The Influence of Maturity, Storage, and Embryo Size on Coconut Callus Induction Success

Round 1

Reviewer 1 Report

Title: “The Effects of Physiological Status of Coconut Plumule 2 Explants on Callus Induction”

 The manuscript investigates the influence of the explant quality and physiological conditions on the initiation of embryogenic callus of Cocos nucifera, a palm tree of great relevance in the global scenario and with significative challenges for maintaining its production and attending to the crescent demand. The results obtained are relevant. However, they are more appropriate for short communication, after a consistent revision in the data presentation (major revisions).

 Major revisions:

Introduction

The introduction is well-founded; however, some information is lacking. I suggest including information about the importance of calluses’ production during the micropropagation process, with an emphasis on somatic embryogenesis.

Line 37: “The coconut (Cocos nucifera L.), belongs”

Lines 46-47: Incomplete sentence = “However, due to the growing population and the 46 substantial rise in the need for both producing and consuming coconuts.”

Material and methods

Lines 124-126: How many embryos were used in this experiment? Did each treatment present how many repetitions?

Lines 127-133: Did each group present how many repetitions?

Figure 2: I suggest changing the order of the images in this figure, according to the calling in the text. Place the letter 'e' in the fifth image and reposition the bar in the letter b. Standardize the location of the letters.

Results

Line 177: ‘emrbryo’

Lines 178, 181: As there are no statistical differences between the treatments tested, we cannot say that the germination rate of tender fruits was higher. The same logic for the browning rate.

Line 185: Table 1 repeats the results presented in the Fig. 4. Unnecessary. Choose one of them to present the data.

Lines 190, 203: Mean values ± standard error for different treatments in each bar,  --- Bars represent standard error, and * sign indicates significative differences (p≤0.05).

Line 194: Replace ‘primary’- ‘embryogenic’.

Line 195: As there are no statistical differences between the treatments tested, we cannot say that the germination rate of storage time (15 d) was higher. The same logic for the other variables.

Table 2 and 4 repeat the results presented in the Fig. 5 and 7, respectively. Unnecessary. Choose one of them to present the data.

Line 207: Table 3 is not presented and is unnecessary, whereas Fig. 6 exhibits the same data.

Line 239: Complete: ‘(...) Cocos nucifera embryos zygotic ...’. Add the scales of the images.

Discussion

Line 249: ‘conditions to on the initiation’

Lines 264-266: This age (12-14 months) is not in the section Material and Methods.

Lines 289, 291, 293, 296, 302, 304, 316, 318: Verify the style of citation. References must be numbered in order of appearance in the text.

References

Put the scientific names in Italics.

Authors did not present in the section References: López‐Villalobos (2002), Quang (2018), Zhihua (2022).

Comments for author File: Comments.pdf

Author Response

Introduction

The introduction is well-founded; however, some information is lacking. I suggest including information about the importance of calluses’ production during the micropropagation process, with an emphasis on somatic embryogenesis.

Thanks for your advice, information is added.

Line 37: “The coconut (Cocos nucifera L.), belongs”

Fixed, thanks.

Lines 46-47: Incomplete sentence = “However, due to the growing population and the 46 substantial rise in the need for both producing and consuming coconuts.”

Fixed, thanks.

Material and methods

Lines 124-126: How many embryos were used in this experiment? Did each treatment present how many repetitions?

Sorry for not making it clear, we have added this part. we used 300 embryos for the experiment, some of them were considered loss because of contaminations and physical damage. We used 5 repetitions.

Lines 127-133: Did each group present how many repetitions?

We used 5 repetitions.

Figure 2: I suggest changing the order of the images in this figure, according to the calling in the text. Place the letter 'e' in the fifth image and reposition the bar in the letter b. Standardize the location of the letters.

Order changed; location standardized.

Results

Line 177: ‘emrbryo’

Fixed, thanks.

Lines 178, 181: As there are no statistical differences between the treatments tested, we cannot say that the germination rate of tender fruits was higher. The same logic for the browning rate.

Phrase changed, thanks.

Line 185: Table 1 repeats the results presented in the Fig. 4. Unnecessary. Choose one of them to present the data.

Table delated, thanks

Lines 190, 203: Mean values ± standard error for different treatments in each bar,  --- Bars represent standard error, and * sign indicates significative differences (p≤0.05).

Fixed, thanks.

Line 194: Replace ‘primary’- ‘embryogenic’.

Fixed, thanks.

Line 195: As there are no statistical differences between the treatments tested, we cannot say that the germination rate of storage time (15 d) was higher. The same logic for the other variables.

Phrase changed, thanks.

Table 2 and 4 repeat the results presented in the Fig. 5 and 7, respectively. Unnecessary. Choose one of them to present the data.

Table delated, thanks

Line 207: Table 3 is not presented and is unnecessary, Fig. 6 exhibits the same data.

Table delated, thanks

Line 239: Complete: ‘(...) Cocos nucifera embryos zygotic ...’. Add the scales of the images.

Scale added, sentence completed.

Discussion

Line 249: ‘conditions to on the initiation’

Fixed, thanks.

Lines 264-266: This age (12-14 months) is not in the section Material and Methods.

Fixed, thanks.

Lines 289, 291, 293, 296, 302, 304, 316, 318: Verify the style of citation. References must be numbered in order of appearance in the text.

Fixed, thanks.

References

Put the scientific names in Italics.

Fixed, thanks.

Authors did not present in the section References: López‐Villalobos (2002), Quang (2018), Zhihua (2022).

Fixed, thanks.

Reviewer 2 Report

This manuscript describes the effects of age and storage time of coconuts and embryo size on callus induction. My comments are as follows:

The title of the manuscript should be changed, since the physiological status of plants refers to the measurement of parameters such as photosynthesis, water status, enzyme activity, etc. Such analyzes were not performed in this study.

Fig. 1 is mentioned in the text after Fig. 2 (L. 137 and L. 75, respectively). Please change the numbering of the figures.

L. 167. “Fig. 1c". Fig. 1c is missing, only Fig. 1a and Fig. 1b are presented.

Table 1 and Fig. 4 present the same results. You should leave only the table or only the figure. Same for Table 2 and Fig. 5, Table 3 and Fig. 6, and Table 4 and Fig. 7.

Table 1. How many replicates were used for statistical analysis? Were Germination and Embryogenic callus rate values the same in all replicates (SE = 0.00)?

L. 198-200. “Our results demonstrated that storing coconut fruits for 15 days significantly elevated the germination and formation of embryogenic callus, and reduced necrosis level of callus.” This phrase is incorrect, since a significant effect was observed only for Embryogenic callus rate (Table 2).

Table 3 is missing in the text.

“Nwite [15]” (L. 66), “Fernando (2004)” (L. 290), “Malaurie (2009)” (L. 316) should be corrected to “Nwite et al. [15]”, etc.

“Zhihua (2022)” (L. 296) should be replaced with “Mu (2022)”.

L. 293, 302. “Quang (2018)” should be replaced with “Nguyen (2018)”.

Author Response

The title of the manuscript should be changed, since the physiological status of plants refers to the measurement of parameters such as photosynthesis, water status, enzyme activity, etc. Such analyzes were not performed in this study.

Changed to The Influence of Maturity, Storage, and Embryo Size on Coconut Callus Induction Success.

Fig. 1 is mentioned in the text after Fig. 2 (L. 137 and L. 75, respectively). Please change the numbering of the figures.

Fixed, thanks.

167. 167. “Fig. 1c". Fig. 1c is missing, only Fig. 1a and Fig. 1b arepresented.

Fixed, thanks.

Table 1 and Fig. 4 present the same results. You should leave only the table or only the figure. Same for Table 2 and Fig. 5, Table 3 and Fig. 6, and Table 4 and Fig. 7.

Table delated, thanks

Table 1. How many replicates were used for statistical analysis? Were Germination and Embryogenic callus rate values the same in all replicates (SE = 0.00)?

Sorry for not making it clear, we have added this description in the text.

200. 198-200. “Our results demonstrated that storing coconut fruits for 15 days significantly elevated the germination and formation of embryogenic callus, and reduced necrosis level of callus.” This phrase is incorrect, since a significant effect was observed only for Embryogenic callus rate (Table 2).

Fixed, thanks.

Table 3 is missing in the text.

Tables were deleted now.

“Nwite [15]” (L. 66), “Fernando (2004)” (L. 290), “Malaurie (2009)” (L. 316) should be corrected to “Nwite et al. [15]”, etc.

Fixed, thanks.

“Zhihua (2022)” (L. 296) should be replaced with “Mu (2022)”.

Fixed, thanks.

302. 293, 302. “Quang (2018)” should be replaced with “Nguyen (2018)”.

Fixed, thanks. 

Reviewer 3 Report

Article The Effects of Physiological Status of Coconut Plumule

Explants on Callus Induction by Zhihua Mu, Shuya Yang, Hang Xu, Mirza Mobashwerul Haque, Binh Minh Tran, Jiepeng Chen, Xingwei Wang, Hui Peng, Jie Luo presented work on coconut propagation through embryonic culture of immature embryos. The topic of the work is relevant and the manuscript is formatted according to the rules.

The manuscript contains the necessary sections and experimental data on introduction into culture, avoiding browning due to the release of metabolites. the work may be interesting, but I was not able to see examples of regeneration, neither shoot formation nor rhizogenesis. It is not clear how many shoots can be obtained and what the micropropagation coefficient of this crop is. This must be specified.

At the end of the introduction (the purpose of the study is placed in a separate paragraph), it needs to be formulated more clearly. What exactly did the authors want to receive, besides an introduction to culture). It is probably necessary to indicate the tasks sequentially, which should be reflected both in the discussion and in the conclusions.

There are comments on the design of the work. There are no rulers in the drawings; they extend beyond the boundaries of the manuscript or are located to the side of the signature (Line 237, 170, 151, 147).

In Figure 2, a collapse apparently occurred, since the ruler is not in place, in addition, the word error is written in two places - check this.

The materials should reflect the method of collection, by whom and how the age of the trees was determined, and other details. Was there a preliminary assessment of the size of the fruits and their quality, and were the most valuable (plus) plants chosen?

After eliminating the shortcomings and expanding the manuscript, the work can be reviewed.

Author Response

Article The Effects of Physiological Status of Coconut Plumule Explants on Callus Induction by Zhihua Mu, Shuya Yang, Hang Xu, Mirza Mobashwerul Haque, Binh Minh Tran, Jiepeng Chen, Xingwei Wang, Hui Peng, Jie Luo presented work on coconut propagation through embryonic culture of immature embryos. The topic of the work is relevant and the manuscript is formatted according to the rules.

Thank you for your comment.

The manuscript contains the necessary sections and experimental data on introduction into culture, avoiding browning due to the release of metabolites. the work may be interesting, but I was not able to see examples of regeneration, neither shoot formation nor rhizogenesis. It is not clear how many shoots can be obtained and what the micropropagation coefficient of this crop is. This must be specified.

Thank you for your comment. This is the next step and we are working on it right now, the data can be obtained soon. In this article we only discuss the callus initiation stage.

At the end of the introduction (the purpose of the study is placed in a separate paragraph), it needs to be formulated more clearly. What exactly did the authors want to receive, besides an introduction to culture). It is probably necessary to indicate the tasks sequentially, which should be reflected both in the discussion and in the conclusions.

This is a good point, we have added tasks and aims in the article.

There are comments on the design of the work. There are no rulers in the drawings; they extend beyond the boundaries of the manuscript or are located to the side of the signature (Line 237, 170, 151, 147).

We have added rulers, and graphs were reformatted by the editors.

In Figure 2, a collapse apparently occurred, since the ruler is not in place, in addition, the word error is written in two places - check this.

Fixed, thanks.

The materials should reflect the method of collection, by whom and how the age of the trees was determined, and other details. Was there a preliminary assessment of the size of the fruits and their quality, and were the most valuable (plus) plants chosen?

The age of the tree will not affect the result of the experiment; however, we selected trees from one coconut farm which means they were planted roughly at the same time, they are all at productive age (10 years old). We collected them and our workers checked all nuts before taking them back, we dehusked them and check the vigour. Our experience showed that fruit size was not related to the quality of the embryo. We kept the size uniform as we can, as shown in the graph. And yes, we only use valuable and productive trees.

Reviewer 4 Report

Introduction.

1) The authors state that little research has been done on coconut micropropagation, but there are publications and reviews on this research topic that have not been considered, for example:

Kalaipandian, S.; Mu, Z.; Kong, E.Y.Y.; Biddle, J.; Cave, R.; Bazrafshan, A.; Wijayabandara, K.; Beveridge, F.C.; Nguyen, Q.; Adkins, S.W. Cloning Coconut via Somatic Embryogenesis: A Review of the Current Status and Future Prospects. Plants 2021, 10, 2050. https:// doi.org/10.3390/plants10102050

Sandoval-Cancino, G. Sáenz, L., Chan, J.L., Oropeza, C. Improved formation of embryogenic callus from coconut immature inflorescence explants. In Vitro Cell.Dev.Biol.—Plant (2016) 52:367–378 DOI 10.1007/s11627-016-9780-7

2) Check the writing, there are several spelling errors, for example:

Line 177. zygotic emrbyo from tender coconut fruits

Results. Immature coconut fruits that generate embryogenic callus with a minimum of darkening are friable or compact, to see the possibility of establishing cell suspension culture.

Discussion. The results suggest that the plumular tissues are the preferred type of explant to produce coconuts on a larger scale over other explants such as rachilla, stem, leaves, immature inflorescences and others; broadly substantiate these results.

Author Response

Introduction.

1) The authors state that little research has been done on coconut micropropagation, but there are publications and reviews on this research topic that have not been considered, for example:

Kalaipandian, S.; Mu, Z.; Kong, E.Y.Y.; Biddle, J.; Cave, R.; Bazrafshan, A.; Wijayabandara, K.; Beveridge, F.C.; Nguyen, Q.; Adkins, S.W. Cloning Coconut via Somatic Embryogenesis: A Review of the Current Status and Future Prospects. Plants 2021, 10, 2050. https:// doi.org/10.3390/plants10102050

Sandoval-Cancino, G. Sáenz, L., Chan, J.L., Oropeza, C. Improved formation of embryogenic callus from coconut immature inflorescence explants. In Vitro Cell.Dev.Biol.—Plant (2016) 52:367–378 DOI 10.1007/s11627-016-9780-7

Dear editor, coconut micropropagation has been studied, but the influence of embryo status was never been considered. We have changed the phrase in the text to make it clear.

2) Check the writing, there are several spelling errors, for example:

Line 177. zygotic emrbyo from tender coconut fruits

Checked and fixed, thanks.

Results. 

Immature coconut fruits that generate embryogenic callus with a minimum of darkening are friable or compact, to see the possibility of establishing cell suspension culture.

Checked and fixed, thanks.

Discussion. 

The results suggest that the plumular tissues are the preferred type of explant to produce coconuts on a larger scale over other explants such as rachilla, stem, leaves, immature inflorescences and others; broadly substantiate these results.

Checked and fixed the phrase, thanks.

Round 2

Reviewer 1 Report

Manuscript

Title: “The Effects of Physiological Status of Coconut Plumule Explants on Callus Induction”

Minor revisions:

Line 72: “which was is directly”.

Line 83: “The initiation of callus and its development conditions”

Line 196: Figure 1 2.

Lines 235-236: The germination data were not presented.

Figure 4: I suggest putting letters indicating each graphic (variable) in all graphics.

Figure 8: Add the scales of the images. Although all figures were obtained on the same scale, the scales must be present in each image, maintaining the correct proportions.

Line 528: López-Villalobos

Line 399: Fernando et al. [26] - Verify the correct way throughout the text.

Author Response

Reviewer 1

Line 72: “which was is directly”.

Fixed, thanks.

Line 83: “The initiation of callus and its development conditions”

Fixed, thanks.

Line 196: Figure 1 2.

Fixed, thanks.

Lines 235-236: The germination data were not presented.

Fixed, thanks.

Figure 4: I suggest putting letters indicating each graphic (variable) in all graphics.

Letter added, thanks.

Figure 8: Add the scales of the images. Although all figures were obtained on the same scale, the scales must be present in each image, maintaining the correct proportions.

Scale added, thanks.

Line 528: López-Villalobos

Fixed, thanks.

Line 399: Fernando et al. [26] - Verify the correct way throughout the text.

Fixed, thanks.

Reviewer 2 Report

The manuscript has been improved and may be published.

Author Response

Thank you for your comments.

Reviewer 3 Report

Article The Effects of Physiological Status of Coconut Plumule Explants on Callus Induction by Zhihua Mu, Shuya Yang, Hang Xu, Mirza Mobashwerul Haque, Binh Minh Tran, Jiepeng Chen, Xingwei Wang, Hui Peng, Jie Luo presented work on coconut propagation through embryonic culture of immature embryos. The topic of the work is relevant and the manuscript is formatted according to the rules.

Thank you for your comment.

The manuscript contains the necessary sections and experimental data on introduction into culture, avoiding browning due to the release of metabolites. the work may be interesting, but I was not able to see examples of regeneration, neither shoot formation nor rhizogenesis. It is not clear how many shoots can be obtained and what the micropropagation coefficient of this crop is. This must be specified.

The authors revised the manuscript and corrected flaws, drawings and design of the manuscript.

The answers are satisfactory.

Author Response

Thank you for your comments.