapters:
2.1 Plant Materials, Experimental Design, and Cultivation System (merge the current 2.1 and 2.2)
2.2 Preparation of Extracts and Nanoemulsions (original 2.3)
2.3 Sampling Methods and Determination Indicators (merge original 2.4 to 2.11 and group them logically)
2.4 Essential Oil Extraction and GC-MS Analysis
2.5 Statistical Analysis (original 2.14, correct the numbering and supplement details)
Answer:The chapters have been revised according to the opinion of the respected referee.
(2) Other details: There is a contradiction between "harvested after 60 days" in Line 206-207 and "90 days after planting" in Line 231.
Answer: The correct duration is 90 days, which has also been corrected and highlighted in the text.
Results:
- The description of parameters is too verbose. It is recommended to uniformly describe parameters with the same change rules.
Answer: Some of the corrections suggested by the esteemed reviewer were implemented in the text due to their similarity to the previous reviewer.
- It is recommended to add significance analysis tests in Figure 2, 3, and 4.
Answer: The results related to Figures 2, 3, and 4 are examined in detail in the article.
Discussion:
The depth of the mechanism is insufficient: it does not thoroughly explain why nano-extracts are superior to traditional extracts.
Answer: According to the reviewer comment, possible mechanisms of how the extract affects physiological responses and stress resistance has been added to the discussion section.
Conclusion:
The potential for technical transformation is not clarified. It is recommended to supplement application suggestions (e.g., "Under 50–100 mM salt stress, foliar spraying of 0.5% Z. multiflora nano-extract three times can maximize yield increase").
Answer: The conclusion was rewritten according to the opinion of the esteemed reviewer.
Author Response File: Author Response.pdf
Reviewer 3 Report
Comments and Suggestions for Authors- Section 2.1 Plant material: Was the O. basilicum plant exposed to light during cultivation?
- Why are some words in the manuscript written in red?
- Table 1: "Extract spary" - please explain
- It might be worth considering including some photos from the O. basilicum cultivation in the manuscript or supplementary data.
Author Response
Reviewer 3
Comments and Suggestions for Authors
- Section 2.1 Plant material: Was the O. basilicum plant exposed to light during cultivation?
Answer: Plants were grown and maintained in greenhouse conditions under natural light, which is added in red in the Materials and Methods section.
- Why are some words in the manuscript written in red?
Answer: The red text was displayed due to an edit in the original version.
- Table 1: "Extract spray" - please explain.
Answer: As is clear from the title of Table 1, "extract spray" refers to the foliar spraying of Z. multiflora extract and nano-extract.
- It might be worth considering including some photos from the O. basilicum cultivation in the manuscript or supplementary data.
Answer: Due to the large volume of tables and figures, adding images to the text of the article was avoided. However, at the request of the esteemed referee, several figures are attached below.
Author Response File: Author Response.pdf
Round 2
Reviewer 2 Report
Comments and Suggestions for AuthorsThe authors have made substantial revisions and enhancements in response to the comments and suggestions provided by the reviewers. I have no further remarks regarding the manuscript.
Author Response
Dear Editor-in-Chief:
I sent the second revised copy of the manuscript " Nano-extract of Zataria multiflora Boiss. enhances salt stress tolerance in hydroponically grown Ocimum basilicum L. var. Genovese" by Shabani et al. Let me first thank the reviewers for their excellent and helpful evaluation.
With reference to some specific comments, please note the following points:
Reviewer 1:
Thank you for your comments and suggestions. We are very grateful for your reflections. Your comments were helpful to further improve clarity of the manuscript. We have revised again the paper according to your comments which try to meet your approval.
- There are still issues with the definition of the samples. In particular, it is confusing to indicate two controls with the same abbreviation "Cont"; therefore, in line 166, it should be defined what the control treatment for the foliar spray with Z. multiflora consists of, perhaps water with no extract? Or without extract spraying (e.g. lines 486-487)? In any case, the abbreviation “Cont” in Table 1 and other part of the MS can be used for only one of the two control treatments to avoid misunderstandings, perhaps using “CoZm” or “CoEx” for the Z. multiflora control. In addition, there are a number of spelling errors in the text (e.g. line 165, Nacl; line 444 bichemical). Answer: The control treatments were redefined according to the opinion of the respected reviewer. Accordingly, the treatment without salinity was named the 0 NaCl treatment (0 NaCl), and the treatment without foliar application of the extract (water with no extract) was named the control treatment (Cont). Throughout the text, tables, and figures were also revised and highlighted in red. The spelling errors noted in the text have been edited and corrected and highlighted in red in the text.
We remain available to clarify any further issues or answer that the Editor or Reviewer may raise.
Best Regards,
The Authors.
Author Response File: Author Response.docx