Developmental, Phytochemical and Enzymatic Changes in Pot Marigold (Calendula officinalis L.) cvs. Hybrid and French with Salicylic Acid (SA) and Polyamine Spermidine (SP) Foliar Spray
, Aftab Jamal 3,*, Muhammad Farhan Saeed 4, Nasir Masood 4, Emanuele Radicetti 5, Muhammad Fawad 6 and Silvana Nicola 7,*Round 1
Reviewer 1 Report
With the growing interest and demand for medicinal products in various industries, a high-throughput approach to increase the quantity and quality of medicinal plants like marigold is considered necessary. The manuscript entitled “Developmental, Phytochemical and Enzymatic Changes in Pot Marigold (Calendula Officinalis L.) cvs. Hybrid and French with Foliar Phytohormone and Polyamine Spray” have a comprehensive background introduction, and the results are clear. The comments and suggestions as follows:
1. The title of the article used “Phytohormone” and “Polyamine” two words, in fact, the test used “SA” and “spermidine” two agents. The topics range a lot. Would it be better if SA and spermidine were directly used instead?
2. It is suggested to indicate in line 114 what the control is, although it is understood in the following illustration that the control is to spray water, it is not indicated here.
3. There is an error in line 125. The unit of stem diameter should be “mm”. At the same time, this section points out that the sample size of assessing vegetative and floral parameters is 3 plants. Is too small a sample size representative ?
4. It can be seen from the results that the experiment analyzes the interaction of two factors, but the analysis method is not pointed out in the materials and methods, and it is suggested to add.
5. Fig.2D flower nodes should be number of flower nodes, the unit should not be “cm”, and this indicator should also be changed to “number of flower nodes”.
6. Image annotation is not standardized. There are individual significant markers deviate from the column. It is recommended to check all pictures redrawn.
7. Figures 4 and 5 should indicate “total phenolic compound content” and “total carotenoids content” on the vertical axis.
8. “Figure 5” and “Fig.5” appear in lines 385 and 390. Should we standardize writing ?
9. The writing of “P” in lines 440 and 449 is also different, and it is recommended to unify the case.
10. The article mentions “Significant variations were found among exogenous application treatments and between the cultivars on …, while interactions between treatments by cultivars (T×C) had non-significant effect”. However, there is no actual data description, so it is suggested to supplement in the text or supplement materials.
Author Response
Reviewer# 1
Comments and Suggestions for Authors
With the growing interest and demand for medicinal products in various industries, a high-throughput approach to increase the quantity and quality of medicinal plants like marigold is considered necessary. The manuscript entitled “Developmental, Phytochemical and Enzymatic Changes in Pot Marigold (Calendula Officinalis L.) cvs. Hybrid and French with Foliar Phytohormone and Polyamine Spray” have a comprehensive background introduction, and the results are clear. The comments and suggestions as follows:
- The title of the article used “Phytohormone” and “Polyamine” two words, in fact, the test used “SA” and “spermidine” two agents. The topics range a lot. Would it be better if SA and spermidine were directly used instead?
Response: Thanks, the title has been revised as directed.
- It is suggested to indicate in line 114 what the control is, although it is understood in the following illustration that the control is to spray water, it is not indicated here.
Response: Thanks so much, this information has been added.
- There is an error in line 125. The unit of stem diameter should be “mm”. At the same time, this section points out that the sample size of assessing vegetative and floral parameters is 3 plants. Is too small a sample size representative ?
Response: We appreciate your remarks; effectively, the stem diameter is measured in "mm.". We have appropriately fixed this error. Furthermore, we have five treatments including control so 15 pots for each cultivar. In total 30 pots. One plant from each pot was taken or 3 plants per treatment. We believe the sample size was appropriate. Thank you for your understanding.
- It can be seen from the results that the experiment analyzes the interaction of two factors, but the analysis method is not pointed out in the materials and methods, and it is suggested to add.
Response: Insightful comment, this information has been stated in section 2.9: Statistical analysis. Thanks so much.
- Fig.2D flower nodes should be number of flower nodes, the unit should not be “cm”, and this indicator should also be changed to “number of flower nodes”.
Response: Done, the change has been implemented. Thanks a lot.
- Image annotation is not standardized. There are individual significant markers deviate from the column. It is recommended to check all pictures redrawn.
Response: Thanks so much, we have taken into account both your remarks and the reviewers' suggestions while updating all the figures. Thanks again for understanding.
- Figures 4 and 5 should indicate “total phenolic compound content” and “total carotenoids content” on the vertical axis.
Response: Done, the aforementioned change has been implemented. Thanks a lot.
- “Figure 5” and “Fig.5” appear in lines 385 and 390. Should we standardize writing ?
Response: Done, Thank you so much.
- The writing of “P” in lines 440 and 449 is also different, and it is recommended to unify the case.
Response: Thanks, it has been corrected.
- The article mentions “Significant variations were found among exogenous application treatments and between the cultivars on …, while interactions between treatments by cultivars (T×C) had non-significant effect”. However, there is no actual data description, so it is suggested to supplement in the text or supplement materials.
Response: A supplement material (Tables) has been included in response to your good idea. Once more, many thanks.
Please let us say again we appreciate all your insightful comments. We have worked hardly to try to cover our deficiencies. Thank you for taking the time and energy to help us improve the paper.
Author Response File: 
Author Response.pdf
Reviewer 2 Report
Thanks to the authors for their effort in this work, but I do have a few notes:
- Salicylic acid was used in concentrations of 1, 2 mM, as well as spermidine in concentrations of 2, 3 mM. What is the reason for choosing these concentrations? Why did you use spermidine with a higher concentration than salicylic acid?
- I expected to use a combined treatment (SA+SP) as well as to show the effect of the treatments on photosynthetic pigments,
- The discussion should include all the phytochemicals mentioned in detail in the Materials and Methods section, including saponins, phytosterols, tannins and diterpenes.
- In Materials and Methods; the section 2.7 (Qualitative analysis of the phytochemicals) does not contain references, please add references
- Line 29; “flower height and diameter (5.32, 8.73 cm)”, while in line 301; the highest flower diameter is (8.28 cm)
- Line 31; “2 mM SA improved the maximum number of leaves and stem thickness in ‘French”, while in the results (line 271, 272) you reported that “‘Hybrid’ marigold recorded the highest value of stem thickness with a mean value of (3.77 mm)”. These are conflicting results.
- Line 255: correct “plant height” to “Leaf length”
- Line 287: delete the repetitive No “3.11 cm”
- Line 402; correct the number “(67.5 mg RE g-1)”
- Line 511; correct “Calendula Arvensis” to “Calendula arvensis”
- Line 543: correct “Akshayat” to “Akshaya”
- Line 605: correct “POX” to “POD”
-In Figure 1D; the French cultivar is not represented
-In figure 2A, B, C, D; the French cultivar is not represented and the chart legend is wrong
- In the list of references: All plant names mentioned should be written in italics
Author Response
Reviewer# 2
Comments and Suggestions for Authors
Thanks to the authors for their effort in this work, but I do have a few notes:
- Salicylic acid was used in concentrations of 1, 2 mM, as well as spermidine in concentrations of 2, 3 mM. What is the reason for choosing these concentrations? Why did you use spermidine with a higher concentration than salicylic acid?
Response: Thank you so much for your insightful comment. The reason is that having a higher content of spermidine (Protein) should impact the cell division and growth process. Furthermore, these concentrations were used by many researchers so we could compare the experiments. For example:
https://doi.org/10.1038/s41598-019-52547-1, https://doi.org/10.1016/j.plaphy.2022.05.011.
- I expected to use a combined treatment (SA+SP) as well as to show the effect of the treatments on photosynthetic pigments.
Response: Effectively, applying the combined treatment (SA+SP) would be interesting, sorry, this is not available, it would be difficult to implement at this stage. Although, we can take into account in our ongoing research project. For photosynthetic pigments, we did not do this due to budgetary constraints, as this study was not externally funded and our resources were limited.
- The discussion should include all the phytochemicals mentioned in detail in the Materials and Methods section, including saponins, phytosterols, tannins and diterpenes.
Response: Done, we have updated this section appropriately and tried to add further information. Thanks a lot.
- In Materials and Methods; the section 2.7 (Qualitative analysis of the phytochemicals) does not contain references, please add references
Response: Thanks for pointing out the missing information, we have added the relevant references in this revised version.
- Line 29; “flower height and diameter (5.32, 8.73 cm)”, while in line 301; the highest flower diameter is (8.28 cm)
Response: Thanks for pointing out this error, this has been corrected. Sorry for the misunderstanding.
- Line 31; “2 mM SA gave the maximum number of leaves and stem thickness in ‘French”, while in the results (line 271, 272) you reported that “‘Hybrid’ marigold recorded the highest value of stem thickness with a mean value of (3.77 mm)”. These are conflicting results.
Response: Agreed, 2 mM SA gave the maximum number of leaves (40.71) and stem thickness (5.76 mm) in ‘French”, this has been corrected in the relevant place. Sorry for the misunderstanding.
- Line 255: correct “plant height” to “Leaf length”
Response: Corrected, thanks.
- Line 287: delete the repetitive No “3.11 cm”
Response: Deleted, thanks.
- Line 402; correct the number “(67.5 mg RE g-1)”
Response: Thanks for pointing out this error, this has been corrected. Sorry for the misunderstanding.
- Line 511; correct “Calendula Arvensis” to “Calendula arvensis”
Response: Thanks, it has been corrected.
- Line 543: correct “Akshayat” to “Akshaya”
Response: Corrected, thanks.
- Line 605: correct “POX” to “POD”
Response: Corrected in all instances, thanks.
-In Figure 1D; the French cultivar is not represented
Response: Thanks for the suggestion, previously, we just used the mean of the cultivars to draw this figure because the interaction (C×T) was not statistically significant. Now, all figures have been double-checked and adjusted as directed. Figures without homogenous groups (lettering) represents that interaction (C×T) was found non-significant.
-In figure 2A, B, C, D; the French cultivar is not represented and the chart legend is wrong
Response: Previously, we just used the mean of the cultivars to draw this figure because the interaction (C×T) was not statistically significant. Now, all figures have been double-checked and adjusted as directed. Thanks so much for understanding.
- In the list of references: All plant names mentioned should be written in italics
Response: Done, all references have been double-checked and adjusted as directed. Thanks again.
Please let us say again we appreciate all your insightful comments. We have worked hardly to try to cover our deficiencies. Thank you for taking the time and energy to help us improve the paper.
Author Response File: Author Response.pdf
Reviewer 3 Report
The manuscript entitled “Developmental, Phytochemical and Enzymatic Changes in Pot Marigold (Calendula Officinalis L.) cvs. Hybrid and French with Foliar Phytohormone and Polyamine Spray” has been reviewed in detail. It indicated that the salicylic acid application constitutes a valuable crop management technique to improve the inflorescences and bioactive ingredient production in the marigold cultivation. The paper is well written and flow logically. I recommend that the manuscript be accepted.
Author Response
Reviewer# 3
Comments and Suggestions for Authors
The manuscript entitled “Developmental, Phytochemical and Enzymatic Changes in Pot Marigold (Calendula Officinalis L.) cvs. Hybrid and French with Foliar Phytohormone and Polyamine Spray” has been reviewed in detail. It indicated that the salicylic acid application constitutes a valuable crop management technique to improve the inflorescences and bioactive ingredient production in the marigold cultivation. The paper is well written and flow logically. I recommend that the manuscript be accepted.
Response: Thank you very much for your appreciating remarks and recommending our work for publication in Agronomy.
Author Response File: Author Response.pdf
