The Effects of Different Auxin–Cytokinin Combinations on Morphogenesis of Fritillaria meleagris Using Bulb Scale Sections In Vitro

Round 1

Reviewer 1 Report

Review comments

 

Line 23-25: More numerical data can be added to this sectionin abstract.

Line 38-41: This sentence needs citation.

Line 67: In the text, the citation [10, 11, 12] can be changed to [10-12].

Line 85, 117: Abbreviations should be written with their full names when first used them.

Line 102-104: Need citation.

Line 139-143: More details can be given about the monitoring method in this section.

Line 156-159: Need citation for statistical analysis.

Line 162-163: “The maximum regeneration percentage (100%) was recorded after only two weeks 162 at 7 °C on medium supplemented with a combination of BAP and 2,4-D (Table 1).” Sentences like these can be supported by adding treatment concentrations in Result section.

In Discussion section, the following sentences can be supported by numerical data:

Line 424-425: “In our study, medium without PGRs as well as medium with low PGR concentrations was very efficient for somatic embryogenesis in F. meleagris.”

Line: 437-438: “In addition to PGR-free medium, low auxin/cytokinin combinations had a positive effect on the number of SEs in snake’s head fritillary.”

Line 465-467: “High cytokinin concentrations initially increased the number of newly formed bulbs during cold treatment, but prolonged cultivation period after transfer to a higher temperature established the lowest BAP concentration as the most effective.” This sentence can be revised.

Latin names should be check in references section. There are some mistakes in the references section:

Line 494: “Fritillaria” should be written italicized.

Line 545: “Lilium longiflorum” should be write italicized.

Line 572: “Lilium” should be written italicized.

Line 376, 412, 436, 497, 508, 533, 545, 565, 570, 606: “In vitro” should be written italicized.

In Materials and Methods section, bracketed table and figure citations should be normal font not bold type in text.

 

 

Author Response

Response to Reviewer 1 Comments

Thank you for reading the manuscript horticulturae-2535650. Your comments and suggestions are greatly appreciated as they have helped us to improve the text. Manuscript has been modified according to the reviewer’s suggestions, and the changes are clearly visible in the Track Changes version of the revised manuscript. Responses to the reviewer’s comments are provided below.

 

 

Line 23-25: More numerical data can be added to this sectionin abstract.

We have omitted (more) numerical data from the section abstract, because the word count is limited to about 200 words according to the instructions of the journal.

However, according to the reviewer’s suggestion, more numerical data has been added to the section Abstract, so that the lines 23-25 have been changed to:

 “Low PGR concentrations were very effective for shoot multiplication, yielding up to 5.5 shoots per explant. 2,4-D (at 2 mg/l) in combination with low BAP (0.25 mg/l) produced the highest number of bulbs (11.00 ± 0.00), while PGR-free medium was extremely effective in somatic embryo formation (13.50 ± 2.90).”

 

 

Line 38-41: This sentence needs citation.

 

Listed below are required citations, which have been added to the main text and the reference list:

 

Tatarenko, I.; Walker, K.; Dyson, M. Biological Flora of Britain and Ireland: Fritillaria meleagris. J. Ecol. 2022, 110, 1704-1726. doi: 10.1111/1365-2745.13886

Jiang, R.; Zou, M.; Qin, Y.; Tan, G.; Huang, S.; Quan, H.; Zhou, J.; Liao, H. Modeling of the Potential Geographical Distribution of Three Fritillaria Species Under Climate Change. Front. Plant Sci. 2022, 12, 749838. doi: 10.3389/fpls.2021.749838

Protopopova, M.; Sandanov, D.; Pavlichenko, V.; Selyutina, I.; Stepanov, N. The Curious Case of Fritillaria sonnikovae (Liliaceae) in South Siberia: New Insights into Its Origin and Phylogeny. Diversity 2023, 15, 193. https://doi.org/10.3390/d15020193

 

 

Line 67: In the text, the citation [10, 11, 12] can be changed to [10-12].

 

We have changed the citations in the text according to the reviewer’s suggestions.

 

 

Line 85, 117: Abbreviations should be written with their full names when first used them.

 

Full names are now given at the first mention of abbreviations in the text.

 

BA (line 85 in the original version) is benzyl adenine, which is another name for 6-benzylaminopurine (BAP). For clarity purposes, we have changed BA to BAP, whose full name is provided earlier in the text (line 62 in the revised version).

 

Full name of MS medium (line 117 in the original text) has been provided.

 

 

Line 102-104: Need citation.

 

Listed below are required citations, which have been added to the main text:

 

Paek, K.Y.; Murthy, H.N. High frequency of bulblet regeneration from bulb scale section of Fritillaria thunbergii. Plant Cell Tissue Organ Cult. 2002, 68, 247-252; https://doi.org/10.1023/A:1013952803887

 

Leshem, B.; Lilien-Kipnis, H.; Steinitz, B. The effect of light and of explant orientation on the regeneration and subsequent growth of bulblets on Lilium longiflorum Thunb, bulb-scale sections cultured in vitro. Sci. Hortic. 1982, 17(2), 129-136. doi: 10.1016/0304-4238(82)90005-X

 

Park, N. Effect of temperature, scale position, and growth regulators on the bulblet formation and growth during scale propagation of Lilium. Acta Hortic. 1996, 414, 257–262; https://doi.org/10.17660/ActaHortic.1996.414.31

 

Niimi, Y.; Nakano, M.; Isogai, N. Effects of temperature and illuminating conditions on regeneration and development of bulblets in scale culture of seven Lilium spp. J. Jpn. Soc. Hortic. Sci. 1999, 68(1), 28–34; https://doi.org/10.2503/jjshs.68.28

 

Tang, N.; Ju, X.; Hu, Y.; Jia, R.; Tang, D. Effects of Temperature and Plant Growth Regulators on the Scale Propagation of Lilium davidii var. unicolor. HortSci. 2020, 55(6), 870-875; https://doi.org/10.21273/HORTSCI14916-20

 

Rice, L.J.; Finnie, J.F.; Van Staden, J. In vitro bulblet production of Brunsvigia undulata from twin-scales. S. Afr. J. Bot. 2011, 77(2), 305–312; https://doi.org/10.1016/j.sajb.2010.08.011

 

Khonakdari, M.R.; Rezadoost, H.; Heydari, R.; Mirjalili, M.H. Effect of photoperiod and plant growth regulators on in vitro mass bulblet proliferation of Narcissus tazzeta L. (Amaryllidaceae), a potential source of galantamine. Plant Cell Tissue Organ Cult. 2020, 142, 187–199. doi: 10.1007/s11240-020-01853-y

 

 

Line 139-143: More details can be given about the monitoring method in this section.

 

The monitoring method is now described in more detail.

“During this time, detailed changes in morphogenetic potential were monitored weekly, with the aid of a stereomicroscope. Any morphogenetic change in an explant (formation of shoot, bulb or SE) was considered regeneration, and each such explant was marked as responsive. At the end of each week, the number of responsive explants, the number of shoots per responsive explant and length of the longest shoot, as well as the number of bulbs and somatic embryos were recorded for each treatment.”

 

Line 156-159: Need citation for statistical analysis.

 

Fisher's least significant difference (LSD) procedure for pairwise comparisons of several treatment groups is a commonly used statistical method in biological studies following analysis of variance. We have never seen a particular paper by Fisher cited in Materials and Methods section of many authors who use this particular test in plant science papers, so we do not know what citation the reviewer had in mind.

As already specified in the text, all statistical analyses were performed using Statistica 10 software (StatSoft, Germany).

 

Line 162-163: “The maximum regeneration percentage (100%) was recorded after only two weeks 162 at 7 °C on medium supplemented with a combination of BAP and 2,4-D (Table 1).” Sentences like these can be supported by adding treatment concentrations in Result section.

 

In this particular paragraph, we wanted to highlight the difference between the effects of two different auxins (in combination with BAP) in the early phase of their action, rather than indicating the most effective concentrations. The reason we did not specify the exact concentrations in this particular sentence is that adding four different concentrations would make it longer and less readable, especially since the corresponding table is positioned very close to the paragraph, and the treatment concentrations are easy to see because the maximum values are in bold.

 

We realize, however, that for clarity some of the claims should be supported by treatment concentrations, and have therefore inserted them elsewhere, where we believe that sentence length and structure permitted such an addition.

 

 

In Discussion section, the following sentences can be supported by numerical data:

Line 424-425: “In our study, medium without PGRs as well as medium with low PGR concentrations was very efficient for somatic embryogenesis in F. meleagris.”

Line: 437-438: “In addition to PGR-free medium, low auxin/cytokinin combinations had a positive effect on the number of SEs in snake’s head fritillary.”

 

We have changed the first sentence (lines 424-425) and supported the latter sentence (lines 437-438) with numerical data.

 

 

Line 465-467: “High cytokinin concentrations initially increased the number of newly formed bulbs during cold treatment, but prolonged cultivation period after transfer to a higher temperature established the lowest BAP concentration as the most effective.” This sentence can be revised.

 

We have rewritten the conclusion, including this sentence. We feel that the current wording better describes our findings, and we hope that you will find the revised version more suitable.

 

 

Latin names should be check in references section. There are some mistakes in the references section:

Line 494: “Fritillaria” should be written italicized.

Line 545: “Lilium longiflorum” should be write italicized.

Line 572: “Lilium” should be written italicized.

 

Latin names in the section References have been italicized.

 

 

Line 376, 412, 436, 497, 508, 533, 545, 565, 570, 606: “In vitro” should be written italicized.

 

According to MDPI Style Guide (https://www.mdpi.com/authors/layout#_bookmark15), the term “in vitro” need not be italicized:

“Foreign words do not need to be highlighted or italicized, including Greek/Latin terms, such as i.e., e.g., etc., et al., vs., ca., cf., in vivo, ex vivo, in situ, ex situ, in vitro, in utero, ad hoc, in silico, ab initio, vice versa, and via.”

This term is also not italicized in already published papers of this special issue ("A New Decade in the Propagation of Horticultural and Medicinal Plants") of Horticulturae.

Regarding the References section, the term “in vitro” is not italicized in the original titles of the papers cited in lines 497, 508, 533, 545, 565, 570, 606, so we would like to keep the original titles.

 

 

In Materials and Methods section, bracketed table and figure citations should be normal font not bold type in text.

Figure citations in Material and Methods section are now changed from bold to regular font.

Author Response File: Author Response.docx

Reviewer 2 Report

Title: The effects of different auxin-cytokinin combinations on morphogenesis of Fritillaria meleagris using bulb scale sections in vitro

Authors: Marija Marković et al. 

 

1. “horticulturally and medicinally valuable”  “horticulturally valuable” may refer to the ornamental value?

 

2. Keywords:  Snake’s Head Fritillary   

“Snake’s Head Fritillary” is only mentioned in the first sentence of the introduction and may not be suitable as a keyword.

 

3. Introduction: There is not sufficient elaboration on the application of PGRs. The author provides extensive information about their research group’s related studies.

What is the progress in studying the effects of different combinations of PGRs in tissue culture systems of other plants?

 

4. After the first occurrence of Fritillaria meleagris L., it should be abbreviated as F. meleagris in the text.

 

5. The units throughout the text need to be verified further. For example, “mg/l” should be “mg/L”.

 

6. Why was the temperature chosen as 7°C for regeneration conditions?

 

7. Table 1 and Table 2: Based on the notation of the significance levels, I suspect there may be errors in the data processing.

 

8. The scale bar size should be indicated in Figure 3 and Figure 4.

 

9. Discussion: The content can be condensed as it is too extensive. Some parts can be introduced in the Introduction.

Minor editing of English language required.

Author Response

 

Response to Reviewer 2 Comments

Thank you for reading the manuscript horticulturae-2535650. Your comments and suggestions are greatly appreciated as they have helped us to improve the text. Manuscript has been modified according to the reviewer’s suggestions, and the changes are clearly visible in the Track Changes version of the revised manuscript. Responses to the reviewer’s comments are provided below.

 

1. “horticulturally and medicinally valuable”  “horticulturally valuable” may refer to the ornamental value?

Yes, the phrase “horticulturally valuable” refers to the ornamental value of this plant.   

We have found the same phrase in other papers, published in journals of highly reputed publishers, e.g. https://link.springer.com/article/10.1007/s12892-011-0113-4 or https://www.sciencedirect.com/science/article/pii/S0254629915311431

 

2. Keywords:  Snake’s Head Fritillary   

“Snake’s Head Fritillary” is only mentioned in the first sentence of the introduction and may not be suitable as a keyword.

This phrase appears 5 times in the text (once in the section Introduction and four times in the section Discussion), but we agree with the reviewer that it may not be suitable as a key word based on the frequency of its appearance. Therefore, we have included the term “fritillary” along with “micropropagation” as more appropriate keywords that better represent the main topics in the paper.

 

3. Introduction: There is not sufficient elaboration on the application of PGRs. The author provides extensive information about their research group’s related studies.

What is the progress in studying the effects of different combinations of PGRs in tissue culture systems of other plants?

We have removed excessive information about our research group’s studies on fritillary, replacing them with more extensive information on the application of PGRs in other plant species, including non-bulbous species and even some woody species (lines 55-63 in revised version of the text).

 

4. After the first occurrence of Fritillaria meleagris L., it should be abbreviated as F. meleagris in the text.

Besides the title, the full name of the species (Fritillaria meleagris L.) appears once in the abstract, once at the beginning of the Introduction section, and once in the first paragraph of the Material and Methods section. In the rest of the text it is abbreviated as F. meleagris.

The occurrence of the full name of the species in the given sections is a common practice found in a number of papers, although in some papers the name is abbreviated in the Material and Methods section. For this reason, we have abbreviated the species name in the Material and Methods section.

 

5. The units throughout the text need to be verified further. For example, “mg/l” should be “mg/L”.

The units “mg/l” have been replaced with “mg/L” throughout the text.

 

6. Why was the temperature chosen as 7°C for regeneration conditions?

The only growth chamber available to us for chilling experiments at the moment was one shared by other research groups in our department, which was set at 7 °C. Our previous experiments at this chilling temperature proved satisfactory for bulb dormancy breaking and sprouting (see doi: 10.3390/plants9111449, doi: 10.3390/plants9111573), when compared with earlier experiments conducted at 4 °C.

 

7. Table 1 and Table 2: Based on the notation of the significance levels, I suspect there may be errors in the data processing.

We have rechecked significance levels between treatments and found typing errors in Table 1 (column Shoot length), Table 5 (columns Number of shoots per explant and Shoot length), and Table 6 (columns Number of shoots per explant and Number of bulbs), which we corrected.

It is noteworthy that the data concerning each PGR combination (BAP/2,4-D and BAP/NAA) were analyzed separately, hence the results are also presented separately (Table 1 and Table 2). This means that the letters denoting significant differences can be different for the same values in different tables (e.g., column Number of SEs, the value found for PGR-free medium, 2.75±1.03).

 

8. The scale bar size should be indicated in Figure 3 and Figure 4.

The scale bars are now inserted in all figures and their size is indicated in the figure legend.

 

9. Discussion: The content can be condensed as it is too extensive. Some parts can be introduced in the Introduction.

The section Discussion was reorganized and its content condensed, according to the reviewer’s suggestion. In addition, some parts of this section were moved to the section Introduction.

Author Response File: Author Response.docx

Reviewer 3 Report

Comments for author File: Comments.docx

The author's English expression ability is quite good

Author Response

Response to Reviewer 3 Comments

Thank you for reading the manuscript horticulturae-2535650. Your comments and suggestions are greatly appreciated. Manuscript has been modified according to the reviewer’s suggestions, and the changes are clearly visible in the Track Changes version of the revised manuscript. Responses to the reviewer’s comments are provided below.

 

1. The authors claimed the content of the manuscript was “The effects of different auxin-cytokinin combinations on morphogenesis of Fritillaria meleagris using bulb scale sections in vitro”, but each concentration gradient only contained two auxin or Cytokinin plant growth regulators. The setting of different Cytokinin and auxin should be at least 3 gradients in results.

 

We are grateful for this comment, especially since we were not aware that the setting should include at least 3 concentration gradients. Although we have come across papers with multiple concentration gradients, we have also seen papers with 2 concentration gradients (e.g., Aasim, M., Ali, S.A., Altaf, M.T. et al. Artificial neural network and decision tree facilitated prediction and validation of cytokinin-auxin induced in vitro organogenesis of sorghum (Sorghum bicolor L.). Plant Cell Tiss Organ Cult 153, 611–624 (2023). https://doi.org/10.1007/s11240-023-02498-3)

However, even though we were unaware of any requirements or limitations when it comes to concentration gradients, we agree that having at least 3 or more would certainly yield more precise results and better contribute to our understanding of combined effect of multiple PGRs on plant morphogenesis. We will certainly keep this in mind for our future experiments and extend the concentration gradients.

 

 

2. A combination containing only Cytokinin or auxin plant growth regulators should be set in the experimental group.

 

We agree that sole cytokinin or auxins should be included in the experimental setup. However, because we investigated the effects of solely administered 2,4-D (at concentrations of 0.1, 0.5, 1.0, 2.0, 5.0, and 10.0 mg/L) in our previous study of somatic embryo induction from bulb scale sections, we thought that there might be too much overlap between that study and the current one.

 

 

3. Why did the author choose a temperature of 7 ℃ instead of 4 ℃ during low-temperature treatment in this experiment.

 

The only growth chamber available to us for chilling experiments at the moment was one shared by other research groups in our department, which was set at 7 °C. Our previous experiments at this chilling temperature proved satisfactory for bulb dormancy breaking and sprouting (see doi: 10.3390/plants9111449, doi: 10.3390/plants9111573), when compared with earlier experiments conducted at 4 °C.

 

 

4. In this experiment, why did not the author choose TDZ as the Cytokinin plant growth regulator or IBA as the auxin plant growth regulator. In the manuscript, the author only used one Cytokinin plant growth regulator, How to explain the effects of different auxin-cytokinin combinations on morphogenesis of Fritillaria meleagris.

 

We have previously investigated the effects of TDZ on somatic embryo induction and bulblet formation (Petrić et al. 2011, doi: 10.5897/AJB09.807). TDZ was not chosen for this study because it is suggested to be a modulator of the endogenous auxin level, and because TDZ was shown to alter bulb formation capacity in tulip, probably related to down-regulation of biosynthesis of trans-zeatin. In addition, TDZ continues to affect cytokinin metabolism in later developmental stages and to perturb cytokinin profiles and signaling long after the explants are removed from TDZ-containing medium because it is very stable and remains in tissue for a long time.

We chose 2,4-D and NAA as the most frequently used auxins in regeneration studies, but it would certainly be useful to also examine others, like IBA.

The effects of different auxin-cytokinin combinations on morphogenesis of Fritillaria meleagris can be explained by the interaction of particular auxin and cytokinin that are exogenously supplied, but also by their interaction with endogenous plant hormones. By modulating the levels of endogenous phytohormones (affecting their biosynthesis and distribution), they can alter developmental pathways under in vitro conditions.

 

 

5. Scale bar should be added in figure 2-6.

 

Scale bars are now added in Figures 2-6, and their size is given in the figure legends.

 

 

6. Why was the concentration of BAP set to 0.25-0.5mg/L, while 2,4-D was set to 1-5mg/L in Table 1, Please provide your reasons.

 

We came across the protocol developed for Lilium by Asmita et al. (2020, https://doi.org/10.56093/ijas.v90i6.104805), who obtained direct organogenesis from tTLC explants using these exact PGR concentrations. In Lilium, both PGR combinations resulted in a significant increase in regeneration percentage and number of adventitious shoots per explant (almost 4-fold). Therefore, we decided to try the same auxin/cytokinin concentrations in our system, with bulb scales as starting explants, and see how different cytokinin:auxin ratios would affect morphogenesis in F. meleagris.

 

 

7. The authors claimed that after chilling bulbs scales were transferred to 24° C and all parameters were recorded again. Low PGR concentrations were very effective for shoot multiplication. 2,4-D (at 2mg/l) in combination with low BAP (0.25 mg/l) produced the highest number of bulbs, while PGR-free medium was extremely effective in somatic embryo formation. The growth of green sprouts in medium with PGRs was not as good as in medium without PGRs at 24℃ in figure 6. How to explain this?

 

The reason why growth of green sprouts on medium with PGRs was not as good as on medium without PGRs in Figure 6 could be that the shoot elongation was inhibited by cytokinin, especially when applied at high concentrations (4 mg/L BAP, Fig. 6E).

Author Response File: Author Response.docx

Reviewer 4 Report

The work focuses on F. meleagris in vitro propagation. The problem is significant because the properly and efficient method of propagation F.m. is unknown. The most of Frittilaria species are generally very difficult in propagation. 

The F.m. is protected in Poland.

The Introduction is in the most part superficial and disordered. Moreover, Authors should precise their goals and short the last paragraph.

 The scale bars should be in Figs. 1-6.

The Tables should be presented in more visible mode, e.g. Figures.

The disscussion is also superficial. 

The conclusions should be more precise without repeating the results.

 

The words / part of sentences repeated oft. It should be corrected stylistically. 

Author Response

Response to Reviewer 4 Comments

Thank you for reading the manuscript horticulturae-2535650. Your comments and suggestions are greatly appreciated as they have helped us to improve the text. Manuscript has been modified according to the reviewer’s suggestions, and all changes are clearly visible in the Track Changes version of the revised manuscript. Responses to the reviewer’s comments are provided below.

 

The work focuses on F. meleagris in vitro propagation. The problem is significant because the properly and efficient method of propagation F.m. is unknown. The most of Frittilaria species are generally very difficult in propagation. 

The F.m. is protected in Poland.

Fritillaria meleagris is a protected species in a number of countries, including Serbia, because it is threatened or extinct in its former range, and like most of Fritillaria species, it is difficult to propagate. Developing a protocol for successful regeneration is therefore of great importance, as a means for germplasm conservation to ensure the survival of this endangered plant.

 

The Introduction is in the most part superficial and disordered. Moreover, Authors should precise their goals and short the last paragraph.

We have added some new information to the Introduction section (at the request of another reviewer), but also removed some. The text has been re-structured; we believe it is now much more orderly and hope its readability is improved. The last paragraph has been shortened, and our goals are more precisely stated.

 

The scale bars should be in Figs. 1-6.

Scale bars are now added to all figures, and their size is given in the figure legends.

 

The Tables should be presented in more visible mode, e.g. Figures.

We initially tried to present the results graphically, but were not satisfied with the clarity because there were 5 measured parameters for each of the 11 treatments. In addition, it was more difficult to highlight statistical significance between treatments with so much data. Therefore, we opted for Tables, in which each measured parameter is more visible, and the cutoff values are even more highlighted.

 

The disscussion is also superficial. 

The Discussion section has also been re-structured and its content modified.  We hope that it is now clearer and less superficial.

 

The conclusions should be more precise without repeating the results.

The conclusion has been rewritten and we hope it is now more precise.

 

Author Response File: Author Response.docx

Round 2

Reviewer 2 Report

This manuscript has been extensively edited according to the comments.

Reviewer 3 Report

Comments for the Author:

In the manuscript of “The effects of different auxin-cytokinin combinations on morphogenesis of Fritillaria meleagris using bulb scale sections in vitro”. The author provides a method for rapid reproduction of Fritillaria meleagris. The review is more interesting, in my opinion, just a few changes are required.

1.     The authors claimed the content of the manuscript was “The effects of different auxin-cytokinin combinations on morphogenesis of Fritillaria meleagris using bulb scale sections in vitro”, but each concentration gradient only contained two auxin or Cytokinin plant growth regulators. The setting of different Cytokinin and auxin should be at least 3 gradients in results.

2.     A combination containing only Cytokinin or auxin plant growth regulators should be set in the experimental group.

3.     Why did the author choose a temperature of 7 ℃ instead of 4 ℃ during low-temperature treatment in this experiment.

4.     In this experiment, why did not the author choose TDZ as the Cytokinin plant growth regulator or IBA as the auxin plant growth regulator. In the manuscript, the author only used one Cytokinin plant growth regulator, How to explain the effects of different auxin-cytokinin combinations on morphogenesis of Fritillaria meleagris.

5.     Scale bar should be added in figure 2-6.

6.     Why was the concentration of BAP set to 0.25-0.5mg/L, while 2,4-D was set to 1-5mg/L in Table 1, Please provide your reasons.

7.     The authors claimed that after chilling bulbs scales were transferred to 24° C and all parameters were recorded again. Low PGR concentrations were very effective for shoot multiplication. 2,4-D (at 2mg/l) in combination with low BAP (0.25 mg/l) produced the highest number of bulbs, while PGR-free medium was extremely effective in somatic embryo formation. The growth of green sprouts in medium with PGRs was not as good as in medium without PGRs at 24℃ in figure 6. How to explain this?

Comments for author File: Comments.docx

 The manuscript was not well written with many typos,grammarerrors, redundancies, and inaccurate statements. A major revision in both science and language is needed to improve readability and clarity.

Reviewer 4 Report

The manuscript were corrected according to the suggestions.

It's sufficient.

It's generally correct.