Putative Spatiotemporal Changes in Inhibitor Activity during Cold Stratification of Sapium sebiferum Seeds

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The manuscript presents the results from a study related to dormancy breaking through cold stratification in seeds from Sapium sebiferum. The methodology used was creative and showed that the results from the cold stratification would be explained by reducing inhibitors in the endosperm. However, there is not much novelty in this study, because the cold stratification effects and the reduction of inhibitors associated with the treatment are already known.

The text could be much shorter and concise, same with the presentation of results (it is not necessary to present values with the effect of a treatment, the percentage of the effect in the same table and in the text).

Several aspects in the text, especially the methodology, that should be improved. Some suggestions:  

In the abstract and last part of the introduction, authors should refer to “Chinese cabbage” (Brassica rapa) instead of “cabbage” (Brassica oleracea), so confusion is avoided. Later in the text, in the Material and Methos section, when you describe that seeds of Brassica rapa were used, you may mention that along the text you will use “cabbage” to refer to “Chinese cabbage”,

Line 50. After “Quercus aliena” said “[5” and should be “[5]”.

Line 93 to 101. How many seeds were stratified?, hoy many for each stratification period?

Line 105: the temperature was constant or changed during the night (16 h dark period)?

Lines 103 to 108: How often did you count germination? It is not clear if germination test was conducted during 30 days or if it “was concluded after three consecutive days without any seed germination”. The formula used for the germination index calculation should be presented.

 

Lines 102 to 114: ISTA reference (“18”) is not correct (it is “19” in the reference list). Additionally, ISTA Rules do not have germination and tetrazolium protocols in a same chapter (Chapter 10 according to reference list), so it should be corrected. Also, the criteria for seed viability should be presented (stain pattern).

 

Line 122: “… Petri dishes with two pieces of standard germination paper… “, what type of Petri dishes were used?, What “standard germination paper” was used?

Line 123: incubator with constant light or an 8 h light period?

Line 126 to 128: “According to the Rules of ISTA [18], the germination was terminated when the germination percentage per day did not exceed of repeated particles for 3 consecutive days”. Where does ISTA Rules say that?

Line 143: “ …5 mL of the extracted solution was added to the cabbage seeds for 3 h …”, how many seeds were used?, inside what type of container?

Later says “The test solution or water was added promptly to the standard germination paper during the entire germination period “, Does it mean that after the initial 3 h imbibition of seed with the extract, seeds were placed to germinate in contact with the same solution?, if so, why you did not put them directly to germinate in the respective solution?

Line 156 to 159. Usually percentages need to be transformed before they are analyzed by ANOVA. Did you check the data followed the requirements for ANOVA?

Lines 169 to 171: “When S. sebiferum seeds were stratified for 90 d, the germination percentage significantly increased to 40.0%, and the viability of ungerminated seeds decreased to 67.4%. Therefore, at this time, 67.4% of viable S. sebiferum seeds still could not germinate successfully”.  First authors mentioned that seed viability was 85%; so, if 40% of the seeds already germinated, means that 45% of viable seeds has not germinated. So, it is not clear why authors says that 67,5% of viable seeds still could not germinate… it should be that 45% of viable seeds still can´t germinate, because 40% of the viable seeds already germinated. 

 

Line 196: “…the most striking result of the table was the GI value of the control, which was 0,…”, but the GI value of the control was 43.8 (Table 3)

Author Response

Response to reviewer#1

Comments and Suggestions for Authors

The manuscript presents the results from a study related to dormancy breaking through cold stratification in seeds from Sapium sebiferum. The methodology used was creative and showed that the results from the cold stratification would be explained by reducing inhibitors in the endosperm. However, there is not much novelty in this study, because the cold stratification effects and the reduction of inhibitors associated with the treatment are already known.

The text could be much shorter and concise, same with the presentation of results (it is not necessary to present values with the effect of a treatment, the percentage of the effect in the same table and in the text).

Response: Thank you for the question, the corresponding results in Table 3, 4 and 5 have been deleted.

Several aspects in the text, especially the methodology, that should be improved. Some suggestions:  

In the abstract and last part of the introduction, authors should refer to “Chinese cabbage” (Brassica rapa) instead of “cabbage” (Brassica oleracea), so confusion is avoided. Later in the text, in the Material and Methos section, when you describe that seeds of Brassica rapa were used, you may mention that along the text you will use “cabbage” to refer to “Chinese cabbage”,

Response: Thank you for pointing out the inconsistency between common name and scientific name. We have checked our manuscript and corrected “cabbage” to “Chinese cabbage”.

Line 50. After “Quercus aliena” said “[5” and should be “[5]”.

Response: Thank you for pointing this out, it has been revised.

Line 93 to 101. How many seeds were stratified? how many for each stratification period?

Response: To ensure that the experiments were carried out, we prepared a minimum of 3,000 seeds, about 600 per stage. The seed supply was replenished in Line 96-98.

Line 105: the temperature was constant or changed during the night (16 h dark period)?

Response: During the germination experiments, the temperature was kept constant while only the light exposure was altered with 8 h of light followed by 16 h of darkness. Line 107 has been updated with the exact information.

Lines 103 to 108: How often did you count germination? It is not clear if germination test was conducted during 30 days or if it “was concluded after three consecutive days without any seed germination”. The formula used for the germination index calculation should be presented.

Response: Nowmally, we counted the germination of Sapium sebiferum seeds every two days, but the germination index was not calculated in table 1 and the GI has been omitted from the manuscript of line 108. The germination test of S. sebiferum seeds was conducted for 30 days, not concluded after three consecutive days without any seed germination, see line 108. In the text only the germination index of Chinese cabbage seeds was calculated, with count every 8 hours and the formula used for the germination index of Chinese cabbage has been presented, see lines 131-133.

Lines 102 to 114: ISTA reference (“18”) is not correct (it is “19” in the reference list).  ISTA Rules do not have germination and tetrazolium protocols in a same chapter (Chapter 10 according to reference list), so it should be corrected. Also, the criteria for seed viability should be presented (stain pattern).

Response: Thanks for pointing this out. it has been revised. Literature related to tetrazolium protocols has been added to line 115. Additionally, the basis for determining viability after staining has been included in lines 115 to 116.

Line 122: “… Petri dishes with two pieces of standard germination paper… “, what type of Petri dishes were used?, What “standard germination paper” was used?

Response: The petri dish was glass petri dishes about 10-cm diameter. The “standard germination paper” was used filter paper about 9-cm diameter. Relevant information was added in Lines 125, 152, 156-157.

Line 123: incubator with constant light or an 8 h light period?

Response: The seeds were exposed to a light-dark cycle of 8 h and 16 h, respectively. Relevant information has been corrected in Lines 107, 127, and 158.

Line 126 to 128: “According to the Rules of ISTA [18], the germination was terminated when the germination percentage per day did not exceed of repeated particles for 3 consecutive days”. Where does ISTA Rules say that?

Response: Thanks for your kind reminder. We ignored that ISTA have regularly updated its guidelines and the version (2020) omitted specific rules for forest tree seed testing. After carefully reviewing the literature. We replaced the reference (Line 130) with the relevant content found in Rules for forest tree seed testing.

Line 143: “ …5 mL of the extracted solution was added to the cabbage seeds for 3 h …”, how many seeds were used?, inside what type of container?

Response: The germination procedures were the same as those described in section 2.4. This information was mentioned in the paper, but it was not clearly described. In the revised version of our manuscript, it has been added in Lines 124 and 149.

Later says “The test solution or water was added promptly to the standard germination paper during the entire germination period “, Does it mean that after the initial 3 h imbibition of seed with the extract, seeds were placed to germinate in contact with the same solution?, if so, why you did not put them directly to germinate in the respective solution?

Response: Since seed imbibition is the initial and important phase during seed germination, it is a standard procedure that seeds should be first soaked in the germination test. That is the reason we did not put them directly to germinate in the respective solution.

Line 156 to 159. Usually percentages need to be transformed before they are analyzed by ANOVA. Did you check the data followed the requirements for ANOVA?

Response: Thanks for your professional comment on ANOVA. ANOVA usually requires data to follow normal distribution, and percentages need to be transformed using the arcsine square root transformation. However, the variance test demonstrates a degree of tolerance towards the normality of the data, accepting data that approximate normality. After careful check, we think our origin data is available for ANOVA.

Lines 169 to 171: “When S. sebiferum seeds were stratified for 90 d, the germination percentage significantly increased to 40.0%, and the viability of ungerminated seeds decreased to 67.4%. Therefore, at this time, 67.4% of viable S. sebiferum seeds still could not germinate successfully”.  First authors mentioned that seed viability was 85%; so, if 40% of the seeds already germinated, means that 45% of viable seeds has not germinated. So, it is not clear why authors says that 67,5% of viable seeds still could not germinate… it should be that 45% of viable seeds still can´t germinate, because 40% of the viable seeds already germinated. 

Response: Thanks for this question. This paper presents the calculation of seed viability of the remaining seeds without germination using the formula  , as shown in Table 1. After stratification for 90 days, the germination percentage of S. sebiferum seeds significantly increased to 40.0%, while 60.0% remained ungerminated. Among the ungerminated seeds, 40.0% (60.0%×67.4%) were viable seeds that could not germinate successfully. Therefore, at this time, the total viability was 80.0%.

Line 196: “…the most striking result of the table was the GI value of the control, which was 0,…”, but the GI value of the control was 43.8 (Table 3).

Response: Thank you for bringing this to my attention. I have corrected the error in line 206.

 

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

1. Line 10: “Sapium sebiferum is an economically important Euphorbiaceae tree species.” change to “Sapium sebiferum is a tree with high socioeconomic, environmental and medicinal value”

 

2. Line 11: Delete “As previously reported“

 

3. Line 18–19: “The germination of non-stratified S. sebiferum seeds was inhibited, with the germination percentage of 0.” change to “The germination of non-stratified S. sebiferum seeds was completely inhibited (0% germination).”

 

4. The authors write the % unit in full at many points in the text. My suggestion is that you make the change to standardize everything using just %. E.g., lines 20–21: “the mean germination percentage was 81.3” This could be changed to “the mean germination was 81.3%”.

Please improve this in similar situations throughout the text.

 

5. At the end of the abstract, I missed the importance of the study for seedling production described more assertively and directly.

 

6. Was it possible to deposit the species in a herbarium? Is there an ID voucher? If there is, insert it in the methodology.

 

7. Seeds were collected from how many trees? Please add a geographic map with the location of the trees that produced the seeds in the study.

​

8. Line 9: Delete percentage.

 

9. The term “Germination Percentage (%)” becomes redundant. Please use “germination (%)” throughout the text. This also applies to tables; for example, Table 1–5.

 

10. Line 274–276: Incomprehensible. Delete.

 

11. Line 180: “The same is 180 below.” I did not understand that.

 

12. Line 286–287. Delete “In other words,” This was not good, as it gave the impression that the authors were unable to explain it previously and are still trying.

 

13. Standardize the text. Sometimes it uses only “d” and in others, it uses “days”.

 

Finally, is there information in the scientific literature on which chemical constituents are present in the aril of the seeds of this species? If there is, insert this into the discussion.

Author Response

Response to reviewer#2

 

Comments and Suggestions for Authors

1. Line 10: “Sapium sebiferum is an economically important Euphorbiaceae tree species.” change to “Sapium sebiferum is a tree with high socioeconomic, environmental and medicinal value”

Response: Thanks for your suggestion. We have revised this sentence in the manuscript (line 10).

2. Line 11: Delete “As previously reported“

Response: The deletion has been made.

3. Line 18–19: “The germination of non-stratified S. sebiferum seeds was inhibited, with the germination percentage of 0.” change to “The germination of non-stratified S. sebiferum seeds was completely inhibited (0% germination).”

Response: The expression has been corrected in line 20.

4. The authors write the % unit in full at many points in the text. My suggestion is that you make the change to standardize everything using just %. E.g., lines 20–21: “the mean germination percentage was 81.3” This could be changed to “the mean germination was 81.3%”.

Please improve this in similar situations throughout the text.

Response: Modifications have been completed line 22 and similar issues.

5. At the end of the abstract, I missed the importance of the study for seedling production described more assertively and directly.

Response: Thanks for your suggestion. A modification has been made to line 32.

6. Was it possible to deposit the species in a herbarium? Is there an ID voucher? If there is, insert it in the methodology.

Response: Unfortunately the species is not preserved in the herbarium. It will be recorded in future studies.

7. Seeds were collected from how many trees? Please add a geographic map with the location of the trees that produced the seeds in the study.

Response: The seeds were collected from the same tree. Its geographic location is 32°4'16'' north latitude and 118°48'10'' east longitude. Additions are made in the manuscript (line 88).

8. Line 9: Delete percentage.

Response: Modifications have been completed.

9. The term “Germination Percentage (%)” becomes redundant. Please use “germination (%)” throughout the text. This also applies to tables; for example, Table 1–5.

Response: Thanks for pointing out the problem. Considering the germination percentage is an  indicator that is the ratio between germinated seeds and total test seeds, we think it is appropriate to retain this technical term. We hope to gain your kind understanding.

10. Line 274–276: Incomprehensible. Delete.

Response: The deletion has been made.

11. Line 180: “The same is 180 below.” I did not understand that.

Response: 180 is a line number, not in the text. In the text, "The same is below".

12. Line 286–287. Delete “In other words,” This was not good, as it gave the impression that the authors were unable to explain it previously and are still trying.

Response: Thank you for your suggestion. We have deleted it.

13. Standardize the text. Sometimes it uses only “d” and in others, it uses “days”.

Response: The word has been standardized.

Finally, is there information in the scientific literature on which chemical constituents are present in the aril of the seeds of this species? If there is, insert this into the discussion.

Response: Thank you for your suggestion. We have added to the content in lines 323-328.

Author Response File: Author Response.pdf

Reviewer 3 Report

Comments and Suggestions for Authors

Dear authors,

This paper addresses the fact that S. sebiferum seeds possess physiological dormancy induced by endogenous inhibitors and could be broken by cold stratification. The authors demonstrate these effects on cabbage seeds using methanolic extracts from the seed coats and endosperm. Germination percentages and germination index increase with the duration of cold stratification. However, the authors did not identify the seed coats and endosperm inhibitors and their degradation under cold stratification. In a previous paper (Shah et al., 2018, Reference 15), the authors identified this internal inhibitor as Proanthocyanidins (PAs), present in S. sebiferum in seed coat and endosperm. 

The authors must read Shah and colleagues' paper carefully and use their approximation to address these questions:

1. What are the concentrations of PAs in the seed coats and endosperms?
2. Is there a relationship between cold stratification and PAs degradation?
3. Does this degradation improve seed germination?
4. Could be reduce the days of stratification?

 

The authors must also check the references section. 11 and 17.

Author Response

Response to reviewer#3

 

Comments and Suggestions for Authors

Dear authors,

This paper addresses the fact that S. sebiferum seeds possess physiological dormancy induced by endogenous inhibitors and could be broken by cold stratification. The authors demonstrate these effects on cabbage seeds using methanolic extracts from the seed coats and endosperm. Germination percentages and germination index increase with the duration of cold stratification. However, the authors did not identify the seed coats and endosperm inhibitors and their degradation under cold stratification. In a previous paper (Shah et al., 2018, Reference 15), the authors identified this internal inhibitor as Proanthocyanidins (PAs), present in S. sebiferum in seed coat and endosperm. 

The authors must read Shah and colleagues' paper carefully and use their approximation to address these questions:

1. What are the concentrations of PAs in the seed coats and endosperms?
2. Is there a relationship between cold stratification and PAs degradation?
3. Does this degradation improve seed germination?
4. Could be reduce the days of stratification?

Response: Thank you for your suggestion. This paper presented a preliminary investigation on the changes of inhibitors in different parts of Sapium sebiferum seeds during dormancy breaking by cold stratification. The study found that the changes of inhibitors in the endosperm and seed coat were different during the dormancy breaking. Really, we will conduct further qualitative and quantitative analyses of the inhibitors to gain a better understanding of their changes. We have also added to the issues you pointed out at Lines 323-328.

 

The authors must also check the references section. 11 and 17.

Response: Thanks for pointing that out. It has been modified.

Author Response File: Author Response.pdf

Round 2

Reviewer 3 Report

Comments and Suggestions for Authors

 This paper could be accept in present form.