In Vitro Plant Regeneration and Bioactive Metabolite Production of Endangered Medicinal Plant Atractylodes lancea (Thunb.) DC

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

Abstract

• Indicate the document or Association that mentions the plant under endangered status.
• What is the reason to put the phrase “Efficient plant regeneration and bioactive metabolite production” in parentheses?

  Introduction

• Please, indicate in this section too, the document or Association that mentions the plant under endangered status.
• What are the references that indicate the decline of A. lancea at the study site?
• The word "bulbus" should be written in lowercase (line 65).

Materials and methods

• Remove the parenthesis (Line 90).
• In the case of the leaves, were they placed on their adaxial or abaxial sides? Please indicate (Line 107).
• Indicate why you chose to work with these concentrations of exogenous hormones.
• Why did you use activated carbon? Indicate in the introduction or discussion section.
• Subheading 2.7 is misspelled. Please correct it. (Line 159)
• The organism A. rhizogenic does not appear earlier in the manuscript. Please provide the full name of the organism (Line 161).
• Please indicate in the methodology with which instrument you measured the optical density (Line 165).
• Indicate the amount of material used from each explant (embryogenic calli, adventitious roots, and hairy roots) to obtain the essential oil (Line 202).

Results and Discussion

• Table 5, 7 and 8, Please use just one significance level. I suggest p<0.05. And indicate in the notes of the table the Test utilized.
• Why the seedlings planted in garden soil exhibited weak growth? Please discuss.
• Figure 1 has several photographs. However, it only mentions that it represents plant regeneration. Please explain in the text what is observed in each photograph (A-I).
• It is necessary to add measuring bars to the photographs in Figure 1, to know the dimensions of the plants at their different stages. Please added.
• Please Add a discussion clarifying why leaf tissue and petioles were not good explants for developing callus on A. lancea?
• In Figure 3, the meaning of the colored circles accompanying each bar in each essential oil is unclear. Please clarify.
• The quality of Figure 4 is low. Please provide a higher quality image.

Conclusions

Could the proposed strategy work for other plants of other genera?

Comments for author File: Comments.pdf

Author Response

Dear Editors,

 

We extend our gratitude to you and the reviewers for the thorough evaluation of our manuscript. We highly value the insightful comments provided, which have significantly contributed to the refinement of our work. We are pleased to resubmit our revised manuscript, titled " In vitro plant regeneration and bioactive metabolite production of endangered medicinal plant Atractylodes lancea" (ID: horticulturae-3656173), for your consideration for publication in Horticulturae.

 

In response to the reviewers' comments, we have diligently revised our manuscript, addressing their concerns comprehensively. Our point-by-point response outlines the specific modifications made to enhance the clarity and scientific merit of our study.

 

We trust that these revisions have significantly improved the overall quality of our manuscript, aligning it with the standards set forth by Medicinal Plant Biology. We remain open to addressing any further concerns and appreciate the opportunity to contribute our research to your esteemed journal.

 

Sincerely,

1. Zhang and S. Wang

 

point-by-point response

 

Reviewer #1

Comments to the Author

Abstract

• Indicate the document or Association that mentions the plant under endangered

status.

• What is the reason to put the phrase “Efficient plant regeneration and bioactive

metabolite production” in parentheses?

Introduction

• Please, indicate in this section too, the document or Association that mentions the

plant under endangered status.

• What are the references that indicate the decline of A. lancea at the study site?
• The word "bulbus" should be written in lowercase (line 65).

Materials and methods

• Remove the parenthesis (Line 90).
• In the case of the leaves, were they placed on their adaxial or abaxial sides? Please

indicate (Line 107).

• Indicate why you chose to work with these concentrations of exogenous hormones.
• Why did you use activated carbon? Indicate in the introduction or discussion section.
• Subheading 2.7 is misspelled. Please correct it. (Line 159)
• The organism A. rhizogenic does not appear earlier in the manuscript. Please provide the full name of the organism (Line 161).
• Please indicate in the methodology with which instrument you measured the optical

density (Line 165).

• Indicate the amount of material used from each explant (embryogenic calli, adventitious roots, and hairy roots) to obtain the essential oil (Line 202).

Results and Discussion

• Table 5, 7 and 8, Please use just one significance level. I suggest p<0.05. And

indicate in the notes of the table the Test utilized.

• Why the seedlings planted in garden soil exhibited weak growth? Please discuss.
• Figure 1 has several photographs. However, it only mentions that it represents plant

regeneration. Please explain in the text what is observed in each photograph (A-I).

• It is necessary to add measuring bars to the photographs in Figure 1, to know the

dimensions of the plants at their different stages. Please added.

• Please Add a discussion clarifying why leaf tissue and petioles were not good

explants for developing callus on A. lancea?

• In Figure 3, the meaning of the colored circles accompanying each bar in each

essential oil is unclear. Please clarify.

• The quality of Figure 4 is low. Please provide a higher quality image.

Conclusions

Could the proposed strategy work for other plants of other genera?

 

Abstract

1. Indicate the document or Association that mentions the plant under endangered status.

Response: Thank you for your positive comments. Atractylodes lancea is listed as a rare and protected plant at the provincial level in Jiangsu Province, and it is also included in the list of four endangered medicinal plants under key protection in Jiangsu Province. The relevant supporting documents are as follows: Jiangsu Provincial List of Key Protected Wild Plants (First Batch), National List of Protected New Plant Varieties (Forestry and Grassland Section, Eighth Batch).

 

 

2. What is the reason to put the phrase “Efficient plant regeneration and bioactive metabolite production” in parentheses?

Response: Because this is a key technical means that this article wants to elaborate on, namely "Efficient plant regeneration and bioactive metabolite production", it is highlighted in double quotation marks. The main function is to emphasize and highlight.

Introduction

1. Please, indicate in this section too, the document or Association that mentions the plant under endangered status.

Response: Corrected.

2. What are the references that indicate the decline of lancea at the study site?

Response: Corrected. Added [5-7], 3 references.

3. The word "bulbus" should be written in lowercase (line 65).

Response: Corrected.

Materials and methods

1. Remove the parenthesis (Line 90).

Response: Removed.

2. In the case of the leaves, were they placed on their adaxial or abaxial sides? Please indicate (Line 107).

Response: It has been marked as the back of the blade

3. Indicate why you chose to work with these concentrations of exogenous hormones.

Response: The concentration range of these exogenous hormones was determined based on our preliminary experimental results. In addition, according to the concentration range of hormones in plant tissue culture, it is shown that 0.1-2.0 mg/L 6-BA promotes shoot proliferation, and the proliferation rate increases with increasing concentration; 6-BA 2.0 mg/L+NAA 0.2~0.5 mg/L is beneficial for inducing adventitious buds in tissue culture.

4. Why did you use activated carbon? Indicate in the introduction or discussion section.

Response: The reason for adding activated carbon has been discussed.

5. Subheading 2.7 is misspelled. Please correct it. (Line 159).

Response: Corrected.

6. The organism A. rhizogenic does not appear earlier in the manuscript. Please provide the full name of the organism (Line 161).

Response: Corrected.

7. Please indicate in the methodology with which instrument you measured the optical density (Line 165).

Response: Already supplemented.

8. Indicate the amount of material used from each explant (embryogenic calli, adventitious roots, and hairy roots) to obtain the essential oil (Line 202).

Response: Corrected.

Results and Discussion

1. Table 5, 7 and 8, Please use just one significance level. I suggest p<0.05. And

indicate in the notes of the table the Test utilized.

Response: Due to significant differences in data, we used two testing methods, p<0.05 and p<0.01, and standardized them in the original table. Please be informed that the data analysis has been indicated in the Materials and Methods section.

2. Why the seedlings planted in garden soil exhibited weak growth? Please discuss.

Response: Improved.

3. Figure 1 has several photographs. However, it only mentions that it represents plant regeneration. Please explain in the text what is observed in each photograph (A-I).

Response: Improved.

4. It is necessary to add measuring bars to the photographs in Figure 1, to know the dimensions of the plants at their different stages. Please added.

Response: Already added.

5. Please Add a discussion clarifying why leaf tissue and petioles were not good

explants for developing callus on A. lancea?

Response: Already added.

6. In Figure 3, the meaning of the colored circles accompanying each bar in each

essential oil is unclear. Please clarify.

Response: Corrected.

7. The quality of Figure 4 is low. Please provide a higher quality image.

Response: Improved.

Conclusions

1. Could the proposed strategy work for other plants of other genera?

Response: The strategy we proposed is optimal for the in vitro regeneration and production of active substances in Atractylodes lancea, which has reference value for other plant species. However, appropriate technical parameter adjustments still need to be made according to different plant species.

Reviewer 1's annotations on the original PDF have been fully revised.

Reviewer #2:

Comments to the Author

The overall content, data, and presentation of the manuscript are good. However, after

careful evaluation, the authors need to address the following comments.

 

Line 2-3: In vitro plant regeneration and bioactive metabolite production of endangered

medicinal plant Atractylodes lancea should be changed to In vitro plant regeneration and bioactive metabolite production of endangered medicinal plant Atractylodes lancea

(Thunb.) DC.

Line 11-12: The rhizome of Atractylodes lancea is a traditional Chinese medicine used

extensively owing to its antimicrobial properties should be changed to the rhizome of

Atractylodes lancea (Thunb.) DC. is a traditional Chinese medicine used extensively owing to its antimicrobial.

In the introduction, you should start with the importance of medicinal plants. 

What specific limitations exist in current lanceatissue culture protocols?

Line 38: hike in market demand should be changed to increase in market demand.

Line 56-57: Since its historical development should be changed to Since its inception.

Line 77: innovate new germplasms should be changed to develop new germplasms.

Light source LED? Or Fluorescent?

How did you control humidity? Was it through sealed containers or vented lids?

Add morphological criteria for callus quality 

Explain why MS, ½MS, ¼MS were tested 

Why transfer to basic medium after 5–7 days? Hormone withdrawal effect?

Why you used ethanol for calli vs. hexane for roots?

Ensure all figures/tables are referenced in order

Why did petioles outperform leaves in callus induction?

In table 5 Why did Treatment 9 (18% induction) fail?

In table 8 Why did NAA activated carbon? Discuss charcoal’s role in adsorbing inhibitors.

Line 239: augments callus proliferation should be changed to enhances callus proliferation.

Line 367: ARF7/19 should be changed to ARF7/19-mediated auxin signaling.

Explicitly state how this study advances beyond prior lancea tissue culture reports In conclusion, States "95 days" but doesn’t clarify if this is from seed to rooted plantlet or metabolite harvest.

 

1. Line 2-3: In vitro plant regeneration and bioactive metabolite production of endangered medicinal plant Atractylodes lancea should be changed to In vitro plant regeneration and bioactive metabolite production of endangered medicinal plant Atractylodes lancea (Thunb.) DC.

Response: Corrected.

2. Line 11-12: The rhizome of Atractylodes lancea is a traditional Chinese medicine used extensively owing to its antimicrobial properties should be changed to the rhizome of Atractylodes lancea (Thunb.) DC. is a traditional Chinese medicine used extensively owing to its antimicrobial.

Response: Corrected.

3. In the introduction, you should start with the importance of medicinal plants.

What specific limitations exist in current A. lancea tissue culture protocols?

Response: Corrected.

4. Line 38: hike in market demand should be changed to increase in market demand.

Response: Already supplemented

5. Line 56-57: Since its historical development should be changed to Since its inception.

Response: Corrected.

6. Line 77: innovate new germplasms should be changed to develop new germplasms. Light source LED? Or Fluorescent?

Response: Corrected. The light source is LED.

7. How did you control humidity? Was it through sealed containers or vented lids?

Add morphological criteria for callus quality. 

Response: Corrected. In this study, the control of humidity in plant tissue culture rooms was achieved through the use of ventilation systems and humidifiers. The humidity of tissue culture seedlings is determined by using tissue culture bottles with breathable membranes. We have added morphological criteria for callus quality.

 

Figure Morphological criteria for callus quality

8. Explain why MS, ½MS, ¼MS were tested.

Response: The reason why MS, ½MS, ¼MS were used as the basic culture media for inducing callus differentiation in this study is to promote the differentiation of A. lancea callus into adventitious buds by reducing inorganic salt concentration, optimizing hormone and nutrient balance, and reducing metabolic burden. Research has shown that high salt medium may accelerate callus proliferation but inhibit differentiation. 1/2MS helps cells to turn towards morphogenesis, and 1/2MS medium can provide a more suitable microenvironment for callus differentiation by regulating salt concentration.

9. Why transfer to basic medium after 5–7 days? Hormone withdrawal effect?

Response: The main reasons for using auxin containing culture medium for 5-7 days before transferring to MS medium during rooting and tissue culture seedling cultivation are as follows: inducing root primordium formation and balancing nutrient and hormone requirements. This strategy optimizes rooting efficiency by regulating hormones and nutrients in stages.

10. Why you used ethanol for calli vs. hexane for roots?

Response: Ethanol (polar solvent) can effectively dissolve polar metabolites (such as phenols, flavonoids, glycosides, etc.) in callus tissue, which accumulate in large quantities during the dedifferentiation process of callus tissue. N-hexane (non-polar solvent) has a higher dissolution efficiency for lipophilic substances such as terpenes, waxes, coenzyme Q10, etc., and the content of such substances is more abundant in the roots.

11. Ensure all figures/tables are referenced in order.

Response: Corrected.

12. Why did petioles outperform leaves in callus induction? In table 5 Why did Treatment 9 (18% induction) fail? In table 8 Why did NAA activated carbon? Discuss charcoal’s role in adsorbing inhibitors.

Response: The leaf explants of A. lancea exhibit significantly lower callus induction efficiency compared to petiole explants, primarily due to high cellular differentiation‌, accumulation of physiological barrier compounds‌, and structural/functional constraints‌. 1.5 mg/L 2,4-D may not be sufficient to initiate cell dedifferentiation, resulting in a low induction rate. The questions raised by the reviewer have been discussed in the original text.

13. Line 239: augments callus proliferation should be changed to enhances callus proliferation.

Response: Corrected.

14. Line 367: ARF7/19 should be changed to ARF7/19-mediated auxin signaling.

Explicitly state how this study advances beyond prior lancea tissue culture reports In conclusion, States "95 days" but doesn’t clarify if this is from seed to rooted plantlet or metabolite harvest.

Response: Corrected. The 95 days period in the conclusion refers to the period from the establishment of sterile lines to the rooting and tissue culture seedlings reaching the stage of seedling refinement and transplantation. On the basis of previous research, this study constructed tissue culture seedlings of A. lancea and induced root cultures such as adventitious roots and hairy roots to systematically establish a technical system for the production of active substances in A. lancea. This is also where our research leads in tissue culture compared to previous studies.

 

Reviewer #3:

Comments to the Author

Thank you for submitting your article for review. The article presents interesting results,

but I believe it requires minor editing before publication.

Below are my specific suggestions:

L 18, 19, 21: Please replace "+" with "and"

L 91: Please briefly describe how the seedlings were obtained - were all explants (seeds,

terminal buds, axillary buds) disinfected in the same way. The terms “buds tips/apical

buds” are more appropriate than “terminal buds”.

L 114 - Please describe what other parameters (color, texture ...) were monitored, besides callus induction.

L126: Please describe what exactly "living callus tissue" means?

L 159 - Missing spaces between words.

L165: Do you have data on the concentration of the bacteria?

L180: Besides morphological analysis, was any other analysis used to confirm bacterial

transformation?

L199: Please, add the missing literature source number.

L288: How many new adventitious buds obtained from each explant?

Authorship Contribution: Please, revise this section according to the journal's requirements.

References: It is impressive that nearly 90% of the cited literature is from the last 5 years.

Please format the refernces according to the journal's requirements.

Typographical errors: There are a number of technical errors in the text that need to be

corrected.

Please, ensure that all cited literature throughout the text is correctly numbered.

* The authors have to revise the manuscript to reduce the amount of wording duplication.

 

1. L 18, 19, 21: Please replace "+" with "and”.

Response: Corrected.

2. L 91: Please briefly describe how the seedlings were obtained - were all explants (seeds, terminal buds, axillary buds) disinfected in the same way. The terms “buds tips/apical buds” are more appropriate than “terminal buds”.

Response: Corrected. The explants of A. lancea are collected from wild plants, while different explants are taken from various organs of A. lancea plants cultured in greenhouses.

3. L 114 - Please describe what other parameters (color, texture ...) were monitored, besides callus induction.

Response: Added. The morphological image representing the quality of callus tissue has been added.

4. L126: Please describe what exactly "living callus tissue" means?

Response: This may indicate the quality of callus tissue

5. L 159 - Missing spaces between words.

Response: Corrected.

6. L165: Do you have data on the concentration of the bacteria?

Response: Corrected. In this study, only the optimal bacterial solution concentration was selected, with an OD₆₀₀ value between approximately 0.6-0.8.

7. L180: Besides morphological analysis, was any other analysis used to confirm bacterial transformation?

Response: If the reviewer is referring to hairy roots of A. lancea, then we still have PCR molecular identification results. However, if it is worth mentioning adventitious roots, then according to literature research, its morphological identification meets the requirements.

This fragment was the same size as a fragment amplified from the pRiA4 plasmid of strain C58C1. We did not observe amplification of this fragment from genomic DNA isolated from NR. These results demonstrated that that the Ri T-DNA region of C58C1 was integrated into the genome of the putative HR from A. lancea, and they confirmed that the roots we obtained were HR.

Figure. Representative PCR analysis for detection of the rolB gene in normal root (NR) and hairy root (HR) of A. rhizogenes-infected A. lancea. Genomic DNA isolated from NR (lane 1) or HR (lane 2) or the pRiA4 plasmid of C58C1 (lane 3) was amplified with Ri-specific primers. Lane M is a 250 bp molecular weight marker. The predicted size of the PCR fragment that positively identifies transgenic HR (250 bp) is indicated by the arrow.

8. L199: Please, add the missing literature source number.

Response: Already added.

9. L288: How many new adventitious buds obtained from each explant?

Response: Generally, the number of newly emerged adventitious buds is between 3-5.

10. Authorship Contribution: Please, revise this section according to the journal's requirements. References: It is impressive that nearly 90% of the cited literature is from the last 5 years. Please format the refernces according to the journal's requirements. Typographical errors: There are a number of technical errors in the text that need to be corrected. Please, ensure that all cited literature throughout the text is correctly numbered. * The authors have to revise the manuscript to reduce the amount of wording duplication.

Response: The above reviewers have made all the necessary modifications to the article format and references as required.

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

The overall content, data, and presentation of the manuscript are good. However, after careful evaluation, the authors need to address the following comments.

Line 2-3: In vitro plant regeneration and bioactive metabolite production of endangered medicinal plant Atractylodes lancea should be changed to In vitro plant regeneration and bioactive metabolite production of endangered medicinal plant Atractylodes lancea (Thunb.) DC.

Line 11-12: The rhizome of Atractylodes lancea is a traditional Chinese medicine used extensively owing to its antimicrobial properties should be changed to The rhizome of Atractylodes lancea (Thunb.) DC. is a traditional Chinese medicine used extensively owing to its antimicrobial.

In the introduction, you should start with the importance of medicinal plants. 

What specific limitations exist in current lanceatissue culture protocols?

Line 38:hike in market demand should be changed to increase in market demand.

Line 56-57:Since its historical development should be changed to Since its inception.

Line 77:innovate new germplasms should be changed to develop new germplasms.

Light source LED? Or Fluorescent?

How did you control humidity? Was it through sealed containers or vented lids?

Add morphological criteria for callus quality 
Explain why MS, ½MS, ¼MSwere tested 

Why transfer to basic medium after 5–7 days? Hormone withdrawal effect?

Why you used ethanol for calli vs. hexane for roots?

Ensure all figures/tables are referenced in order
Why did petioles outperform leaves in callus induction?

In table 5 Why did Treatment 9 (18% induction) fail?

In table 8 Why did NAA activated carbon? Discuss charcoal’s role in adsorbing inhibitors.

Line 239: augments callus proliferation should be changed to enhances callus proliferation.

Line 367: ARF7/19 should be changed to ARF7/19-mediated auxin signalling.

Explicitly state how this study advances beyond prior lancea tissue culture reports

In conclusion, States "95 days" but doesn’t clarify if this is from seed to rooted plantlet or metabolite harvest.

Author Response

Dear Editors,

 

We extend our gratitude to you and the reviewers for the thorough evaluation of our manuscript. We highly value the insightful comments provided, which have significantly contributed to the refinement of our work. We are pleased to resubmit our revised manuscript, titled " In vitro plant regeneration and bioactive metabolite production of endangered medicinal plant Atractylodes lancea" (ID: horticulturae-3656173), for your consideration for publication in Horticulturae.

 

In response to the reviewers' comments, we have diligently revised our manuscript, addressing their concerns comprehensively. Our point-by-point response outlines the specific modifications made to enhance the clarity and scientific merit of our study.

 

We trust that these revisions have significantly improved the overall quality of our manuscript, aligning it with the standards set forth by Medicinal Plant Biology. We remain open to addressing any further concerns and appreciate the opportunity to contribute our research to your esteemed journal.

 

Sincerely,

1. Zhang and S. Wang

 

point-by-point response

 

Reviewer #1

Comments to the Author

Abstract

• Indicate the document or Association that mentions the plant under endangered

status.

• What is the reason to put the phrase “Efficient plant regeneration and bioactive

metabolite production” in parentheses?

Introduction

• Please, indicate in this section too, the document or Association that mentions the

plant under endangered status.

• What are the references that indicate the decline of A. lancea at the study site?
• The word "bulbus" should be written in lowercase (line 65).

Materials and methods

• Remove the parenthesis (Line 90).
• In the case of the leaves, were they placed on their adaxial or abaxial sides? Please

indicate (Line 107).

• Indicate why you chose to work with these concentrations of exogenous hormones.
• Why did you use activated carbon? Indicate in the introduction or discussion section.
• Subheading 2.7 is misspelled. Please correct it. (Line 159)
• The organism A. rhizogenic does not appear earlier in the manuscript. Please provide the full name of the organism (Line 161).
• Please indicate in the methodology with which instrument you measured the optical

density (Line 165).

• Indicate the amount of material used from each explant (embryogenic calli, adventitious roots, and hairy roots) to obtain the essential oil (Line 202).

Results and Discussion

• Table 5, 7 and 8, Please use just one significance level. I suggest p<0.05. And

indicate in the notes of the table the Test utilized.

• Why the seedlings planted in garden soil exhibited weak growth? Please discuss.
• Figure 1 has several photographs. However, it only mentions that it represents plant

regeneration. Please explain in the text what is observed in each photograph (A-I).

• It is necessary to add measuring bars to the photographs in Figure 1, to know the

dimensions of the plants at their different stages. Please added.

• Please Add a discussion clarifying why leaf tissue and petioles were not good

explants for developing callus on A. lancea?

• In Figure 3, the meaning of the colored circles accompanying each bar in each

essential oil is unclear. Please clarify.

• The quality of Figure 4 is low. Please provide a higher quality image.

Conclusions

Could the proposed strategy work for other plants of other genera?

 

Abstract

1. Indicate the document or Association that mentions the plant under endangered status.

Response: Thank you for your positive comments. Atractylodes lancea is listed as a rare and protected plant at the provincial level in Jiangsu Province, and it is also included in the list of four endangered medicinal plants under key protection in Jiangsu Province. The relevant supporting documents are as follows: Jiangsu Provincial List of Key Protected Wild Plants (First Batch), National List of Protected New Plant Varieties (Forestry and Grassland Section, Eighth Batch).

 

 

2. What is the reason to put the phrase “Efficient plant regeneration and bioactive metabolite production” in parentheses?

Response: Because this is a key technical means that this article wants to elaborate on, namely "Efficient plant regeneration and bioactive metabolite production", it is highlighted in double quotation marks. The main function is to emphasize and highlight.

Introduction

1. Please, indicate in this section too, the document or Association that mentions the plant under endangered status.

Response: Corrected.

2. What are the references that indicate the decline of lancea at the study site?

Response: Corrected. Added [5-7], 3 references.

3. The word "bulbus" should be written in lowercase (line 65).

Response: Corrected.

Materials and methods

1. Remove the parenthesis (Line 90).

Response: Removed.

2. In the case of the leaves, were they placed on their adaxial or abaxial sides? Please indicate (Line 107).

Response: It has been marked as the back of the blade

3. Indicate why you chose to work with these concentrations of exogenous hormones.

Response: The concentration range of these exogenous hormones was determined based on our preliminary experimental results. In addition, according to the concentration range of hormones in plant tissue culture, it is shown that 0.1-2.0 mg/L 6-BA promotes shoot proliferation, and the proliferation rate increases with increasing concentration; 6-BA 2.0 mg/L+NAA 0.2~0.5 mg/L is beneficial for inducing adventitious buds in tissue culture.

4. Why did you use activated carbon? Indicate in the introduction or discussion section.

Response: The reason for adding activated carbon has been discussed.

5. Subheading 2.7 is misspelled. Please correct it. (Line 159).

Response: Corrected.

6. The organism A. rhizogenic does not appear earlier in the manuscript. Please provide the full name of the organism (Line 161).

Response: Corrected.

7. Please indicate in the methodology with which instrument you measured the optical density (Line 165).

Response: Already supplemented.

8. Indicate the amount of material used from each explant (embryogenic calli, adventitious roots, and hairy roots) to obtain the essential oil (Line 202).

Response: Corrected.

Results and Discussion

1. Table 5, 7 and 8, Please use just one significance level. I suggest p<0.05. And

indicate in the notes of the table the Test utilized.

Response: Due to significant differences in data, we used two testing methods, p<0.05 and p<0.01, and standardized them in the original table. Please be informed that the data analysis has been indicated in the Materials and Methods section.

2. Why the seedlings planted in garden soil exhibited weak growth? Please discuss.

Response: Improved.

3. Figure 1 has several photographs. However, it only mentions that it represents plant regeneration. Please explain in the text what is observed in each photograph (A-I).

Response: Improved.

4. It is necessary to add measuring bars to the photographs in Figure 1, to know the dimensions of the plants at their different stages. Please added.

Response: Already added.

5. Please Add a discussion clarifying why leaf tissue and petioles were not good

explants for developing callus on A. lancea?

Response: Already added.

6. In Figure 3, the meaning of the colored circles accompanying each bar in each

essential oil is unclear. Please clarify.

Response: Corrected.

7. The quality of Figure 4 is low. Please provide a higher quality image.

Response: Improved.

Conclusions

1. Could the proposed strategy work for other plants of other genera?

Response: The strategy we proposed is optimal for the in vitro regeneration and production of active substances in Atractylodes lancea, which has reference value for other plant species. However, appropriate technical parameter adjustments still need to be made according to different plant species.

Reviewer 1's annotations on the original PDF have been fully revised.

Reviewer #2:

Comments to the Author

The overall content, data, and presentation of the manuscript are good. However, after

careful evaluation, the authors need to address the following comments.

 

Line 2-3: In vitro plant regeneration and bioactive metabolite production of endangered

medicinal plant Atractylodes lancea should be changed to In vitro plant regeneration and bioactive metabolite production of endangered medicinal plant Atractylodes lancea

(Thunb.) DC.

Line 11-12: The rhizome of Atractylodes lancea is a traditional Chinese medicine used

extensively owing to its antimicrobial properties should be changed to the rhizome of

Atractylodes lancea (Thunb.) DC. is a traditional Chinese medicine used extensively owing to its antimicrobial.

In the introduction, you should start with the importance of medicinal plants. 

What specific limitations exist in current lanceatissue culture protocols?

Line 38: hike in market demand should be changed to increase in market demand.

Line 56-57: Since its historical development should be changed to Since its inception.

Line 77: innovate new germplasms should be changed to develop new germplasms.

Light source LED? Or Fluorescent?

How did you control humidity? Was it through sealed containers or vented lids?

Add morphological criteria for callus quality 

Explain why MS, ½MS, ¼MS were tested 

Why transfer to basic medium after 5–7 days? Hormone withdrawal effect?

Why you used ethanol for calli vs. hexane for roots?

Ensure all figures/tables are referenced in order

Why did petioles outperform leaves in callus induction?

In table 5 Why did Treatment 9 (18% induction) fail?

In table 8 Why did NAA activated carbon? Discuss charcoal’s role in adsorbing inhibitors.

Line 239: augments callus proliferation should be changed to enhances callus proliferation.

Line 367: ARF7/19 should be changed to ARF7/19-mediated auxin signaling.

Explicitly state how this study advances beyond prior lancea tissue culture reports In conclusion, States "95 days" but doesn’t clarify if this is from seed to rooted plantlet or metabolite harvest.

 

1. Line 2-3: In vitro plant regeneration and bioactive metabolite production of endangered medicinal plant Atractylodes lancea should be changed to In vitro plant regeneration and bioactive metabolite production of endangered medicinal plant Atractylodes lancea (Thunb.) DC.

Response: Corrected.

2. Line 11-12: The rhizome of Atractylodes lancea is a traditional Chinese medicine used extensively owing to its antimicrobial properties should be changed to the rhizome of Atractylodes lancea (Thunb.) DC. is a traditional Chinese medicine used extensively owing to its antimicrobial.

Response: Corrected.

3. In the introduction, you should start with the importance of medicinal plants.

What specific limitations exist in current A. lancea tissue culture protocols?

Response: Corrected.

4. Line 38: hike in market demand should be changed to increase in market demand.

Response: Already supplemented

5. Line 56-57: Since its historical development should be changed to Since its inception.

Response: Corrected.

6. Line 77: innovate new germplasms should be changed to develop new germplasms. Light source LED? Or Fluorescent?

Response: Corrected. The light source is LED.

7. How did you control humidity? Was it through sealed containers or vented lids?

Add morphological criteria for callus quality. 

Response: Corrected. In this study, the control of humidity in plant tissue culture rooms was achieved through the use of ventilation systems and humidifiers. The humidity of tissue culture seedlings is determined by using tissue culture bottles with breathable membranes. We have added morphological criteria for callus quality.

 

Figure Morphological criteria for callus quality

8. Explain why MS, ½MS, ¼MS were tested.

Response: The reason why MS, ½MS, ¼MS were used as the basic culture media for inducing callus differentiation in this study is to promote the differentiation of A. lancea callus into adventitious buds by reducing inorganic salt concentration, optimizing hormone and nutrient balance, and reducing metabolic burden. Research has shown that high salt medium may accelerate callus proliferation but inhibit differentiation. 1/2MS helps cells to turn towards morphogenesis, and 1/2MS medium can provide a more suitable microenvironment for callus differentiation by regulating salt concentration.

9. Why transfer to basic medium after 5–7 days? Hormone withdrawal effect?

Response: The main reasons for using auxin containing culture medium for 5-7 days before transferring to MS medium during rooting and tissue culture seedling cultivation are as follows: inducing root primordium formation and balancing nutrient and hormone requirements. This strategy optimizes rooting efficiency by regulating hormones and nutrients in stages.

10. Why you used ethanol for calli vs. hexane for roots?

Response: Ethanol (polar solvent) can effectively dissolve polar metabolites (such as phenols, flavonoids, glycosides, etc.) in callus tissue, which accumulate in large quantities during the dedifferentiation process of callus tissue. N-hexane (non-polar solvent) has a higher dissolution efficiency for lipophilic substances such as terpenes, waxes, coenzyme Q10, etc., and the content of such substances is more abundant in the roots.

11. Ensure all figures/tables are referenced in order.

Response: Corrected.

12. Why did petioles outperform leaves in callus induction? In table 5 Why did Treatment 9 (18% induction) fail? In table 8 Why did NAA activated carbon? Discuss charcoal’s role in adsorbing inhibitors.

Response: The leaf explants of A. lancea exhibit significantly lower callus induction efficiency compared to petiole explants, primarily due to high cellular differentiation‌, accumulation of physiological barrier compounds‌, and structural/functional constraints‌. 1.5 mg/L 2,4-D may not be sufficient to initiate cell dedifferentiation, resulting in a low induction rate. The questions raised by the reviewer have been discussed in the original text.

13. Line 239: augments callus proliferation should be changed to enhances callus proliferation.

Response: Corrected.

14. Line 367: ARF7/19 should be changed to ARF7/19-mediated auxin signaling.

Explicitly state how this study advances beyond prior lancea tissue culture reports In conclusion, States "95 days" but doesn’t clarify if this is from seed to rooted plantlet or metabolite harvest.

Response: Corrected. The 95 days period in the conclusion refers to the period from the establishment of sterile lines to the rooting and tissue culture seedlings reaching the stage of seedling refinement and transplantation. On the basis of previous research, this study constructed tissue culture seedlings of A. lancea and induced root cultures such as adventitious roots and hairy roots to systematically establish a technical system for the production of active substances in A. lancea. This is also where our research leads in tissue culture compared to previous studies.

 

Reviewer #3:

Comments to the Author

Thank you for submitting your article for review. The article presents interesting results,

but I believe it requires minor editing before publication.

Below are my specific suggestions:

L 18, 19, 21: Please replace "+" with "and"

L 91: Please briefly describe how the seedlings were obtained - were all explants (seeds,

terminal buds, axillary buds) disinfected in the same way. The terms “buds tips/apical

buds” are more appropriate than “terminal buds”.

L 114 - Please describe what other parameters (color, texture ...) were monitored, besides callus induction.

L126: Please describe what exactly "living callus tissue" means?

L 159 - Missing spaces between words.

L165: Do you have data on the concentration of the bacteria?

L180: Besides morphological analysis, was any other analysis used to confirm bacterial

transformation?

L199: Please, add the missing literature source number.

L288: How many new adventitious buds obtained from each explant?

Authorship Contribution: Please, revise this section according to the journal's requirements.

References: It is impressive that nearly 90% of the cited literature is from the last 5 years.

Please format the refernces according to the journal's requirements.

Typographical errors: There are a number of technical errors in the text that need to be

corrected.

Please, ensure that all cited literature throughout the text is correctly numbered.

* The authors have to revise the manuscript to reduce the amount of wording duplication.

 

1. L 18, 19, 21: Please replace "+" with "and”.

Response: Corrected.

2. L 91: Please briefly describe how the seedlings were obtained - were all explants (seeds, terminal buds, axillary buds) disinfected in the same way. The terms “buds tips/apical buds” are more appropriate than “terminal buds”.

Response: Corrected. The explants of A. lancea are collected from wild plants, while different explants are taken from various organs of A. lancea plants cultured in greenhouses.

3. L 114 - Please describe what other parameters (color, texture ...) were monitored, besides callus induction.

Response: Added. The morphological image representing the quality of callus tissue has been added.

4. L126: Please describe what exactly "living callus tissue" means?

Response: This may indicate the quality of callus tissue

5. L 159 - Missing spaces between words.

Response: Corrected.

6. L165: Do you have data on the concentration of the bacteria?

Response: Corrected. In this study, only the optimal bacterial solution concentration was selected, with an OD₆₀₀ value between approximately 0.6-0.8.

7. L180: Besides morphological analysis, was any other analysis used to confirm bacterial transformation?

Response: If the reviewer is referring to hairy roots of A. lancea, then we still have PCR molecular identification results. However, if it is worth mentioning adventitious roots, then according to literature research, its morphological identification meets the requirements.

This fragment was the same size as a fragment amplified from the pRiA4 plasmid of strain C58C1. We did not observe amplification of this fragment from genomic DNA isolated from NR. These results demonstrated that that the Ri T-DNA region of C58C1 was integrated into the genome of the putative HR from A. lancea, and they confirmed that the roots we obtained were HR.

Figure. Representative PCR analysis for detection of the rolB gene in normal root (NR) and hairy root (HR) of A. rhizogenes-infected A. lancea. Genomic DNA isolated from NR (lane 1) or HR (lane 2) or the pRiA4 plasmid of C58C1 (lane 3) was amplified with Ri-specific primers. Lane M is a 250 bp molecular weight marker. The predicted size of the PCR fragment that positively identifies transgenic HR (250 bp) is indicated by the arrow.

8. L199: Please, add the missing literature source number.

Response: Already added.

9. L288: How many new adventitious buds obtained from each explant?

Response: Generally, the number of newly emerged adventitious buds is between 3-5.

10. Authorship Contribution: Please, revise this section according to the journal's requirements. References: It is impressive that nearly 90% of the cited literature is from the last 5 years. Please format the refernces according to the journal's requirements. Typographical errors: There are a number of technical errors in the text that need to be corrected. Please, ensure that all cited literature throughout the text is correctly numbered. * The authors have to revise the manuscript to reduce the amount of wording duplication.

Response: The above reviewers have made all the necessary modifications to the article format and references as required.

Author Response File: Author Response.pdf

Reviewer 3 Report

Comments and Suggestions for Authors

Thank you for submitting your article for review. The article presents interesting results, but I believe it requires minor editing before publication.

Below are my specific suggestions:

L 18, 19, 21: Please replace "+" with "and"

L 91: Please briefly describe how the seedlings were obtained - were all explants (seeds, terminal buds, axillary buds) disinfected in the same way. The terms “buds tips/apical buds” are more appropriate than “terminal buds”.

L 114 - Please describe what other parameters (color, texture ...) were monitored, besides callus induction.

L126: Please describe what exactly "living callus tissue" means?

L 159 - Missing spaces between words.

L165: Do you have data on the concentration of the bacteria?

L180: Besides morphological analysis, was any other analysis used to confirm bacterial transformation?

L199: Please, add the missing literature source number.

L288: How many new adventitious buds obtained from each explant?

Authorship Contribution: Please, revise this section according to the journal's requirements.

References: It is impressive that nearly 90% of the cited literature is from the last 5 years. Please format the refernces according to the journal's requirements.

Typographical errors: There are a number of technical errors in the text that need to be corrected.

Please, ensure that all cited literature throughout the text are correctly numbered.

* The authors have to revise the manuscript to reduce the amount of wording duplication.

Author Response

Dear Editors,

 

We extend our gratitude to you and the reviewers for the thorough evaluation of our manuscript. We highly value the insightful comments provided, which have significantly contributed to the refinement of our work. We are pleased to resubmit our revised manuscript, titled " In vitro plant regeneration and bioactive metabolite production of endangered medicinal plant Atractylodes lancea" (ID: horticulturae-3656173), for your consideration for publication in Horticulturae.

 

In response to the reviewers' comments, we have diligently revised our manuscript, addressing their concerns comprehensively. Our point-by-point response outlines the specific modifications made to enhance the clarity and scientific merit of our study.

 

We trust that these revisions have significantly improved the overall quality of our manuscript, aligning it with the standards set forth by Medicinal Plant Biology. We remain open to addressing any further concerns and appreciate the opportunity to contribute our research to your esteemed journal.

 

Sincerely,

1. Zhang and S. Wang

 

point-by-point response

 

Reviewer #1

Comments to the Author

Abstract

• Indicate the document or Association that mentions the plant under endangered

status.

• What is the reason to put the phrase “Efficient plant regeneration and bioactive

metabolite production” in parentheses?

Introduction

• Please, indicate in this section too, the document or Association that mentions the

plant under endangered status.

• What are the references that indicate the decline of A. lancea at the study site?
• The word "bulbus" should be written in lowercase (line 65).

Materials and methods

• Remove the parenthesis (Line 90).
• In the case of the leaves, were they placed on their adaxial or abaxial sides? Please

indicate (Line 107).

• Indicate why you chose to work with these concentrations of exogenous hormones.
• Why did you use activated carbon? Indicate in the introduction or discussion section.
• Subheading 2.7 is misspelled. Please correct it. (Line 159)
• The organism A. rhizogenic does not appear earlier in the manuscript. Please provide the full name of the organism (Line 161).
• Please indicate in the methodology with which instrument you measured the optical

density (Line 165).

• Indicate the amount of material used from each explant (embryogenic calli, adventitious roots, and hairy roots) to obtain the essential oil (Line 202).

Results and Discussion

• Table 5, 7 and 8, Please use just one significance level. I suggest p<0.05. And

indicate in the notes of the table the Test utilized.

• Why the seedlings planted in garden soil exhibited weak growth? Please discuss.
• Figure 1 has several photographs. However, it only mentions that it represents plant

regeneration. Please explain in the text what is observed in each photograph (A-I).

• It is necessary to add measuring bars to the photographs in Figure 1, to know the

dimensions of the plants at their different stages. Please added.

• Please Add a discussion clarifying why leaf tissue and petioles were not good

explants for developing callus on A. lancea?

• In Figure 3, the meaning of the colored circles accompanying each bar in each

essential oil is unclear. Please clarify.

• The quality of Figure 4 is low. Please provide a higher quality image.

Conclusions

Could the proposed strategy work for other plants of other genera?

 

Abstract

1. Indicate the document or Association that mentions the plant under endangered status.

Response: Thank you for your positive comments. Atractylodes lancea is listed as a rare and protected plant at the provincial level in Jiangsu Province, and it is also included in the list of four endangered medicinal plants under key protection in Jiangsu Province. The relevant supporting documents are as follows: Jiangsu Provincial List of Key Protected Wild Plants (First Batch), National List of Protected New Plant Varieties (Forestry and Grassland Section, Eighth Batch).

 

 

2. What is the reason to put the phrase “Efficient plant regeneration and bioactive metabolite production” in parentheses?

Response: Because this is a key technical means that this article wants to elaborate on, namely "Efficient plant regeneration and bioactive metabolite production", it is highlighted in double quotation marks. The main function is to emphasize and highlight.

Introduction

1. Please, indicate in this section too, the document or Association that mentions the plant under endangered status.

Response: Corrected.

2. What are the references that indicate the decline of lancea at the study site?

Response: Corrected. Added [5-7], 3 references.

3. The word "bulbus" should be written in lowercase (line 65).

Response: Corrected.

Materials and methods

1. Remove the parenthesis (Line 90).

Response: Removed.

2. In the case of the leaves, were they placed on their adaxial or abaxial sides? Please indicate (Line 107).

Response: It has been marked as the back of the blade

3. Indicate why you chose to work with these concentrations of exogenous hormones.

Response: The concentration range of these exogenous hormones was determined based on our preliminary experimental results. In addition, according to the concentration range of hormones in plant tissue culture, it is shown that 0.1-2.0 mg/L 6-BA promotes shoot proliferation, and the proliferation rate increases with increasing concentration; 6-BA 2.0 mg/L+NAA 0.2~0.5 mg/L is beneficial for inducing adventitious buds in tissue culture.

4. Why did you use activated carbon? Indicate in the introduction or discussion section.

Response: The reason for adding activated carbon has been discussed.

5. Subheading 2.7 is misspelled. Please correct it. (Line 159).

Response: Corrected.

6. The organism A. rhizogenic does not appear earlier in the manuscript. Please provide the full name of the organism (Line 161).

Response: Corrected.

7. Please indicate in the methodology with which instrument you measured the optical density (Line 165).

Response: Already supplemented.

8. Indicate the amount of material used from each explant (embryogenic calli, adventitious roots, and hairy roots) to obtain the essential oil (Line 202).

Response: Corrected.

Results and Discussion

1. Table 5, 7 and 8, Please use just one significance level. I suggest p<0.05. And

indicate in the notes of the table the Test utilized.

Response: Due to significant differences in data, we used two testing methods, p<0.05 and p<0.01, and standardized them in the original table. Please be informed that the data analysis has been indicated in the Materials and Methods section.

2. Why the seedlings planted in garden soil exhibited weak growth? Please discuss.

Response: Improved.

3. Figure 1 has several photographs. However, it only mentions that it represents plant regeneration. Please explain in the text what is observed in each photograph (A-I).

Response: Improved.

4. It is necessary to add measuring bars to the photographs in Figure 1, to know the dimensions of the plants at their different stages. Please added.

Response: Already added.

5. Please Add a discussion clarifying why leaf tissue and petioles were not good

explants for developing callus on A. lancea?

Response: Already added.

6. In Figure 3, the meaning of the colored circles accompanying each bar in each

essential oil is unclear. Please clarify.

Response: Corrected.

7. The quality of Figure 4 is low. Please provide a higher quality image.

Response: Improved.

Conclusions

1. Could the proposed strategy work for other plants of other genera?

Response: The strategy we proposed is optimal for the in vitro regeneration and production of active substances in Atractylodes lancea, which has reference value for other plant species. However, appropriate technical parameter adjustments still need to be made according to different plant species.

Reviewer 1's annotations on the original PDF have been fully revised.

Reviewer #2:

Comments to the Author

The overall content, data, and presentation of the manuscript are good. However, after

careful evaluation, the authors need to address the following comments.

 

Line 2-3: In vitro plant regeneration and bioactive metabolite production of endangered

medicinal plant Atractylodes lancea should be changed to In vitro plant regeneration and bioactive metabolite production of endangered medicinal plant Atractylodes lancea

(Thunb.) DC.

Line 11-12: The rhizome of Atractylodes lancea is a traditional Chinese medicine used

extensively owing to its antimicrobial properties should be changed to the rhizome of

Atractylodes lancea (Thunb.) DC. is a traditional Chinese medicine used extensively owing to its antimicrobial.

In the introduction, you should start with the importance of medicinal plants. 

What specific limitations exist in current lanceatissue culture protocols?

Line 38: hike in market demand should be changed to increase in market demand.

Line 56-57: Since its historical development should be changed to Since its inception.

Line 77: innovate new germplasms should be changed to develop new germplasms.

Light source LED? Or Fluorescent?

How did you control humidity? Was it through sealed containers or vented lids?

Add morphological criteria for callus quality 

Explain why MS, ½MS, ¼MS were tested 

Why transfer to basic medium after 5–7 days? Hormone withdrawal effect?

Why you used ethanol for calli vs. hexane for roots?

Ensure all figures/tables are referenced in order

Why did petioles outperform leaves in callus induction?

In table 5 Why did Treatment 9 (18% induction) fail?

In table 8 Why did NAA activated carbon? Discuss charcoal’s role in adsorbing inhibitors.

Line 239: augments callus proliferation should be changed to enhances callus proliferation.

Line 367: ARF7/19 should be changed to ARF7/19-mediated auxin signaling.

Explicitly state how this study advances beyond prior lancea tissue culture reports In conclusion, States "95 days" but doesn’t clarify if this is from seed to rooted plantlet or metabolite harvest.

 

1. Line 2-3: In vitro plant regeneration and bioactive metabolite production of endangered medicinal plant Atractylodes lancea should be changed to In vitro plant regeneration and bioactive metabolite production of endangered medicinal plant Atractylodes lancea (Thunb.) DC.

Response: Corrected.

2. Line 11-12: The rhizome of Atractylodes lancea is a traditional Chinese medicine used extensively owing to its antimicrobial properties should be changed to the rhizome of Atractylodes lancea (Thunb.) DC. is a traditional Chinese medicine used extensively owing to its antimicrobial.

Response: Corrected.

3. In the introduction, you should start with the importance of medicinal plants.

What specific limitations exist in current A. lancea tissue culture protocols?

Response: Corrected.

4. Line 38: hike in market demand should be changed to increase in market demand.

Response: Already supplemented

5. Line 56-57: Since its historical development should be changed to Since its inception.

Response: Corrected.

6. Line 77: innovate new germplasms should be changed to develop new germplasms. Light source LED? Or Fluorescent?

Response: Corrected. The light source is LED.

7. How did you control humidity? Was it through sealed containers or vented lids?

Add morphological criteria for callus quality. 

Response: Corrected. In this study, the control of humidity in plant tissue culture rooms was achieved through the use of ventilation systems and humidifiers. The humidity of tissue culture seedlings is determined by using tissue culture bottles with breathable membranes. We have added morphological criteria for callus quality.

 

Figure Morphological criteria for callus quality

8. Explain why MS, ½MS, ¼MS were tested.

Response: The reason why MS, ½MS, ¼MS were used as the basic culture media for inducing callus differentiation in this study is to promote the differentiation of A. lancea callus into adventitious buds by reducing inorganic salt concentration, optimizing hormone and nutrient balance, and reducing metabolic burden. Research has shown that high salt medium may accelerate callus proliferation but inhibit differentiation. 1/2MS helps cells to turn towards morphogenesis, and 1/2MS medium can provide a more suitable microenvironment for callus differentiation by regulating salt concentration.

9. Why transfer to basic medium after 5–7 days? Hormone withdrawal effect?

Response: The main reasons for using auxin containing culture medium for 5-7 days before transferring to MS medium during rooting and tissue culture seedling cultivation are as follows: inducing root primordium formation and balancing nutrient and hormone requirements. This strategy optimizes rooting efficiency by regulating hormones and nutrients in stages.

10. Why you used ethanol for calli vs. hexane for roots?

Response: Ethanol (polar solvent) can effectively dissolve polar metabolites (such as phenols, flavonoids, glycosides, etc.) in callus tissue, which accumulate in large quantities during the dedifferentiation process of callus tissue. N-hexane (non-polar solvent) has a higher dissolution efficiency for lipophilic substances such as terpenes, waxes, coenzyme Q10, etc., and the content of such substances is more abundant in the roots.

11. Ensure all figures/tables are referenced in order.

Response: Corrected.

12. Why did petioles outperform leaves in callus induction? In table 5 Why did Treatment 9 (18% induction) fail? In table 8 Why did NAA activated carbon? Discuss charcoal’s role in adsorbing inhibitors.

Response: The leaf explants of A. lancea exhibit significantly lower callus induction efficiency compared to petiole explants, primarily due to high cellular differentiation‌, accumulation of physiological barrier compounds‌, and structural/functional constraints‌. 1.5 mg/L 2,4-D may not be sufficient to initiate cell dedifferentiation, resulting in a low induction rate. The questions raised by the reviewer have been discussed in the original text.

13. Line 239: augments callus proliferation should be changed to enhances callus proliferation.

Response: Corrected.

14. Line 367: ARF7/19 should be changed to ARF7/19-mediated auxin signaling.

Explicitly state how this study advances beyond prior lancea tissue culture reports In conclusion, States "95 days" but doesn’t clarify if this is from seed to rooted plantlet or metabolite harvest.

Response: Corrected. The 95 days period in the conclusion refers to the period from the establishment of sterile lines to the rooting and tissue culture seedlings reaching the stage of seedling refinement and transplantation. On the basis of previous research, this study constructed tissue culture seedlings of A. lancea and induced root cultures such as adventitious roots and hairy roots to systematically establish a technical system for the production of active substances in A. lancea. This is also where our research leads in tissue culture compared to previous studies.

 

Reviewer #3:

Comments to the Author

Thank you for submitting your article for review. The article presents interesting results,

but I believe it requires minor editing before publication.

Below are my specific suggestions:

L 18, 19, 21: Please replace "+" with "and"

L 91: Please briefly describe how the seedlings were obtained - were all explants (seeds,

terminal buds, axillary buds) disinfected in the same way. The terms “buds tips/apical

buds” are more appropriate than “terminal buds”.

L 114 - Please describe what other parameters (color, texture ...) were monitored, besides callus induction.

L126: Please describe what exactly "living callus tissue" means?

L 159 - Missing spaces between words.

L165: Do you have data on the concentration of the bacteria?

L180: Besides morphological analysis, was any other analysis used to confirm bacterial

transformation?

L199: Please, add the missing literature source number.

L288: How many new adventitious buds obtained from each explant?

Authorship Contribution: Please, revise this section according to the journal's requirements.

References: It is impressive that nearly 90% of the cited literature is from the last 5 years.

Please format the refernces according to the journal's requirements.

Typographical errors: There are a number of technical errors in the text that need to be

corrected.

Please, ensure that all cited literature throughout the text is correctly numbered.

* The authors have to revise the manuscript to reduce the amount of wording duplication.

 

1. L 18, 19, 21: Please replace "+" with "and”.

Response: Corrected.

2. L 91: Please briefly describe how the seedlings were obtained - were all explants (seeds, terminal buds, axillary buds) disinfected in the same way. The terms “buds tips/apical buds” are more appropriate than “terminal buds”.

Response: Corrected. The explants of A. lancea are collected from wild plants, while different explants are taken from various organs of A. lancea plants cultured in greenhouses.

3. L 114 - Please describe what other parameters (color, texture ...) were monitored, besides callus induction.

Response: Added. The morphological image representing the quality of callus tissue has been added.

4. L126: Please describe what exactly "living callus tissue" means?

Response: This may indicate the quality of callus tissue

5. L 159 - Missing spaces between words.

Response: Corrected.

6. L165: Do you have data on the concentration of the bacteria?

Response: Corrected. In this study, only the optimal bacterial solution concentration was selected, with an OD₆₀₀ value between approximately 0.6-0.8.

7. L180: Besides morphological analysis, was any other analysis used to confirm bacterial transformation?

Response: If the reviewer is referring to hairy roots of A. lancea, then we still have PCR molecular identification results. However, if it is worth mentioning adventitious roots, then according to literature research, its morphological identification meets the requirements.

This fragment was the same size as a fragment amplified from the pRiA4 plasmid of strain C58C1. We did not observe amplification of this fragment from genomic DNA isolated from NR. These results demonstrated that that the Ri T-DNA region of C58C1 was integrated into the genome of the putative HR from A. lancea, and they confirmed that the roots we obtained were HR.

Figure. Representative PCR analysis for detection of the rolB gene in normal root (NR) and hairy root (HR) of A. rhizogenes-infected A. lancea. Genomic DNA isolated from NR (lane 1) or HR (lane 2) or the pRiA4 plasmid of C58C1 (lane 3) was amplified with Ri-specific primers. Lane M is a 250 bp molecular weight marker. The predicted size of the PCR fragment that positively identifies transgenic HR (250 bp) is indicated by the arrow.

8. L199: Please, add the missing literature source number.

Response: Already added.

9. L288: How many new adventitious buds obtained from each explant?

Response: Generally, the number of newly emerged adventitious buds is between 3-5.

10. Authorship Contribution: Please, revise this section according to the journal's requirements. References: It is impressive that nearly 90% of the cited literature is from the last 5 years. Please format the refernces according to the journal's requirements. Typographical errors: There are a number of technical errors in the text that need to be corrected. Please, ensure that all cited literature throughout the text is correctly numbered. * The authors have to revise the manuscript to reduce the amount of wording duplication.

Response: The above reviewers have made all the necessary modifications to the article format and references as required.

Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

Accept in present form