Consecutive Pruning Enhances Leaf Flavonoids, Leaf Yield, and Cutting Rooting in Ginkgo biloba

Round 1

Reviewer 1 Report

Experience confirms existing views on the researched topic or extends them. The abstract does not contain any information about the results obtained and is too general. There are many inaccuracies in the methodology that need to be corrected. In the discussion, it is not known why the authors did not focus on the studied species (gingiko) in the context of reporting the results of other authors. This is a widely researched topic, especially regarding the rooting of cuttings. How can you compare a tomato with a perennial tree. No conclusions or future research directions. Many editorial errors pointed out in the text.

Comments for author File: Comments.pdf

The level of English is sufficient.

Author Response

Reply to Reviewer #1

Experience confirms existing views on the researched topic or extends them. The abstract does not contain any information about the results obtained and is too general. There are many inaccuracies in the methodology that need to be corrected. In the discussion, it is not known why the authors did not focus on the studied species (gingiko) in the context of reporting the results of other authors. This is a widely researched topic, especially regarding the rooting of cuttings. How can you compare a tomato with a perennial tree. No conclusions or future research directions. Many editorial errors pointed out in the text.

Reply: We thank you for reviewing our manuscript and providing many valuable comments and suggestions. The questions you raised were very helpful for our revisions. Over the past two weeks, we have worked hard to revise the original manuscript. In the revised manuscript, all issues and suggestions have been taken into consideration, with modifications highlighted in red. Below, we provide corresponding responses one by one.

1. The abstract does not provide any information regarding the results obtained, especially regarding the most significant differences between treatments, this form is unacceptable.

Reply: We apologize for this. We have already provided detailed results in the abstract, including information on the most significant differences between the various treatments.

2. by what percentage.

Reply: The number of branches increased by 150%, the leaf weight increased by 130%, and the flavonoid content increased by 40.6%. We have added these specific details in the revised manuscript.

3. by what percentage.

Reply: Leaf area increased by 22.3%, leaf lobule depth increased by 61.1%, and leaf weight increased by 130%. We have added these specific details in the revised manuscript.

4. do not repeat terms from the title.

Reply: We have revised the title, according to another reviewer's suggestion. Based on the new title, we have changed the keywords to “pruning intensity; cutting rooting; gene expression; flavonoid compounds; plant regeneration”.

5. provide the initials of the author of the Latin name of the species, this is a scientific publication.

Reply: As suggested, we have provided the initials of the author of the Latin name of the species (Ginkgo biloba L.).

6. provide information confirming this thesis, how important is the cultivation of this species in China, area of ​​cultivation, value in yuan.

Reply: Due to area of cultivation and value in yuan, there are no detailed and accurate statistical data, and it fluctuates every year, therefore we have cited three references (Zhang et al., 2016; Akaberi et al., 2023; Mao et al., 2023) to demonstrate the economic importance of this tree species.

7. 1-4

Reply: As suggested, due to the insertion of two new references at the beginning, we have changed [1,2,3,4] into [4-7].

8. name which ones.

Reply: The name of active compounds are flavonoids (e.g., kaempferol, quercetin, isorhamnetin) and terpenoids (e.g., ginkgolides, bilobalides). We have added these specific details in the revised manuscript.

9. remove not needed.

Reply: We have removed “(Vellas et al., 2012; Herrschaft et al., 2012)”.

10. what percentage of survival.

Reply: The percentage of survival is about 40%. We have added this in the revised manuscript.

11. The cuttings are not years old, only the mother plants from which they are taken, these are always the youngest sections of the shoots.

Reply: Age refers to the age of the scion mother plant, which is subject to age effects. The younger the scion mother plant, the stronger the rooting ability of its branches. We have revised this in the revised manuscript.

12. 13-15.

Reply: We have changed [13, 14, 15] into [15-17].

13. provide the English and Latin names in brackets with the author for all species.

Reply: As suggested, we have provided the English and Latin names in brackets with the author for all species.

14. 20-22

Reply: We have changed [20, 21, 22] into [22-24].

15. the English name and the Latin name with the author in brackets

Reply: We have added the English name and the Latin name with the author in brackets, yellowhorn (Xanthoceras sorbifolium Bunge).

16. wrong font size, author

Reply: As suggested, we have unified font size and added the author.

17. as above

Reply: As suggested, we have added the author.

18. can be used interchangeably with the English name

Reply: As suggested, we have consistently utilized the English name "ginkgo" for “G. biloba” interchangeably.

19. reverse order

Reply: As suggested, we have adjusted the order, shifting “[26, 5]” to “[8, 29]”.

20. what was the experimental setup, how many repetitions to reduce the influence of soil variability

Reply: The experimental setup initially performed topping, light pruning, and heavy pruning on 10 five-year-old ginkgo trees on February 5, 2022. The corresponding indicators for each treatment were then measured on May 1, 2022. Subsequently, the best pruning method was chosen and applied for a second pruning on May 5, 2022, with the corresponding indicators being measured on June 5, 2022. There are ten repetitions to reduce the impact of soil variability.

21. provide units of measurement.

Reply: We have added units of measurement (cm).

22. you can't write it here only in the results.

Reply: As suggested, we have deleted this sentence in the materials and methods section.

23. Lowercase

Reply: We have already changed Secondary to secondary.

24. combine this with previous information about the experimental setup.

Reply: Here is the information about leaf sampling methods, so we kept this information.

25. how it was downloaded randomly is not everything.

Reply: For each treatment, we chose three trees that are similar in growth, and selected ten leaves from the middle part of the branches of each tree that are essentially the same size.

26. how samples were taken, from how many trees, from what place in the trees.

Reply: We select three trees with similar growth from each treatment, and took 10 leaves from the middle part of the middle branch of each plant for measurement.

27. based on what parameter, better the length of the shoots.

Reply: The new shoot growth is based on the length of new branches grown in the current year.

28. the same beginning of the sentence repeated many times, change it.

Reply: As suggested, we have changed the “after” of the sentence.

29. how much percentage is difficult to read from the figure

Reply: The rooting rate of the branches increased by 12.5% after first pruning, and by 16.6% after the second pruning. We have added these specific details in the revised manuscript.

30. the results of rooting ginkgo stem cuttings obtained by other authors are available, please compare them

Reply: As suggested, we have already compared the results with those obtained by other authors on ginkgo stem cutting propagation.

31. see previous notes

Reply: We have added the English name and the Latin name with the author in brackets, Eucommia (Eucommia ulmoides Oliv.).

32. If possible, state by what percentage

Reply: Compared to light pruning, mild pruning increased branch and leaf biomass by 41.2%, while heavy pruning increased it by 61.2%. We have added these specific details in the revised manuscript.

33. provide what species were tested, e.g. apple tree, and what results were obtained, there are many studies on the rooting of ginkgo cuttings, so why do the authors give examples of tomatoes, a completely different group of plants

Reply: As suggested, we have provided the information of species and related results. We deleted the examples of tomatoes. The rooting rate of apple branches is 24.02% higher than that of mature branches; the highest rooting rate of African blackwood cuttings is 71.11%, while the rooting rate of mature cuttings is only 24.42%. We have added these specific details in the revised manuscript.

34. reduce the distance

Reply: We have reduced the distance.

35. English name

Reply: As suggested, we have used the English name "ginkgo" for “G. biloba” interchangeably.

36. by what percentage, by what value

Reply: The content of flavonoids in the leaves increased by 24%, while the content of terpenoids increased by 8%.

37. wrong size

Reply: After careful checking by us, the font size may appear incorrect due to display issues.

38. summarize the most important results at the end, and especially provide research proposals for the future

Reply: As suggested, we have added a conclusion as follows.

Pruning treatments resulted in a significant increase in branch number, leaf lobe depth, leaf area, leaf weight, and leaf flavonoids. Among the treatments, light pruning had the most positive effect on increasing branch number, leaf area, and leaf flavonoids. Secondary light pruning also led to an increase in branch number. Additionally, cuttings taken from secondary pruning showed a higher rate of rooting. Further research is needed to understand the molecular mechanisms behind these improved economic traits.

Reviewer 2 Report

The title does not entirely correspond with the study - study on flavonoids and leaf yield is not in the title - please revise it and consider a change.

Introduction is straight to the point - great quality. Citations are adequate.

Material and Methods - I would appreciate greater number of trees, at least double, ten for each variant is in my opinion below minimum for corresponding statistical analyses.

Graphical content is great, adds to the clarity and makes the manuscript easier to use for non-scientific readers.

I miss Conclusion, maybe you can name the last chapter Discussion and Conclusion, but it is not very suitable, as you describe a partial problem in the last part of Discussion (and quite widely). It would be desirable to point out the most important things in separate Conclusion.

I recommend the manuscript for minor revision, even though I have problem with the number of trees used.

On the other hand, the manuscript is well-crafted and clearly describes the experiment in all necessary detail with suitable graphical content.

Author Response

Reply to Reviewer #2

1. The title does not entirely correspond with the study - study on flavonoids and leaf yield is not in the title - please revise it and consider a change.

Reply: Thank you for pointing out this question, we have revised it to “Consecutive pruning enhances leaf flavonoids, leaf yield, and cutting rooting in Ginkgo biloba”.

      2. Introduction is straight to the point - great quality. Citations are adequate.

Reply: Thanks for your positive comments.

3. Material and Methods - I would appreciate greater number of trees, at least double, ten for each variant is in my opinion below minimum for corresponding statistical analyses

Reply: Thank you for pointing out this issue. In the first pruning, we pruned ten trees of each treatment, and since the second pruning was based on the first pruning, we selected five trees for each treatment.

4. Graphical content is great, adds to the clarity and makes the manuscript easier to use for non-scientific readers

Reply: Thanks for your positive comments.

5. I miss Conclusion, maybe you can name the last chapter Discussion and Conclusion, but it is not very suitable, as you describe a partial problem in the last part of Discussion (and quite widely). It would be desirable to point out the most important things in separate Conclusion.

Reply: Thanks for your suggestion. As suggested, we have added a separate Conclusion as follows.

Pruning treatments resulted in a significant increase in branch number, leaf lobe depth, leaf area, leaf weight, and leaf flavonoids. Among the treatments, light pruning had the most positive effect on increasing branch number, leaf area, and leaf flavonoids. Secondary light pruning also led to an increase in branch number. Additionally, cuttings taken from secondary pruning showed a higher rate of rooting. Further research is needed to understand the molecular mechanisms behind these improved economic traits.

6. I recommend the manuscript for minor revision, even though I have problem with the number of trees used

Reply: Thanks for your reviewing and suggestions. The number of trees used was explained in our reply to question 3.

Reviewer 3 Report

The author has presented about consecutive pruning enhances plant rejuvenation and promote the rooting of cuttings in Ginkgo biloba. However, there are some concerns.

In Introduction

This introduction should include information that is consistent with the analysis used in the experiment. In order to make the content in the introduction section more complete.

Page 2 line 71: “oil (Camellia oleifera) tea” should be “oil tea (Camellia oleifera)”

In Materials and methods

Page 3 2.2. Morphological Observation: The measurement method should be presented with illustrations and explanations for clearer understanding.

In Results

Page 5 Figure 1: The sequence of images should be presented the same throughout the manuscript. Ex. (A) Control plant  (B) Top pruning plant  (C) Light pruning plant (D) Heavy pruning plant

Page 8 Figure 4A: Images should be presented at the same scale. * 5 cm

Page 11 line 297: “rot development” should be “rot development”

Page 12 line 305: “The root development of cuttings after hormone treatments.” The hormone treatments should be explained more clearly.

In Discussion

The content should be arranged in order to be consistent with the content in the experimental results section for ease of understanding.

Discussion should include relevant research that explains why this experiment obtained the results as presented, rather than simply presenting the same results.

Some content should be moved to the introduction section. Such as the contents in page 12 lines 311-321, page 13 line 329-339, 347-349 and 351-355.

The review section still lacks content regarding to

-        Effects of various pruning methods on plant growth and leaf development

-        Changes of in morphology, water content, and flavonoids in leaves after pruning

The results of the experiment should be concluded in Conclusion section.

Comments for author File: Comments.pdf

Author Response

Please see the attachment.

Author Response File: Author Response.pdf

Reviewer 4 Report

The problem identified and addressed by the authors is highly useful for rapid multiplication of the selected medicinal plant, as there is huge demand for cultivation of such medicinal plants to avoid over exploitation of the wild or naturally grown plants.

The authors have used different terms, like first pruning, second pruning. pruning, multiple pruning, and primary and secondary pruning at different places, creating confusion for the readers; hence, it is recommended that they use the same terms, viz., primary and secondary pruning, throughout the manuscript.

In the results section, the authors clearly stated the significance of the treatments by percentage comparisons which is highly appreciable as it gives a clearer picture of the results to the readers.

In Figure 1, the sequence of pruned images A-B-C-D is not matching with the sequence of grown plant images E-F-G-H. Same sequence may be maintained for both the series for easy comparison and understanding.

Also, in Figure 1, the authors may please confirm that the standard error bars used in graphic images (I-J-K) are specific to the replication data of the respective treatments.

Lane 120: water content percentage formula needs to be multiplied by 100 to ensure the given data results in per cent.

Lane 176: The statistical design used for this experiment may be clearly stated in this section.

Lane 217: Figure 02. The caption title would be appropriate to have the term ‘first /primary pruning’ instead of general pruning word.

Lane 248: Figure 04. The caption title would be appropriate to have the term ‘secondary pruning’ instead of multiple pruning word.

Lane 276: Figure 06. The caption title would be appropriate to have the term ‘multiple pruning’ instead of different pruning word.

A brief summary of the study may be added after the discussion section for better understanding and conclusion of the topic.

Author Response

Reply to Reviewer #4

1. The problem identified and addressed by the authors is highly useful for rapid multiplication of the selected medicinal plant, as there is huge demand for cultivation of such medicinal plants to avoid over exploitation of the wild or naturally grown plants.

Reply: We thank you for reviewing our manuscript and providing many valuable comments and suggestions. Over the past two weeks, we have revised the original manuscript according to the comments and suggestions raised by you and the other three reviewers. In the revised manuscript, all modifications were marked in red.

2. The authors have used different terms, like first pruning, second pruning. pruning, multiple pruning, and primary and secondary pruning at different places, creating confusion for the readers; hence, it is recommended that they use the same terms, viz., primary and secondary pruning, throughout the manuscript

Reply: As suggested, we have already unified the “first pruning” and “second pruning” into “primary pruning” and “secondary pruning” throughout the manuscript.

3. In the results section, the authors clearly stated the significance of the treatments by percentage comparisons which is highly appreciable as it gives a clearer picture of the results to the readers.

Reply: Thank you for your positive comments.

4. In Figure 1, the sequence of pruned images A-B-C-D is not matching with the sequence of grown plant images E-F-G-H. Same sequence may be maintained for both the series for easy comparison and understanding.

Reply: Thank you for your comments. There were 10 trees for each treatment at that time, and the data measurement was taken on different trees of the same treatment. The images presented typical plants of each treatment. Due to our incomplete consideration, we did not conduct the concessive photography on the same plant.

5. Also, in Figure 1, the authors may please confirm that the standard error bars used in graphic images (I-J-K) are specific to the replication data of the respective treatments.

Reply: For each treatment, we did 10 biological replications (10 trees). For each biological replication, we measured 10 values and took the average of these 10 values. We may confirm that the standard error value is specific to the 10 biological replications data of the respective treatments.

6. Lane 120: water content percentage formula needs to be multiplied by 100 to ensure the given data results in per cent

Reply: Thank you for your suggestion, we have already added *100 there.

7. Lane 176: The statistical design used for this experiment may be clearly stated in this section

Reply: As suggested, we have already supplemented the statistical analysis in detail.

8. Lane 217: Figure 02. The caption title would be appropriate to have the term ‘first /primary pruning’ instead of general pruning word

Reply: As suggested, we have changed “pruning” to “primary pruning”.

9. Lane 248: Figure 04. The caption title would be appropriate to have the term ‘secondary pruning’ instead of multiple pruning word

Reply: As suggested, we have changed “multiple pruning” to “secondary pruning”.

10. Lane 276: Figure 06. The caption title would be appropriate to have the term ‘multiple pruning’ instead of different pruning word

Reply: As suggested, we have changed “different pruning” to “multiple prunings”.

11. A brief summary of the study may be added after the discussion section for better understanding and conclusion of the topic

Reply: As suggested, we have added a conclusion as the brief summary of the study as follows.

Pruning treatments resulted in a significant increase in branch number, leaf lobe depth, leaf area, leaf weight, and leaf flavonoids. Among the treatments, light pruning had the most positive effect on increasing branch number, leaf area, and leaf flavonoids. Secondary light pruning also led to an increase in branch number. Additionally, cuttings taken from secondary pruning showed a higher rate of rooting. Further research is needed to understand the molecular mechanisms behind these improved economic traits.

Round 2

Reviewer 1 Report

The information I requested has still not been provided, e.g. the economic value of ginkgo cultivation, units of measurement, end of subsection 2.1. The discussion did not compare the results of publications by other authors regarding the species under study.

Author Response

1. The economic value of ginkgo cultivation.

Reply: Ginkgo biloba L. is an economically important tree species, with a very wide planting area in China, about more than 400,000 hectares, and the total output value of the ginkgo industry has reached more than 18 billion RMB. We have added these specific details in the revised manuscript. Please see line 57-59.

2. Units of measurement, end of subsection 2.1.

Reply: We have added units of measurement, the shoot length (cm), number of lateral branches (pcs), stem diameter (mm), the lobing number (pcs), lobing depth (cm), thickness (mm), area (cm²), fresh weight (g), dry weight (g), and water content (%). Please see line 139-141.

3. The discussion did not compare the results of publications by other authors regarding the species under study.

Reply: Thank you for pointing out this question, we have already compared the results with those obtained by other authors on ginkgo stem cutting propagation. Please see line 441-449.