Differential Bio-Elicitor Effects on Bioactive Compound Production in Cichorium intybus Root Callus Cultures

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

Dear authors,

the paper represent an interesting subjects regarding the effect of elicitation in  root callus culture. I write some suggestions and comments, necessary in my opinion.

Thank you very much for the opportunity to discover your work.

1. Introduction

Line 55:  the sentences begin with ‘Because’ likewise line 62. May be can reformulates the topic of the sentences.

I consider that the introduction part from 114 line and 124 line could be included (but not mandatory) to Discussion.

2. Materials and methods

At this chapter is not specified the origin of fungal strains (are from international collection?) and the provenance of the seeds (are commercial products?)

It is not specified the manner in which the fresh callus is processed to obtained dry callus. In general, the method used to dehydrate the callus and determine the dry weight is that of repetitive drying in an oven for several hours until the callus has a constant weight, in fact a constant mass.

Another method to calculated the dry weight is to use the fresh callus to be lyophilized. This method is very expensive and need specific equipment. The lyophilized callus can be mixed with ethanol or other extraction buffer, especially the samples used for HPLC analyses.

2.4.2 Callus biomass production is not related to the amount of medium in the culture vessel, because the volume of medium on which the callus is grown must be the same, standard in each culture vessel.

2.4.5 The author did not specify the method for obtaining the callus powder used for determination of chlorogenic acid, caffeic acid and catechin.

Line 207 between word “acid” and “and” is a comma to be deleted

3. Results

The type of light treatment is confusing. I think it would have been more suitable to treat with light for 24h and dark for 24h respectively, the ratio 16/8 is confusing in determining the type of treatment. It is known in vitro cultures the alternating illumination is ratio 16h light/8 h dark but in the experiment, I consider that it was more appropriate either only light or either dark. The callus cultures do not necessarily require light.

How did you exploit the adventitiuos root and adventitious shoot? There were processed for determination of content in phenolic and flavonoids or chlorogenic acid, caffeic acid and catechin production?

In table 3 the callus biomass was expressed as a concentration which is not correct (g/l).

Line 353 Table 4 moved after paragraph 3.4 or paragraphs 3.3 and 3.4 combined than continue with table 4. The title of the paragraph 3.3 can be ‘Effect of fungal elicitors on total phenolics and total flavonoids productivity (or content)’

 

Comments for author File: Comments.pdf

Author Response

Date: June 4, 2025

Manuscript ID: horticulturae-3674024

Title: Differential Bio-Elicitor Effects on Bioactive Compound Production in Cichorium intybus Root Callus Cultures

Journal: Horticulturae

Dear Reviewer,

We sincerely appreciate your time and effort in reviewing our manuscript and providing constructive and valuable feedback. Your insightful comments have significantly improved the quality of our work. We have carefully addressed each of your suggestions, and all modifications made in the revised manuscript are highlighted in Red for easy reference.

Below, we provide a detailed point-by-point response to your comments, outlining the changes implemented in the revised version.

We are grateful for your thoughtful recommendations and believe that the manuscript has been strengthened as a result. We hope that the revisions meet your expectations, and that the manuscript is now suitable for publication in Horticulturae.

Thank you once again for your guidance and support.

Kind regards,

The authors

Response to Reviewer 1 Comments

1. Questions for General Evaluation	Reviewer’s Evaluation	Response and Revisions
Does the introduction provide sufficient background and include all relevant references?	Yes
Is the research design appropriate?	Yes
Are the methods adequately described?	Must be improved	We have carefully revised the section to provide a more detailed and clear description of the methodologies used in the study. Please find the updated version highlighted in the manuscript for your convenience.
Are the results clearly presented?	Can be improved	We have carefully revised the results section to improve clarity, organization, and data presentation, ensuring better readability and interpretation.
Are the conclusions supported by the results?	Yes
Are all figures and tables clear and well-presented?	Can be improved	We have carefully revised all figures and tables to enhance clarity, labeling, and visual presentation, ensuring they effectively support the results and improve reader comprehension.

 

2. Point-by-point response to Comments and Suggestions for Authors

Comments 1: Line 55:  the sentences begin with ‘Because’ likewise line 62. May be can reformulates the topic of the sentences.

Response 1: We acknowledge the reviewer's valuable suggestion regarding the sentence structure on lines 55 and 62.  Please see the rephrased sentences in the revised manuscript (lines 55-56, 62-63)

Comments 2: I consider that the introduction part from 114 line and 124 line could be included (but not mandatory) to Discussion.

Response 2: Upon careful consideration, we agree with the reviewer's insightful suggestion. We have rephrased and relocated the detailed discussion of previous studies on C. intybus callus induction, plant growth regulators, and light conditions (originally presented in lines 114-124 of the Introduction) to the Discussion section (lines 508-513). We believe this placement now provides a more appropriate and direct context for interpreting and comparing our experimental findings with existing literature, thereby improving the logical flow of the manuscript. We also rephrased this paragraph in a non-detailed manner to maintain the meaning derived from it in the introduction as a background for the study (lines 114-116).

Comments 3: At this chapter (Materials and methods) is not specified the origin of fungal strains (are from international collection?) and the provenance of the seeds (are commercial products?)

Response 3: We thank the reviewer for pointing out these important omissions. We have now added the following details to the 'Materials and Methods' section:

• Seeds: The Cichorium intybus seeds were kindly provided by the Horticultural Research Institute, Agricultural Research Center, Giza, Egypt (lines 127-128).
• Fungal Strains: The fungal strains used in this study were obtained from the Microbiology Laboratory, Botany Department, Faculty of Agriculture, Al-Azhar University, Cairo, Egypt (lines 146-147).

Comments 4: It is not specified the manner in which the fresh callus is processed to obtained dry callus. In general, the method used to dehydrate the callus and determine the dry weight is that of repetitive drying in an oven for several hours until the callus has a constant weight, in fact a constant mass.

Another method to calculated the dry weight is to use the fresh callus to be lyophilized. This method is very expensive and need specific equipment. The lyophilized callus can be mixed with ethanol or other extraction buffer, especially the samples used for HPLC analyses.

Response 4: We thank the reviewer for their valuable comment regarding the callus drying process. We would like to clarify that the method used to obtain dry callus has been specified in the 'Materials and Methods' section (lines 178-179).

 Comments 5: 2.4.2 Callus biomass production is not related to the amount of medium in the culture vessel, because the volume of medium on which the callus is grown must be the same, standard in each culture vessel.

Response 5: We thank the reviewer for this important comment and for highlighting the necessity of standardized culture conditions. We would like to clarify that in our study, a standard volume of 30 ml of medium was consistently used for each culture vessel, and three explants were planted in each vessel (lines 185-186). This method of calculation is followed in many published studies, including, for example, the most recent ones: Bai, X.; Lee, H.-S.; Han, J.-E.; Murthy, H.N.; Park, S.-Y. Enhancement of Phenolic and Polyacetylene Accumulation in Lobelia chinensis (Chinese lobelia) Plantlet Cultures Through Yeast Extract and Salicylic Acid Elicitation. Horticulturae 2025, 11, 612. https://doi.org/10.3390/horticulturae11060612

Comments 6: 2.4.5 The author did not specify the method for obtaining the callus powder used for determination of chlorogenic acid, caffeic acid and catechin.

Response 6: This was actioned. We have added to section 2.4.2 in Materials and Methods (lines 187-188) that the dried callus was ground into a fine powder using a regular laboratory blender. This powder was then used for further analysis.

Comments 7: Line 207 between word “acid” and “and” is a comma to be deleted

Response 7: This was corrected (line 215)

Comments 8: The type of light treatment is confusing. I think it would have been more suitable to treat with light for 24h and dark for 24h respectively, the ratio 16/8 is confusing in determining the type of treatment. It is known in vitro cultures the alternating illumination is ratio 16h light/8 h dark but in the experiment, I consider that it was more appropriate either only light or either dark. The callus cultures do not necessarily require light.

Response 8: We thank the reviewer for their valuable comments regarding the light treatment conditions. We understand the concern about clarity and the suggestion for a 24h light or 24h dark regimen.

We would like to clarify that our experimental design indeed included both complete darkness and a 16h light/8h dark photoperiod as distinct treatments. These conditions were deliberately chosen to investigate the differential responses of C. intybus callus under varying light exposures, including a standard photoperiod commonly used in in vitro plant tissue culture and a complete dark condition.

Our results, as presented in the manuscript (Table 1, 2 and Figure 1), clearly demonstrated distinct responses in callus color, texture, adventitious organ formation (roots and shoots), and weight under these specific incubation conditions. Since the experiment has already been completed and these differential responses provide meaningful insights, the chosen light treatments were instrumental in achieving our study's objectives.

We have ensured that the methodology clearly outlines the application of both these photoperiods. We appreciate the reviewer's attention to the experimental details.

Comments 9: How did you exploit the adventitiuos root and adventitious shoot? There were processed for determination of content in phenolic and flavonoids or chlorogenic acid, caffeic acid and catechin production?

Response 9: The formation of adventitious roots and shoots was indeed recorded as one of the key morphogenetic responses observed in the callus cultures under the influence of different plant growth regulators and light conditions. This observation highlights the varied developmental pathways induced in the callus.

While these adventitious organs hold significant biotechnological importance, the primary focus of our current study was specifically on the callus's response to fungal elicitors and its subsequent production of target compounds. Therefore, for all analyses, including the determination of phenolic acids and flavonoids, the callus material was carefully trimmed to remove any adventitious root or shoot growths (line 156) to ensure that the analytical results directly reflect the callus biomass itself.

Comments 10: In table 3 the callus biomass was expressed as a concentration which is not correct (g/l).

Response 10: We thank the reviewer for pointing out the initial ambiguity in expressing callus biomass in Table 3. To ensure clarity and precision, we have revised the unit in Table 3 to grams per liter of culture medium (g/l medium) (line 346). This unit accurately reflects the biomass yield of callus produced per unit volume of the culture medium, which is a standard method for representing productivity in plant tissue culture.

Comments 11: Line 353 Table 4 moved after paragraph 3.4 or paragraphs 3.3 and 3.4 combined than continue with table 4. The title of the paragraph 3.3 can be ‘Effect of fungal elicitors on total phenolics and total flavonoids productivity (or content)’

Response 11: We are very grateful for this insightful suggestion. We agree that the proposed changes enhance the logical flow of the manuscript. In response to this comment, we have combined paragraphs 3.3 and 3.4 under a more comprehensive heading: ‘Effect of fungal elicitors on total phenolics and total flavonoids production’ (lines 360-390)

Reviewer 2 Report

Comments and Suggestions for Authors

Overall, I believe the study was conducted appropriately. However, certain variables need clarification regarding their importance to the study. Enhancing their analysis and discussion would significantly improve the manuscript.

Lines 152–166 Although the preparation of the fungal elicitor is described in detail, I believe there is no clear way to standardize the compounds responsible for the elicitation. Therefore, the reproducibility of this assay over time—particularly when preparing new batches of elicitors—may be quite low.

Lines 499–501 The analysis and discussion regarding the different coloration of the calli are very limited. This variable is barely addressed, so I do not see the relevance of including it in the manuscript (neither in the results nor in the discussion). If a deeper analysis is provided, its inclusion could be justified.

It is not clear what the purpose is for registering fresh weight, dry weight, and dry matter content. If these variables were indeed measured and showed treatment effects, it would be beneficial to discuss them more thoroughly, rather than simply reporting the values. The explanation provided (lines 520–533) lacks depth and does not explore the relationships between these variables.

In the discussion on fungal elicitors, it would be helpful to clarify whether the concentration used in this study is considered high, medium, or low compared to the literature cited. While the authors mention that the elicitor’s effect depends on fungal mycelium components (such as chitin, cellulose, and proteins), the methodology does not include any standardization or quantification of the mycelium or the extract used. This makes the assay difficult to reproduce.

Author Response

Date: June 4, 2025

Manuscript ID: horticulturae-3674024

Title: Differential Bio-Elicitor Effects on Bioactive Compound Production in Cichorium intybus Root Callus Cultures

Journal: Horticulturae

Dear Reviewer,

We sincerely appreciate your time and effort in reviewing our manuscript and providing constructive and valuable feedback. Your insightful comments have significantly improved the quality of our work. We have carefully addressed each of your suggestions, and all modifications made in the revised manuscript are highlighted in Red for easy reference.

Below, we provide a detailed point-by-point response to your comments, outlining the changes implemented in the revised version.

We are grateful for your thoughtful recommendations and believe that the manuscript has been strengthened as a result. We hope that the revisions meet your expectations, and that the manuscript is now suitable for publication in Horticulturae.

Thank you once again for your guidance and support.

Kind regards,

The authors

Response to Reviewer 2 Comments

1. Questions for General Evaluation	Reviewer’s Evaluation		Response and Revisions
Does the introduction provide sufficient background and include all relevant references?	Yes
Is the research design appropriate?	Yes
Are the methods adequately described?	Can be improved	We have carefully revised the section to provide a more detailed and clear description of the methodologies used in the study. Please find the updated version highlighted in the manuscript for your convenience.
Are the results clearly presented?	Yes
Are the conclusions supported by the results?	Yes
Are all figures and tables clear and well-presented?	Yes

 

2. Point-by-point response to Comments and Suggestions for Authors

Comments 1: Lines 152–166 Although the preparation of the fungal elicitor is described in detail, I believe there is no clear way to standardize the compounds responsible for the elicitation. Therefore, the reproducibility of this assay over time—particularly when preparing new batches of elicitors—may be quite low.

Response 1: We appreciate your perspective on the standardization challenges of fungal elicitors. While it is true that natural elicitors can exhibit variability due to differences in composition, certain approaches, such as concentration adjustments and determined sourcing, defining bioactivity assays and reference controls, can help mitigate this issue and may enhance consistency over time. Moreover, we have taken measures such as maintaining a consistent fungal strain and growth conditions to minimize variability. We believe that despite this, the observed effects within the scope of this study are robust and provide valuable insights into the elicitation process. For future studies, we aim to explore more defined elicitor fractions or purified compounds to enhance standardization and ensure higher long-term reproducibility.

Comments 2: Lines 499–501 The analysis and discussion regarding the different coloration of the calli are very limited. This variable is barely addressed, so I do not see the relevance of including it in the manuscript (neither in the results nor in the discussion). If a deeper analysis is provided, its inclusion could be justified.

Response 2: We appreciate the reviewer's insightful comment regarding the limited analysis and discussion of callus coloration in the manuscript. We agree that this aspect deserves a more detailed exploration to justify its inclusion. In the revised manuscript, we have provided an expanded discussion on the significance of callus coloration and its relevance to the study (lines 515-519, 521-526).

Comments 3: It is not clear what the purpose is for registering fresh weight, dry weight, and dry matter content. If these variables were indeed measured and showed treatment effects, it would be beneficial to discuss them more thoroughly, rather than simply reporting the values. The explanation provided (lines 520–533) lacks depth and does not explore the relationships between these variables.

Response 3: We sincerely appreciate the reviewer’s valuable feedback regarding the discussion of fresh weight (FW), dry weight (DW), and dry matter content (DM) in our study. We provided a more thorough explanation of their significance, the observed treatment effects, and their relationships with other variables (lines 547-549, 554-558, 562-563, 571-572, 575-578, 584-586). 

Comments 4: In the discussion on fungal elicitors, it would be helpful to clarify whether the concentration used in this study is considered high, medium, or low compared to the literature cited.

Response 4: This was addressed in the revised manuscript (lines 580-584, 605-617).

Comments 5: While the authors mention that the elicitor’s effect depends on fungal mycelium components (such as chitin, cellulose, and proteins), the methodology does not include any standardization or quantification of the mycelium or the extract used. This makes the assay difficult to reproduce.

Response 5: This was corrected in the Materials and Methods section (lines 150-154)

Reviewer 3 Report

Comments and Suggestions for Authors

This manuscript reports the induction of Cichorium intybus callus using different concentrations and combinations of NAA (an auxin) and BA (a cytokinin), in the presence and absence of light, using roots as the explant source. The authors also studied the effect of different biotic elicitors (fungi) on biomass production, antioxidant activity, and phenolics accumulation in C. intybus root-derived callus. The results confirmed the influence of PGRs and light conditions on the callus induction and in the morphological characteristics of callus, as well as the effect of the biotic elicitors used on callus growth and on secondary metabolites production.

Callus culture as well as cell suspension culture are biotechnological tools that can be used to produce bioactive compounds of interest through a sustainable and renewable way because. These techniques allow the modulation of plant metabolite synthesis, with continuous production occurring in a controlled environment, in a smaller space, and in less time. In addition, the use of elicitors during in vitro culture constitutes a strategy that can favor the increase in biomass and the production and accumulation of bioactive compounds.

Scientific investigations with a focus on increasing the productivity of important medicinal compounds in agricultural crops are welcome, because they generally assume an important character related to the clarification of important physiological and chemical aspects involved in this process. The aims of this manuscript are interesting, and the experimental stage was apparently well conducted. The results are consistent, robust and well detailed. In addition, authors also used many current references to discuss the results. However, there are some points that need explanation and/or correction. Please see the comments below.

1- Abstract:

- line 24: “… [yeast extract, Fusarium oxysporum, and Aspergillus niger]...” – please rephrase to: “… [yeast extract (YE), Fusarium oxysporum, and Aspergillus niger]...”

2- Material and methods:

- line 136: please provide details on the origin of the C. intybus seeds (brand, manufacturer...);

- line 152 – Fungal elicitors – please provide details on obtaining/origin of YE;

- lines 162-163: please provide details on how the fungal elicitors were solubilized (what solvent was used?);

- line 168: I think it is important to indicate that the callus induction rate was evaluated in each treatment (% induction) and add this result to Table 1;

- lines 171: “... and the degree of adventitious root or adventitious shoot...” – How was this measured? Were they counted? Please specify;

3- Results

- line 251: please begin the presentation of results with the report described in lines 299-302;

- line 284: Does the degree (+) indicate how much adventitious root or adventitious shoot formation? Please specify these degrees in the methodology (from line 171);

- line 325: “Callus formed in ...” – please rephrase to “Callus induced in...”;

- line 326: “mg/l NAA + 1 mg/l BA medium was chosen ...” – please rephrase to: “mg/l NAA + 1 mg/l BA medium under the 16/8 h light/dark cycle was chosen ...”;

- lines 365-368 – this sentence is confusing – please rephrase it;

- Figure 2: should be presented before Table 5 because it was mentioned first in the text;

- Figure 2: please identify the peaks of the patterns shown in figure A, as was done for the other figures;

4- Conclusion:

The conclusion is too long and provides a summary of the results found. Please provide a shorter conclusion and highlight the perspectives for the advancement of knowledge in the area studied – please review.

In my final comments, I recommend that the manuscript should be reviewed by the authors, especially about the points highlighted above. I would like to congratulate the authors for this valuable contribution and the promising results presented here.

Author Response

Date: June 4, 2025

Manuscript ID: horticulturae-3674024

Title: Differential Bio-Elicitor Effects on Bioactive Compound Production in Cichorium intybus Root Callus Cultures

Journal: Horticulturae

Dear Reviewer,

We sincerely appreciate your time and effort in reviewing our manuscript and providing constructive and valuable feedback. Your insightful comments have significantly improved the quality of our work. We have carefully addressed each of your suggestions, and all modifications made in the revised manuscript are highlighted in Red for easy reference.

Below, we provide a detailed point-by-point response to your comments, outlining the changes implemented in the revised version.

We are grateful for your thoughtful recommendations and believe that the manuscript has been strengthened as a result. We hope that the revisions meet your expectations, and that the manuscript is now suitable for publication in Horticulturae.

Thank you once again for your guidance and support.

Kind regards,

The authors

Response to Reviewer 3 Comments

1. Questions for General Evaluation	Reviewer’s Evaluation	Response and Revisions
Does the introduction provide sufficient background and include all relevant references?	Yes
Is the research design appropriate?	Yes
Are the methods adequately described?	Can be improved	We have carefully revised the section to provide a more detailed and clear description of the methodologies used in the study. Please find the updated version highlighted in the manuscript for your convenience.
Are the results clearly presented?	Can be improved	We have carefully revised the results section to improve clarity, organization, and data presentation, ensuring better readability and interpretation.
Are the conclusions supported by the results?	Can be improved	To strengthen the alignment between our results and conclusions, we have carefully revised the discussion and conclusion sections to ensure all claims are robustly supported by the data.
Are all figures and tables clear and well-presented?	Yes

 

2. Point-by-point response to Comments and Suggestions for Authors

Comments 1: - line 24: “… [yeast extract, Fusarium oxysporum, and Aspergillus niger]...” – please rephrase to: “… [yeast extract (YE), Fusarium oxysporum, and Aspergillus niger]...”

Response 1: This was corrected in the revised manuscript (line 24).

Comments 2: - line 136: please provide details on the origin of the C. intybus seeds (brand, manufacturer...);

Response 2: We thank the reviewer for pointing out these important omissions. We have now added the following details to the 'Materials and Methods' section:

• Seeds: The Cichorium intybus seeds were kindly provided by the Horticultural Research Institute, Agricultural Research Center, Giza, Egypt (lines 127-128).

Comments 3: - line 152 – Fungal elicitors – please provide details on obtaining/origin of YE;

Response 3: We appreciate the reviewer's request for clarification regarding the origin of the yeast extract (YE) used in our study. We have revised the Materials and Methods section (Page 4, Section 2.3) to include these important details (lines 146-154, 159): The YE used in this study was obtained from Sigma Chemical Co., St. Louis, MO. The fungal strains used in this study (A. niger and F. oxysporum) were obtained from the Microbiology Laboratory, Botany Department, Faculty of Agriculture, Al-Azhar University, Cairo, Egypt.

Comments 4: - lines 162-163: please provide details on how the fungal elicitors were solubilized (what solvent was used?);

Response 4: We thank the reviewer for this important technical clarification. We have revised the Materials and Methods section (Page 4, Section 2.3) to provide complete details about the solubilization process (lines 161-164): ‘The dried mycelium and YE powders were first solubilized in distilled water with constant stirring until completely dissolved. This solution was then added to the culture media before being autoclaved at 121°C for 20 minutes.’

Comments 5: - line 168: I think it is important to indicate that the callus induction rate was evaluated in each treatment (% induction) and add this result to Table 1;

Response 5: We appreciate the reviewer's suggestion to clarify the callus induction rate. Since all treatments showed 100% induction, we have added this important information to the Results section (lines 266-267) and Table 1 (line 286).

Comments 6: - lines 171: “... and the degree of adventitious root or adventitious shoot...” – How was this measured? Were they counted? Please specify;

Response 6: We appreciate the reviewer's request for clarification regarding our assessment of adventitious organ formation. The degree of adventitious root or shoot formation was evaluated using a semi-quantitative scoring system based on visual assessment: (-) no formation, (+) low, (++) moderate, (+++) high, (++++) very high (lines 173-176, 287-288).

Comments 7: - line 251: please begin the presentation of results with the report described in lines 299-302;

Response 7: We appreciate this suggestion to improve the logical flow of our results section. We have reorganized and rephrased the presentation (lines 261-263).

Comments 8: - line 284: Does the degree (+) indicate how much adventitious root or adventitious shoot formation? Please specify these degrees in the methodology (from line 171);

Response 8: The degree of adventitious root or shoot formation was evaluated using a semi-quantitative scoring system based on visual assessment: (-) no formation, (+) low, (++) moderate, (+++) high, (++++) very high. This was added to the revised 'Materials and Methods' section (lines 173-176, 287-288).

Comments 9: - line 325: “Callus formed in ...” – please rephrase to “Callus induced in...”;

Response 9: This was corrected (line 332).

Comments 10: - line 326: “mg/l NAA + 1 mg/l BA medium was chosen ...” – please rephrase to: “mg/l NAA + 1 mg/l BA medium under the 16/8 h light/dark cycle was chosen ...”;

Response 10: The sentence was rephrased (line 333).

Comments 11: - lines 365-368 – this sentence is confusing – please rephrase it;

Response 11: The original confusing sentence has been revised in lines 368-372.

Comments 12: - Figure 2: should be presented before Table 5 because it was mentioned first in the text;

Response 12: This was actioned. We appreciate this important observation regarding manuscript flow (lines 412 and 418).

Comments 13: - Figure 2: please identify the peaks of the patterns shown in figure A, as was done for the other figures;

Response 13: We have revised Figure 2 to include comprehensive peak labeling in all chromatograms (line 412). 

Comments 14: The conclusion is too long and provides a summary of the results found. Please provide a shorter conclusion and highlight the perspectives for the advancement of knowledge in the area studied – please review.

Response 14: We have carefully revised the conclusion to be more concise, highlighting the study's contribution to knowledge advancement and outlining future research perspectives, rather than merely summarizing the results (lines 723-736).

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

The propose suggestions were made it and the suggested changes have been carried out. Regarding the method of calculation of callus biomass I consider that the medium volume is standard for all  culture vessel , solidified with 7g/l, while in the reference you exemplified the authors used liquid medium: "Cultures were established in 500 mL conical flasks containing 200 mL of MS medium with 3% sucrose (w/v). Explants (3 g L⁻¹) were cultured and maintained on a rotary shaker (OS 4000 orbital shaker, Jeio Tech, Daejon, Republic of Korea) at 100 rpm. The cultures were maintained at 25 ± 2 °C with 70% relative humidity, a 16/8 h photoperiod, and 60 µmol m⁻² s⁻¹ light intensity provided by red + blue + green light-emitting diodes (LEDs) equipped with a red 20 stick/400 tip, blue 20 stick/400 tip, and green 20 stick/400 tp to arrange the panel (Itswell Co., Incheon, Republic of Korea). Bai, X.; Lee, H.-S.; Han, J.-E.; Murthy, H.N.; Park, S.-Y. Enhancement of Phenolic and Polyacetylene Accumulation in Lobelia chinensis (Chinese lobelia) Plantlet Cultures Through Yeast Extract and Salicylic Acid Elicitation. Horticulturae 2025, 11, 612. https://doi.org/10.3390/horticulturae11060612

Sorry if I misunderstood the experiment in your article.

Author Response

Date: June 6, 2025

Manuscript ID: horticulturae-3674024

Title: Differential Bio-Elicitor Effects on Bioactive Compound Production in Cichorium intybus Root Callus Cultures

Journal: Horticulturae

Dear Reviewer,

We sincerely appreciate your time and effort in reviewing our manuscript and providing constructive and valuable feedback.

Below, we provide a response to your comment.

We are grateful for your thoughtful recommendations and believe that the manuscript has been strengthened as a result. We hope that the revisions meet your expectations and that the manuscript is now suitable for publication in Horticulturae.

Thank you once again for your guidance and support.

Kind regards,

The authors

Response to Reviewer 1 Comments

1. Questions for General Evaluation	Reviewer’s Evaluation	Response and Revisions
Does the introduction provide sufficient background and include all relevant references?	Yes
Is the research design appropriate?	Yes
Are the methods adequately described?	Yes
Are the results clearly presented?	Yes
Are the conclusions supported by the results?	Yes
Are all figures and tables clear and well-presented?	Yes

 

2. Point-by-point response to Comments and Suggestions for Authors

Comments 1: The propose suggestions were made it and the suggested changes have been carried out. Regarding the method of calculation of callus biomass I consider that the medium volume is standard for all  culture vessel , solidified with 7g/l, while in the reference you exemplified the authors used liquid medium: "Cultures were established in 500 mL conical flasks containing 200 mL of MS medium with 3% sucrose (w/v). Explants (3 g L⁻¹) were cultured and maintained on a rotary shaker (OS 4000 orbital shaker, Jeio Tech, Daejon, Republic of Korea) at 100 rpm. The cultures were maintained at 25 ± 2 °C with 70% relative humidity, a 16/8 h photoperiod, and 60 µmol m⁻² s⁻¹ light intensity provided by red + blue + green light-emitting diodes (LEDs) equipped with a red 20 stick/400 tip, blue 20 stick/400 tip, and green 20 stick/400 tp to arrange the panel (Itswell Co., Incheon, Republic of Korea). Bai, X.; Lee, H.-S.; Han, J.-E.; Murthy, H.N.; Park, S.-Y. Enhancement of Phenolic and Polyacetylene Accumulation in Lobelia chinensis (Chinese lobelia) Plantlet Cultures Through Yeast Extract and Salicylic Acid Elicitation. Horticulturae 2025, 11, 612. https://doi.org/10.3390/horticulturae11060612

Sorry if I misunderstood the experiment in your article.

Response 1: First, we sincerely apologize for not providing sufficient details when responding to your comment regarding calculating callus production relative to the volume of the culture medium used.

We provided an example of a study that calculated the productivity of in vitro cultures (plantlet) relative to the volume of the medium used in liters. That study actually used liquid media. In our current study, we use solid media. We simply wanted to cite a very recent study (Bai et al., 2025: https://doi.org/10.3390/horticulturae11060612).

We would like to clarify that it is indeed possible to calculate the productivity of in vitro cultures, whether callus, suspended cells, adventitious organs, or plantlets, relative to the volume known in in vitro experiments, which is liters. This is accurate as long as a constant volume of culture medium for each explant (lines 185-186) and a very close weight of inoculums of tissues or organs are used (line 157). Therefore, the productivity or weight (lines 184-185) as well as the productivity of bioactive compounds (lines 201-202, 213-214) can be calculated relative to the volume in liters.

Numerous studies have supported this, regardless of the type of medium—solid, liquid, or semi-liquid—and also regardless of the type of cultures, whether callus, cell suspensions, or organs. Examples include, but are not limited to:

1. Bai, X.; Lee, H.-S.; Han, J.-E.; Murthy, H.N.; Park, S.-Y. Enhancement of Phenolic and Polyacetylene Accumulation in Lobelia chinensis(Chinese lobelia) Plantlet Cultures Through Yeast Extract and Salicylic Acid Elicitation. Horticulturae 2025, 11, 612. https://doi.org/10.3390/horticulturae11060612
2. Elateeq,A.; Gabr, A.M.M.; Abdelkawy, A.M.; Toaima, N.M.; Bosila, H.A.; Zarad, M.M.; Ebrahim, H.S.; Jiao, J.; Pan, H.; Ullah, S.; Fu, Y. Establishment of Gypsophila paniculata root culture for biomass, saponin, and flavonoid production. Not. Bot. Horti Agrobot. Cluj-Napoca 2022, 50, 12886. https://doi.org/10.15835/nbha50312886
3. Elshahawy, O.A.M.; Zeawail, M.E.-F.; Hamza, M.A.; Elateeq, A.A.; Omar, M.A. Improving the Production of Total Phenolics and Flavonoids and the Antioxidant Capacity of Echinacea purpurea Callus through Biotic Elicitation. J. Chem. 2022, 65, 137–149. https://doi.org/10.21608/ejchem.2022.145210.6328
4. Günter, E.A.; Ovodov, Y.S. Production of Polysaccharides by Silene vulgaris Callus Culture Depending on Carbohydrates of the Medium. Biochemistry (Moscow) 2003, 68, 882–889. https://doi.org/10.1023/A:1025751015684
5. Kapoor, S.; Raghuvanshi, R.; Bhardwaj, P.; Sood, H.; Saxena, S.; Chaurasia, O.P. Influence of light quality on growth, secondary metabolites production and antioxidant activity in callus culture of Rhodiola imbricata Edgew. J. Photochem. Photobiol. B: Biol. 2018, 183, 258-265. https://doi.org/10.1016/j.jphotobiol.2018.04.018
6. Mohammad, S.; Khan, M.A.; Ali, A.; Khan, L.; Khan, M.S.; Mashwani, Z.U.R. Feasible production of biomass and natural antioxidants through callus cultures in response to varying light intensities in olive (Olea europaea. L) cult. Arbosana.  Photochem. Photobiol. B: Biol. 2019, 193, 140-147. https://doi.org/10.1016/j.jphotobiol.2019.03.001
7. Rattan, S.; Kumar, D.; Warghat, A.R. Growth kinetics, metabolite yield, and expression analysis of biosynthetic pathway genes in friable callus cell lines of Rhodiola imbricata (Edgew). Plant Cell Tiss. Organ Cult. 2021, 146, 149–160. https://doi.org/10.1007/s11240-021-02057-8

Reviewer 3 Report

Comments and Suggestions for Authors

The authors satisfactorily addressed all previously indicated suggestions and corrections.

Author Response

Date: June 6, 2025

Manuscript ID: horticulturae-3674024

Title: Differential Bio-Elicitor Effects on Bioactive Compound Production in Cichorium intybus Root Callus Cultures

Journal: Horticulturae

Response to Reviewer 3 Comments

1. Questions for General Evaluation	Reviewer’s Evaluation	Response and Revisions
Does the introduction provide sufficient background and include all relevant references?	Yes
Is the research design appropriate?	Yes
Are the methods adequately described?	Yes
Are the results clearly presented?	Yes
Are the conclusions supported by the results?	Yes
Are all figures and tables clear and well-presented?	Yes

 

2. Point-by-point response to Comments and Suggestions for Authors

Comments 1: The authors satisfactorily addressed all previously indicated suggestions and corrections.

Response 1: We sincerely appreciate your time and effort in reviewing our manuscript and providing constructive and valuable feedback.