Combining Diluted Seawater and Fertilizer in an Ion-Based Multivariate Approach as an Effective Assay of Salt Tolerance in Brassica juncea Seedlings

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

1. Why did the authorschoose silicon, calcium, and total nitrogen as the main ions that affect the salt tolerance of plant seedlings? Please explain this in the ms.
2. The authorsmeasured the changes of fresh weight under salt treatment. Why didn’t measured the dry weight? That should be important too.
3. In the ms, the authorsmentioned that silicon element ions have certain positive effects on the growth of plants under salt stress. It is  I suggest the authors further explain the mechanism of its action?
4. Only determine the new leaves in the plants after stress is too limited, Arethere any changes in leaf color or MDA and other indicators?

Comments on the Quality of English Language

The language needs to be further standardized.

Author Response

Comment 1: Why did the authors choose silicon, calcium, and total nitrogen as the main ions that affect the salt tolerance of plant seedlings? Please explain this in the ms.

Response 1: Line 93 in the manuscripts mentioned that nitrogen is a large contributor of dry biomass. Line 103 mentioned that calcium is a macronutrient and has been reported to increase salt tolerance in literature. Line 115 describes that silicon may be an overlooked contributor to salt tolerance because it is not typically regarded as an essential plant element, although it too has been shown to increase salt tolerance.

Comment 2: The authorsmeasured the changes of fresh weight under salt treatment. Why didn’t measured the dry weight? That should be important too.

Response 2: In our assay scale experiment, dry weight are low and that data was fraught with some error. Dry weight was recorded and analyzed but data was not sufficient for modeling, the authors did not find this response useful (Figure 3).

Comment 3: In the ms, the authorsmentioned that silicon element ions have certain positive effects on the growth of plants under salt stress. It is  I suggest the authors further explain the mechanism of its action?

Response 3: Dr. Jeong’s review citation (Etesami, H., & Jeong, B. R. (2023). How does silicon help alleviate biotic and abiotic stresses in plants? Mechanisms and future prospects. In Plant Stress Mitigators: Types, Techniques and Functions (pp. 359–402). Academic Press. https://doi.org/10.1016/B978-0-323-89871-3.00031-8) suggests mechanisms of silicon alleviation to salt stress and the authors did not want to risk excessive self-citation. In the discussion, lines 418-426 draw more from this review where it is directly applicable to the results in this study.

Comment 4: Only determine the new leaves in the plants after stress is too limited, Arethere any changes in leaf color or MDA and other indicators?

Response 4: The assay scale hydroponic system was used as a tool for discovery and change in leaf color is being captured as a response in a larger scale experiment in deep water culture (currently underway). In DWC high-quality images of each treatment combination were recorded throughout the duration of the experiment (28 days).  Furthermore, the leaves after 10 days in treatment conditions were too small for MDA analysis.

Reviewer 2 Report

Comments and Suggestions for Authors

I reviewed the manuscript, "Combining Diluted Seawater and Fertilizer in an Ion-based Multivariate Approach as an Effective Assay of Salt Tolerance in Brassica juncea Seedlings," for possible publication in Horticulturae.

• Line 24, what do you mean by Hydroponics assay.
• You add reference Soilless Agricultural Systems: Opportunities, Challenges, and Applications for Enhancing Horticultural Resilience to Climate Change and Urbanization. Horticulturae 2025, 11, 568. With: Line 50 to 51. (More highly controlled management of nutrient solutions, greater water availability, and a single liquid phase are advantages of hydroponic solutions over soil-based systems.)
• line 41 to 53: The paragraph presents several essential points regarding freshwater scarcity, desalinated and brackish water use, and hydroponic cultivation. However, the technical flow between these ideas is somewhat fragmented. I suggest improving the logical progression by clearly linking the challenges of saline irrigation to the emergence of hydroponics as a potential solution. A more structured transition would help the reader understand how each issue builds on the previous one. This will enhance the coherence and overall readability of the paragraph.
• Line 55: Soilless or soil-based (crop cultivation), please specify. In the same line, what is NPK?
• Line 86, which studies?
• The introduction contains relevant and informative content but lacks a clear scientific tone and technical flow. I recommend revising the introduction to strengthen the manuscript to ensure a more cohesive progression of ideas and a consistent academic tone. Clearer transitions between topics such as freshwater scarcity, desalination challenges, and the role of hydroponics would help establish a stronger foundation for the study's objectives.
• Line 154- 156: "Polyphenolic foam" (like Aeromax horticubes) is not a hydroponic system itself but rather a soilless rooting medium or substrate used within hydroponic systems, such as nutrient film technique (NFT), deep water culture (DWC), ebb and flow, etc. So, the phrase "The hydroponic substrate ‘Aeromax’..." is not technically wrong if "substrate" is the intended focus. Still, it becomes misleading if the sentence implies that polyphenolic foam is the hydroponic system.
• There are multiple types of hydroponic systems (e.g., NFT, DWC, ebb and flow, aeroponics), each with distinct characteristics and implications for plant growth and nutrient delivery. The current manuscript's specific hydroponic system is not clearly identified.
• Line 156 to 158: The description of the substrate preparation is appreciated; however, the manuscript lacks clarity regarding the specific material composition of the substrate itself. While it is referred to as “polyphenolic foam,” this term is broad and does not provide enough technical detail for reproducibility or for understanding its interaction with hydroponic conditions. I recommend specifying the exact material type, manufacturer product name, or relevant properties (e.g., porosity, pH stability, water retention capacity) of the substrate.
• While the manuscript presents relevant scientific content, several instances of awkward phrasing and grammatical inconsistencies affect readability. I recommend a thorough language revision to improve sentence structure, clarity, and scientific tone. Consider working with a professional English editing service or a native speaker familiar with academic writing to ensure the manuscript meets the journal’s language standards. Please see, for example, lines 160 to 162.
• Line 164: The manuscript mentions using a shelving unit in the experimental setup. However, details are missing regarding its structure, material, dimensions, number of shelves, or arrangement relative to the light source and other environmental controls. For reproducibility and better understanding of the growth conditions, I recommend providing more detailed information about the shelving unit and its role in the experimental design.
• Using “in the lab” in this sentence is vague and unsuitable for a scientific manuscript. Line 170.
• Section 2.1: The methodology section provides useful procedural information but lacks clarity, scientific tone, and technical detail in several areas. I recommend revising this section to specify the type of hydroponic system used, the exact materials and dimensions of trays and shelving units, accurate environmental conditions (e.g., light intensity, temperature settings), and the role of the substrate in the system. Improving the structure and language will enhance reproducibility and align the section with scientific writing standards.
• There are two reference styles; please consider one or follow the journal guidance. Line 188.
• The current description of the experimental setup is somewhat confusing and could be improved for clarity and coherence. I recommend reorganizing the section to follow a more logical sequence (e.g., seed handling, substrate preparation, nutrient application, germination conditions, and transfer to treatment). Additionally, please clarify the type of hydroponic system used and provide consistent details on environmental conditions, materials, and procedures to ensure reproducibility.
• To strengthen your discussion and contextualize your findings, please incorporate references to recent studies that have reported similar or contrasting results. Including up-to-date literature—ideally from recent years—will help justify your conclusions and demonstrate how your work advances current understanding.

Author Response

Comment 1: Line 24, what do you mean by Hydroponics assay.

Response 1: Lines 66-70 state that the hydroponic assay system utilizes a chemically inert soilless substrate and includes additional details that justify how we characterized the hydroponics assay system.

Comment 2: You add reference Soilless Agricultural Systems: Opportunities, Challenges, and Applications for Enhancing Horticultural Resilience to Climate Change and Urbanization. Horticulturae 2025, 11, 568. With: Line 50 to 51. (More highly controlled management of nutrient solutions, greater water availability, and a single liquid phase are advantages of hydroponic solutions over soil-based systems.)

Response 2: This excellent review serves as a great citation for lines 40-42 and will be useful in repository of literature for future experiments and publications.

Comment 3: line 41 to 53: The paragraph presents several essential points regarding freshwater scarcity, desalinated and brackish water use, and hydroponic cultivation. However, the technical flow between these ideas is somewhat fragmented. I suggest improving the logical progression by clearly linking the challenges of saline irrigation to the emergence of hydroponics as a potential solution. A more structured transition would help the reader understand how each issue builds on the previous one. This will enhance the coherence and overall readability of the paragraph.

Response 3: The second paragraph in the manuscript (starting on line 39) has been re-arranged for a more logical flow of ideas.

Comment 4: Line 55: Soilless or soil-based (crop cultivation), please specify. In the same line, what is NPK?

Response 4: In line 43: the colligative effects of ions are critical in both hydroponic and soil-based systems. Also, NPK definition has been explained in line 44.

Comment 5: Line 86, which studies?

Response 5: Lines 77-79 have been amended to cite studies quantifying salt tolerance in mature crops whose methods require greater water nutrient and space when compared to our experiment.

Comment 6: The introduction contains relevant and informative content but lacks a clear scientific tone and technical flow. I recommend revising the introduction to strengthen the manuscript to ensure a more cohesive progression of ideas and a consistent academic tone. Clearer transitions between topics such as freshwater scarcity, desalination challenges, and the role of hydroponics would help establish a stronger foundation for the study's objectives.

Response 6: The introduction has been revised to improve the flow of ideas.

Comment 7: Line 154- 156: "Polyphenolic foam" (like Aeromax horticubes) is not a hydroponic system itself but rather a soilless rooting medium or substrate used within hydroponic systems, such as nutrient film technique (NFT), deep water culture (DWC), ebb and flow, etc. So, the phrase "The hydroponic substrate ‘Aeromax’..." is not technically wrong if "substrate" is the intended focus. Still, it becomes misleading if the sentence implies that polyphenolic foam is the hydroponic system.

Response 7: Lines 150-161 are explaining how the substrate for the hydroponic assay system was prepared as the nutrients and seeds were prepared before being placed in the microcosm with 150 mL treatment solution. The authors believe that although the horticubes system can be used in a “substrate culture system”, similar to passive rockwool soilless systems, the substrate was being used in a system that is more accurately characterized as a hydroponic system akin to the “Kratky DWC” opposed to a substrate system (see lines 66-70).

Comment 8: There are multiple types of hydroponic systems (e.g., NFT, DWC, ebb and flow, aeroponics), each with distinct characteristics and implications for plant growth and nutrient delivery. The current manuscript's specific hydroponic system is not clearly identified.

Response 8: The answer to the previous comment addresses how the authors classified the soilless plant system. The picture added to section 2.3. further illustrates the hydroponic system utilized in the experiment.

Comment 9: Line 156 to 158: The description of the substrate preparation is appreciated; however, the manuscript lacks clarity regarding the specific material composition of the substrate itself. While it is referred to as “polyphenolic foam,” this term is broad and does not provide enough technical detail for reproducibility or for understanding its interaction with hydroponic conditions. I recommend specifying the exact material type, manufacturer product name, or relevant properties (e.g., porosity, pH stability, water retention capacity) of the substrate.

Response 9: Line 149 includes a link to the manufacturers website where the physical parameters (porosity, pH stability, water retention capacity) are specified. Referral to manufacturer specifications are appropriate since we did not confirm these specifications first-hand.

Comment 10: While the manuscript presents relevant scientific content, several instances of awkward phrasing and grammatical inconsistencies affect readability. I recommend a thorough language revision to improve sentence structure, clarity, and scientific tone. Consider working with a professional English editing service or a native speaker familiar with academic writing to ensure the manuscript meets the journal’s language standards. Please see, for example, lines 160 to 162.

Response 10: Many co-authors of this manuscript are well-published in academic journals and great care has been taken in preparing this submission.

Comment 11: line 164: The manuscript mentions using a shelving unit in the experimental setup. However, details are missing regarding its structure, material, dimensions, number of shelves, or arrangement relative to the light source and other environmental controls. For reproducibility and better understanding of the growth conditions, I recommend providing more detailed information about the shelving unit and its role in the experimental design.

Response 11: Seed germination occurred on vertically stacked metal shelving with supplementary lights 40 cm above work surface in a climate controlled positive pressure clean room.

Comment 12: Using “in the lab” in this sentence is vague and unsuitable for a scientific manuscript. Line 170.

Response 12: Lines 160-168 were amended to more accurately define the growing conditions of the seeds and emergent seedlings.

Comment 13: Section 2.1: The methodology section provides useful procedural information but lacks clarity, scientific tone, and technical detail in several areas. I recommend revising this section to specify the type of hydroponic system used, the exact materials and dimensions of trays and shelving units, accurate environmental conditions (e.g., light intensity, temperature settings), and the role of the substrate in the system. Improving the structure and language will enhance reproducibility and align the section with scientific writing standards.

Response 13: Refer to lines 159-167 for further information regarding the laboratory shelves used during the germination and emergence phases. Lines 209-226 describe in detail the microcosm construction as well as solution management.

Comment 14: There are two reference styles; please consider one or follow the journal guidance. Line 188.

Response 14: This has been fixed to reflect journal guidelines.

Comment 15: The current description of the experimental setup is somewhat confusing and could be improved for clarity and coherence. I recommend reorganizing the section to follow a more logical sequence (e.g., seed handling, substrate preparation, nutrient application, germination conditions, and transfer to treatment). Additionally, please clarify the type of hydroponic system used and provide consistent details on environmental conditions, materials, and procedures to ensure reproducibility.

Response 15: The method subsections were organized chronologically although some subsection titles were less clear. We have modified the sub-titles to contain many of your suggestions to increase clarity.

Comment 16: To strengthen your discussion and contextualize your findings, please incorporate references to recent studies that have reported similar or contrasting results. Including up-to-date literature—ideally from recent years—will help justify your conclusions and demonstrate how your work advances current understanding.

Response 16: When starting this project, we were disappointed how the published literature handled ion confounding in these mixed milieus (synthetic seawater and hydroponics) but before launching full scale investigation we wanted to explore the feasibility of these statistical models in assay scale. The assay starts to address this lack of information in published literature. If you know of other relevant publications we would enjoy reading and including them.

 

Reviewer 3 Report

Comments and Suggestions for Authors

The manuscript “Combining Diluted Seawater and Fertilizer in an Ion-based Multivariate Approach as an Effective Assay of Salt Tolerance in Brassica juncea Seedlings” presents an interesting investigation into salt tolerance mechanisms in Brassica juncea seedlings through rigorously controlled nutrient management. The multivariate ion-based approach leveraging synthetic seawater and specific nutrients (total inorganic nitrogen, potassium, calcium, silicon, and zinc) provides valuable insights. While the methodology is robust, I have two minor suggestions to further strengthen the work:

1. Materials and Methods Section:

Including an additional experimental group treated with natural seawater (diluted to equivalent salinity levels) would provide critical context. Comparing seedling responses between synthetic and real seawater conditions could reveal the influence of unaccounted ions (e.g., trace elements, organics) present in natural environments. This comparison would enhance the ecological relevance of the assay.

2. Results Section (Table 3 -ANOVA):

The notation "Si*Si" in the summarized ANOVA table requires clarification. Typically, terms like "Factor*Factor" denote interaction effects (e.g., Si × Zn).

Please explicitly define whether "Si*Si" represents:

1. A quadratic term for silicon concentration (i.e., a non-linear effect),
2. b) An error in notation (e.g., intended as Si ×Zn or another interaction).

Author Response

Comment 1:

1. Materials and Methods Section:

Including an additional experimental group treated with natural seawater (diluted to equivalent salinity levels) would provide critical context. Comparing seedling responses between synthetic and real seawater conditions could reveal the influence of unaccounted ions (e.g., trace elements, organics) present in natural environments. This comparison would enhance the ecological relevance of the assay.

Response 1: Our research group has compared average surface seawater (Pilson, M. E. Q. (2013). An Introduction to the Chemistry of the Sea (2nd ed.). Cambridge University Press) to Instant Ocean formulation. Instant Ocean formulation is highly similar to average sweater ionic constituents excluding the presence of boron. Boron was present in inorganic fertilizer utilized for the experiment (~0.023128 mM when Hoagland’s H353 is applied at 0.816 g/L). Seawater constituents and their concentrations will vary based on location and climate and is amply available in databases. We used Instant Ocean as a proxy for brackish water so our conclusions regarding salt tolerance and ionic interactions would be robust to many saline water sources.

Comment 2: 

1. Results Section (Table 3 -ANOVA):

The notation "Si*Si" in the summarized ANOVA table requires clarification. Typically, terms like "Factor*Factor" denote interaction effects (e.g., Si × Zn).

Please explicitly define whether "Si*Si" represents:

1. A quadratic term for silicon concentration (i.e., a non-linear effect),
2. b) An error in notation (e.g., intended as Si ×Zn or another interaction).

Response 2: In lines 248-249 this denotation is now explicitly defined.

Reviewer 4 Report

Comments and Suggestions for Authors

The present study is very interesting and useful for dealing with the ever-increasing problem of salt stress. The manuscript is well structured and written. Improvements are needed to make it publishable and understandable even for readers who are not experts in the field.

Below there are suggestions for authors regarding the various parts of the manuscript.

ABSTRACT

The section is to long, please summarized the abstract according to the journal instructions (max. 200 words).

INTRODUCTION

Line 55: “…cultivation (e.g., NPK, pH, …”. Please add the explanation of NPK.

Lines 81-85: please add the citations.

Lines 118-121: please add the citations.

I suggest the authors include a paragraph stating the threshold of salt tolerance of the species or, even better, of the 2 varieties used in the study and the reason why Brassica juncea was chosen for this research.

MATERIAL AND METHODS

Lines 215-219: Please rewrite the sentence because it is confused. I suggest the authors improve the section regarding the “size of the leaves”, specifying whether they are real leaves or cotyledons.

Lines 230-231: Please add the citation about the formula reported (Equation 1).

I suggest the authors add the number of replicates (n) used for each trial in Data analysis section.

It would be very interesting for the readers, if pictures were included in the Materials and Methods section or as Supplementary Materials, showing the microcosms and seedlings, used during the research.

RESULTS

Line 253: B. juncea should be italic. Please check the entire manuscript.

Figure 1: The error bars of the third boxplot (CB 15% seawater) are very high; is it possible that mistakes occurred during data processing?

DISCUSSION

Lines 413-442: I suggest the authors merge the paragraphs 4.3 and 4.4 and create one bigger paragraph, improving the discussion concerning the future use of Assay-Scale Hydroponic Systems, also adding more citations related to this particular type of research.

Author Response

Comment 1: The section is to long, please summarized the abstract according to the journal instructions (max. 200 words).

Response 1: We are at 200 words currently (309 in the original submission)

Comment 2: Line 55: “…cultivation (e.g., NPK, pH, …”. Please add the explanation of NPK.

Response 2: NPK definition has been explained in line 44.

Comment 3: Lines 81-85: please add the citations.

Response 3: Appropriate citations have been added.

Comment 4: Lines 118-121: please add the citations.

Response 4: Appropriate citations have been added.

Comment 5: I suggest the authors include a paragraph stating the threshold of salt tolerance of the species or, even better, of the 2 varieties used in the study and the reason why Brassica juncea was chosen for this research.

Response 5: Crops that are harvested as vegetative rosettes (lettuce L. sativa and mustard B. juncea) are easier to model than those that go through phase transitions to produce fruit as harvested portions (tomato S. lycopersicum) since preferential nutrient uptake and partitioning within the plant changes with maturation. B. juncea was chosen because oil seed juncea has moderate salt tolerance within the brassica species (Ashraf, M., & McNeilly, T. (2004). Salinity tolerance in Brassica oilseeds. Critical reviews in plant Sciences, 23(2), 157-174.).

Comment 6: Lines 215-219: Please rewrite the sentence because it is confused. I suggest the authors improve the section regarding the “size of the leaves”, specifying whether they are real leaves or cotyledons.

Response 6: Lines 210-216 were added to increase clarity for seedling uniformity during selection.

Comment 7:Lines 230-231: Please add the citation about the formula reported (Equation 1).

Response 7: The formula reported (Equation 1) was created by authors. The formula utilizes measurements (number of leaves, length and width of longest leaf) to create an index for growth, not as an estimation of leaf area, which correlates with fresh mass and new leaves per day.

Comment 8: I suggest the authors add the number of replicates (n) used for each trial in Data analysis section.

Response 8: Since the number of replicates differs among the treatment combinations, the numbers of replicates are represented in Table 1 as repeated rows of the treatment combinations.  We have added a sentence about this in the paragraph below Table 1 (lines 201-203). The authors thought this addition would be most appropriate in section 2.2.

Comment 9: It would be very interesting for the readers, if pictures were included in the Materials and Methods section or as Supplementary Materials, showing the microcosms and seedlings, used during the research.

Response 9: Figure 1 was added to section 2.3 to display the microcosms and seedlings during the research.

Comment 10: Line 253: B. juncea should be italic. Please check the entire manuscript.

Response 10: All B. juncea written in italics.

Comment 11: Figure 1: The error bars of the third boxplot (CB 15% seawater) are very high; is it possible that mistakes occurred during data processing?

Response 11: All systems have some error, but much of this variation is caused by mineral elements that are not the instant ocean variable. The other elements (main and interactive effects) are not being regressed to the x-axis and this great variance is the purpose of the figure, and subject of the paper as stated in lines 257-265 as well the Figure 2 caption.

Comment 12: Lines 413-442: I suggest the authors merge the paragraphs 4.3 and 4.4 and create one bigger paragraph, improving the discussion concerning the future use of Assay-Scale Hydroponic Systems, also adding more citations related to this particular type of research.

Response 12: Thank you for the suggestion- now included.

 

Round 2

Reviewer 2 Report

Comments and Suggestions for Authors

The authors have revised the manuscript in accordance with the suggestions and recommendations.

Reviewer 4 Report

Comments and Suggestions for Authors

After the revisions, the manuscript is now ready for the publication.