Effect of Jasmonic Acid on the Elicitation of Phenolic Compounds and Naphthodianthrones in Hypericum perforatum L. Callus and Shoot Cultures

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

Dear Authors,

It has been a pleasure to read such a nice and properly written manuscript.

I have just few suggestions or questions:

Key words: they should be different than in the title so all of them should be replaced.

Introduction: page 1/line 49 - richness in instead of richness of

M&M: 

• the MS/B5 should be explained
• provide the exact vitamins types and concentrations
• provide producers of chemicals
• it is advised to use one units in a whole manuscript, I recommend to change JA concentration into mg L^-1
• there should be no space between temperature and Celsius degrees sign
• The methods of TP, TF, TFL content determination should be briefly described and the authors' or name of the methods should be given as well.

Results: 

• page 6, lins 248-250: the information that 'JA doses was no changed until the day 14 ...' is in contrast with the figure - it was increasing from day 4.
• the '(Fig. 2a)' should be inserted after 'control calli'
• line 251 - should be: 'showed a 1.2-fold increase'
• page 8, lines 297-298 - 250 uM JA after 14 and 21 days was not statistically significant
• page 10, line 355- I propose: 'Interestingly, in the case of shoots, PHYP production...'
• page 10, lines 366 - 367 - no in the case of 250 uM JA

Discussion: 

It is recommended to use simple tenses in scientific manuscripts to make it easier to read. Just a remark for the future. 

• page 13, line 500 - 'Those' instead of 'These'
• page 15, line 593 - 'Many researchers', instead of 'Many studies'

Conclusions: 

page 16, line 635 - 'those' instead of 'these'.

Author Response

Please see the attachment.

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

Dear authors,

The manuscript entitled "Effect of jasmonic acid on the elicitation of phenolic compounds and naphthodianthrones in Hypericum perforatum L. callus and shoot cultures" is well written, clearly structured, and scientifically sound. The results are comprehensively presented, supported with adequate statistical analysis, and thoroughly discussed in comparison with previous findings. The technical quality of the MS meets the requirements of the journal, with only minor corrections of technical nature suggested in the attached document.

The significance of this study lies in the fact that it reports for the first time the influence of different concentrations of JA on growth, phenylpropanoid and naphthodianthrones production, as well as on the antioxidant state of H. perforatum callus and shoot cultures. It is demonstrated that low concentrations of JA stimulate the biomass production, while higher concentrations inhibit growth but enhance accumulation of phenolics, flavonoids and naphthodianthrones. The observed elicitation effects also revealed the complex interplay between enzymatic and non-enzymatic antioxidant mechanisms, with distinct responses in callus vs. shoot cultures.

Altogether, the present findings provide valuable insights into JA-mediated metabolic regulation and highlight H. perforatum cultures as promising biotechnological system for the production of bioactive phenolic compounds and naphthodianthrones. 

I recommend your MS for publication after minor technical revisions.

Comments for author File: Comments.pdf

Author Response

Please see the attachment.

Author Response File: Author Response.pdf

Reviewer 3 Report

Comments and Suggestions for Authors

Dear authors,

I have reviewed the current version of your manuscript, "Effect of jasmonic acid on the elicitation of phenolic compounds and naphthodianthrones in Hypericum perforatum L. callus and shoot cultures." Your work reflects arduous and valuable research into the biosynthesis of phenolic compounds and naphthodianthrones in tissue cultures, which represents an area of ​​great interest for the production of high-value molecules.

To enhance the presentation, clarity, and scientific rigor of the article, I suggest making the following revisions, with a focus on integrating your findings with the proposed objectives and the existing scientific literature.

Detailed Feedback by Section
Introduction and Methods
Lines 100-108 (Objective): Please expand and refine the description of the central objective of the research, ensuring that the specific contribution of the study is apparent.

Lines 133-138 (Harvest): Describe the rationale (kinetic, phenological, or experimental) that justifies the selection of the harvest days mentioned.

Lines 140-165 (Reagents): Include the purity and brand name of all reagents, chemicals, and solvents used in the methodology to ensure reproducibility.

Results and Discussion
Lines 354-363 (Confusing Paragraph): Completely rewrite this paragraph. The current wording is confusing. Emphasize and present the results obtained after applying each dose of jasmonic acid (JA) concisely and sequentially in a continuous text.

Figure 6: The Y-axis scales are markedly different, making direct visual interpretation difficult. I suggest separating the information into two separate figures to improve clarity (e.g., Figures a and c in one, and b and d in the other).

Lines 398-441 (Discussion I): Rewrite this discussion section. You should compare your findings with other studies that have used the same elicitor (JA) on species from the same botanical family. Describe any significant similarities or differences regarding the most effective micromolar concentrations of JA.

Lines 443-463 (Discussion II): Reorganize and rewrite this section. The primary focus of the discussion should be on the results obtained with JA. You must discuss the differences between the elicitor-free cultures (control) and the elicited cultures, directly relating these results to the fulfillment of your research objectives.

Lines 536-560 (Deep Dive): Elaborate on your arguments. The claims made in lines 536-538 are not supported by the results or discussions presented above. Establish a clear and direct relationship between your experimental findings and the arguments you present, linking them to the objectives of the work.

Conclusions and Final Revision
Conclusions: Completely rewrite your conclusions. They should emphasize the most relevant findings that directly relate to the title of the work. In addition, include the benefits of your results for the field of tissue culture or the production of the compound of interest, highlight future applications (medium- or long-term) of the research, and mention the specific limitations of the study.

Language Review: A thorough and professional language review is strongly recommended to ensure fluent, accurate, and high-level technical writing.

With these revisions, your manuscript will be significantly better positioned for acceptance.

Comments on the Quality of English Language

It is recommended that authors conduct a thorough review of their language.

Author Response

Please see the attachment.

Author Response File: Author Response.pdf

Reviewer 4 Report

Comments and Suggestions for Authors

This is a valuable manuscript that explores jasmonic acid elicitation in Hypericum perforatum callus and shoot cultures, presenting detailed data on phenolic compounds, naphthodianthrones, and antioxidant responses. The introduction is well-written and supported with relevant references, and the study design with multiple concentrations and time points is appropriate. Before publication, several clarifications and improvements are needed: (1) Specify in Section 2.6 whether the reported triplicates are biological or technical replicates. In Sections 2.2 – 2.5, confirm explicitly that all quantitative results (phenolic compounds, flavonoids, enzyme activities, hypericin/pseudohypericin) are expressed per dry weight, as this is currently stated only in figure legends. For the HPLC method (Section 2.4), please include basic validation parameters such as limits of detection and quantification and method repeatability. (2) The statistical analysis uses one-way ANOVA with SNK test, but since the design includes two factors (dose × time), a two-way ANOVA would provide a more rigorous test of interaction effects. Figures 2 – 6 could be improved by enlarging axis labels, clarifying what the asterisks denote (e.g., significance vs. control at the same time point), and possibly summarizing complex datasets in a table or heatmap. In the results, consider reporting exact p-values or at least ranges (p < 0.01, p < 0.001) for vetter transparency.

Author Response

Please see the attachment.

Author Response File: Author Response.pdf

Round 2

Reviewer 3 Report

Comments and Suggestions for Authors

Dear authors,
Your manuscript presents notable changes that facilitate a clear understanding of your findings. I don't have any additional comments. I wish you much success in your publication.