Neodymium Nitrate Improves the Germination of Aged Wheat Seeds by Increasing Soluble Substances and Activating Antioxidative and Metabolic Enzymes in Seeds
Round 1
Reviewer 1 Report
The study is dedicated to improving quality of aged wheat seeds. The results reported in the text show that the method of improving germination of aged seeds is promising. However, the proposed method is potentially expensive and can lead to soil pollution, which must be discussed in the text. Furthermore, some of the references provided by the authors are inaccurate (the results in the papers cited are opposite from what is claimed in the text).
Ln 35 " normal storage conditions, post-harvest wheat seeds consume stored nutrients to provide energy, thereby maintaining metabolism " - there seems to be no such information in the cited article, and as far as I know, dry orthodox seeds do not maintain active metabolism during storage. Correction is required.
Ln 66 reference [20] states the opposite to what is claimed (the results were that rare earth elements inhibit germination)
Ln 71 the meaning is not clear (what is " non-commutative character " in relation to a chemical element?). The cited references are in Chinese.
It can be argued that the best strategy for seed producers is to delay seed ageing as long as possible using appropriate storage methods, perhaps the authors can comment what causes aged seed lots to accumulate?
Methods
Ln 130-136 The procedure for the analysis of photosynthetic pigments is inadequate, absorbance, not fluorescence is measured and in no case can samples be allowed to 'fade.' Possibly a translation mistake, the authors must check the description of this and other methods.
Ln 239 the significance level is p = 0.05 (not < 0.05) (therefore, treatments where p < 0.05 are considered significantly different)
The same abbreviation (GP) is used differently in Figure 1 and Table 1. I would suggest "germination percentage" in both cases. In Table 1 germination percentage at Day 4 is used, for better clarity this can be indicated as GP4 (an index, to avoid confusing it with GP at the final day of the germination test). There is also a term "germination energy" (germination percentage at a fixed interval, which is shorter than the total length of the germination test).
It is not conventional to present data with different units in the same figure.
Ln 288 this probably refers to the GP, but not other parameters presented in the Table1
The quality of the figures must be improved, because the text (axis labels, axis titles) is poorly visible.
Discussion
In general, the length of the Discussion section should be reduced (in some paragraphs the results are repeated). Instead, the authors should focus on the agreement and disagreement of their findings to the results of previous research, consider practical applicability of the results and direction of further studies.
Ln 427 Repetition of the results is not necessary. The increase in germination percentage and velocity is compared to seedling growth (Ln 428), which is not correct. What is meant by the "experimental gradient design"?
Photosynthetic pigments in the seedlings are synthesized after germination, so seed soaking can only have indirect effect.
I see a potential problem in the method used by the authors to test the effect of Nd nitrate: nitrate ions are known to stimulate seed germination. While the concentrations used were quite low, there still is a possibility that nitrate, rather than Nd caused the positive effects observed in the experiments. This could be discussed as well.
Conclusions: this section is supposed to be more concise.
Generally text is good, in some cases grammar correction is required
Author Response
The study is dedicated to improving quality of aged wheat seeds. The results reported in the text show that the method of improving germination of aged seeds is promising. However, the proposed method is potentially expensive and can lead to soil pollution, which must be discussed in the text. Furthermore, some of the references provided by the authors are inaccurate (the results in the papers cited are opposite from what is claimed in the text).
Answer :That's good advice. Thank you for your guidance. The price of neodymium nitrate is about $50 for 100 g, the recommended concentration is 20uM, and generally 1 kg of seed uses 3 kg of water, so the production cost is about $1.8 per hectare of wheat. The content about the dosage of neodymium nitrate recommended in this test and environmental pollution has been added in the discussion section.The author has also proofread the references.
Ln 35 " normal storage conditions, post-harvest wheat seeds consume stored nutrients to provide energy, thereby maintaining metabolism " - there seems to be no such information in the cited article, and as far as I know, dry orthodox seeds do not maintain active metabolism during storage. Correction is required.
Answer:This is a good question. To quote this sentence, the author wants to express that seed aging and vitality decline are inevitable normal events, which provides the background for this study, because aging will lead to the decline of seed germination potential, and it is necessary to improve seed germination potential in production.
Ln 66 reference [20] states the opposite to what is claimed (the results were that rare earth elements inhibit germination)
Answer : This is a good question. The author consulted the original literature again, and the original lanthanum has different effects on different organs at different concentrations, promoting effect in roots and inhibiting effect in stems. This illustrates the complexity of the effects of lanthanum, which the authors cite primarily as having an effect on antioxidant protective enzymes. It is necessary to study the effect of rare earth elements on seed germination. The effects of different concentrations of rare earth elements are different, and the specific effects of each element need to be studied in detail.
Ln 71 the meaning is not clear (what is " non-commutative character " in relation to a chemical element?). The cited references are in Chinese.
It can be argued that the best strategy for seed producers is to delay seed ageing as long as possible using appropriate storage methods, perhaps the authors can comment what causes aged seed lots to accumulate?
Answer:This is a good question, thank you for your guidance.The author here is to explain the reasons for choosing to do neodymium treatment, but also to provide ideas for analyzing the experimental results.Lanthanum and neodymium belong to the same rare earth element with the same number of electrons in the outermost layer, but lanthanum has 3 electron layers and neodymium has 4 layers, which leads to the chemical properties of neodymium and lanthanum are similar, but there are differences. Predecessors have done many studies on the influence of lanthanum on seed germination, and neodymium is selected in this experiment, which may also have an effect on seed germination.
Methods
Ln 130-136 The procedure for the analysis of photosynthetic pigments is inadequate, absorbance, not fluorescence is measured and in no case can samples be allowed to 'fade.' Possibly a translation mistake, the authors must check the description of this and other methods.
Answer : This is a good question. thank you for your guidance. The description here is wrong. The description phenomenon is that the leaf residue becomes white after homogenization, and this part has been changed to “the leaves color had turned white”.
Ln 239 the significance level is p = 0.05 (not < 0.05) (therefore, treatments where p < 0.05 are considered significantly different)
The same abbreviation (GP) is used differently in Figure 1 and Table 1. I would suggest "germination percentage" in both cases. In Table 1 germination percentage at Day 4 is used, for better clarity this can be indicated as GP4 (an index, to avoid confusing it with GP at the final day of the germination test). There is also a term "germination energy" (germination percentage at a fixed interval, which is shorter than the total length of the germination test).
It is not conventional to present data with different units in the same figure.
Answer: This is a good question, thanks for your guidance, "the p ≤ 0.05 significance level" has been changed to" the p = 0.05 significance level"; The acronym GP in Table 1 and Figure 1 has been changed to GP4.
Ln 288 this probably refers to the GP, but not other parameters presented in the Table1
The quality of the figures must be improved, because the text (axis labels, axis titles) is poorly visible.
Answer :This is a good question, "GP" here also refers to the germination potential as before, according to your suggestion, has been changed to" GP4 ", the quality of this table has been modified and improved.
Discussion
In general, the length of the Discussion section should be reduced (in some paragraphs the results are repeated). Instead, the authors should focus on the agreement and disagreement of their findings to the results of previous research, consider practical applicability of the results and direction of further studies.
Ln 427 Repetition of the results is not necessary. The increase in germination percentage and velocity is compared to seedling growth (Ln 428), which is not correct. What is meant by the "experimental gradient design"?
Photosynthetic pigments in the seedlings are synthesized after germination, so seed soaking can only have indirect effect.
I see a potential problem in the method used by the authors to test the effect of Nd nitrate: nitrate ions are known to stimulate seed germination. While the concentrations used were quite low, there still is a possibility that nitrate, rather than Nd caused the positive effects observed in the experiments. This could be discussed as well.
Conclusions: this section is supposed to be more concise.
Answer :According to your suggestion, the duplicate part has been deleted. This experiment is a seed soaking experiment, while Lu Ninghai's experiment is about the effect of spraying on wheat at seedling stage. The original purpose is to show that the effect of neodymium concentration has similar differences, but there are differences, which may be because the concentration used in Lu Ninghai's experiment design is not 20uM, and perhaps the concentration of neodymium 20uM is appropriate. For brevity, modify to remove the reference.
Author Response File: 
Author Response.pdf
Reviewer 2 Report
The submitted manuscript is very current, with relatively significant potential for subsequent research and citation, the manuscript focused on the study of the effect of neodymium nitrate on older seeds. The manuscript is relatively carefully written, but still needs editing. The methodology is described accordingly. What were the seed storage conditions? What was the source of the seed? The results are a description of the graphs and tables, but the bars must be supplemented with the SE and not just the statistical deviation. The graphs are small and unreadable. Their adjustment is necessary. It is not entirely clear from the text why the pigment content was monitored. So what is the relationship of pigment content to germination, seed emergence and enzymatic activity? It is a pity that the individual results were not connected within the framework of correlation relations. The discussion is descriptive in places. I recommend adding a summary. It is necessary to unify the review of the literature, as some sources are cited in abbreviated form.
Author Response
The submitted manuscript is very current, with relatively significant potential for subsequent research and citation, the manuscript focused on the study of the effect of neodymium nitrate on older seeds. The manuscript is relatively carefully written, but still needs editing. The methodology is described accordingly. What were the seed storage conditions? What was the source of the seed?
Answer：This is a good suggestion, and the authors have supplemented the geographical location and temperature data of the experiment site.The wheat varieties used are cultivated by Henan Institute of Science and Technology and approved by the state.This wheat variety is a winter variety.
The results are a description of the graphs and tables, but the bars must be supplemented with the SE and not just the statistical deviation. The graphs are small and unreadable. Their adjustment is necessary.
Answer：This is a good suggestion, thanks for your guidance, this figure has been adjusted.
It is not entirely clear from the text why the pigment content was monitored. So what is the relationship of pigment content to germination, seed emergence and enzymatic activity?
Answer :This is a good suggestion, thank you for your guidance. Seed emergence becomes weak after aging, showing slower metabolism, smaller leaves and lighter color. This paper discusses the effects of neodymium immersion on seed germination. Photosynthetic pigment and enzyme activities are related to aging, so it is necessary to detect changes in photosynthetic pigment and metabolic enzyme activities.
It is a pity that the individual results were not connected within the framework of correlation relations. The discussion is descriptive in places. I recommend adding a summary.
Answer：This is a good suggestion, but this paper focuses on the effects of neodymium immersion on physiological, biochemical and metabolic enzymes during seed germination, and the author thinks that it is not appropriate to increase the correlation analysis of each index.
It is necessary to unify the review of the literature, as some sources are cited in abbreviated form.
Answer :Thanks for your suggestion, the author has checked and corrected the abbreviation entry errors in the literature citations
Author Response File: Author Response.pdf
Reviewer 3 Report
Dear Authors,
Nice manuscript, well-written, and well-presented research results.
I just have questions about the natural neodymium presence in the soil where wheat is growing in China. Do you have some data about that? Also, rare earth elements are considered toxic, and would be good to discuss this issue more deeply in the Discussion section.
Also, I am wondering about using mercuric chloride in your work for the seed sterilization. This compound has been forbidden for many years to be used in laboratory experiments!
These comments or questions are still not preventing me from recommending your manuscript for publishing.
25.8.2023
Author Response
Nice manuscript, well-written, and well-presented research results.
I just have questions about the natural neodymium presence in the soil where wheat is growing in China. Do you have some data about that? Also, rare earth elements are considered toxic, and would be good to discuss this issue more deeply in the Discussion section.
Answer:That's a good question. Thank you for your guidance. The distribution trend of rare earth content in the farming area of China is slightly higher in the south and slightly lower in the north, with an average of 200ug/g. The content of neodymium in the soil layer ranges from 7.5 -162ug/g, with an average of 32.5ug/g, Under this concentration, rare earth is not enough to produce biological effects on crops, so it is possible to increase crop yield by using supplemental rare earth in the farming area with low rare earth concentration.This paper only discusses the suitable concentration of neodymium for wheat seed soaked, The experiment recommended using a lower dose of neodymium nitrate, wheat seed soaked will not cause super fatigue and environmental pollution, the discussion section has added relevant content.
Also, I am wondering about using mercuric chloride in your work for the seed sterilization. This compound has been forbidden for many years to be used in laboratory experiments!
Amnswer :This is a good question, the seed disinfection methods are 70% ethanol, 2% sodium hypochlorite and 0.1% mercury chloride soaking seeds, etc. The soaking time of 0.1% mercury chloride was the shortest and the effect was the best. After soaking the seeds with 0.1% mercuric chloride for 15 minutes and washing them with distilled water 5 times, the germination of the seeds was not affected. If mercury chloride is banned, 2% sodium hypochlorite can be considered as an alternative.
Author Response File: Author Response.pdf
Reviewer 4 Report
Research is important for the advancement of scientific knowledge and application in practice. I suggest that the manuscript be published after minor corrections
Author Response
Research is important for the advancement of scientific knowledge and application in practice. I suggest that the manuscript be published after minor corrections
Answer :Thank you for your evaluation of the author's work. The author carefully read through the whole text and tried his best to revise some contents.
Author Response File: Author Response.pdf
Round 2
Reviewer 1 Report
The comments and suggestions in the review were not adequately addressed.
The quality of newly added paragraphs is considerably lower than the rest of the text
Author Response
Open Review
(x) I would not like to sign my review report
( ) I would like to sign my review report
Quality of English Language
( ) I am not qualified to assess the quality of English in this paper
( ) English very difficult to understand/incomprehensible
( ) Extensive editing of English language required
( ) Moderate editing of English language required
(x) Minor editing of English language required
( ) English language fine. No issues detected
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Comments and Suggestions for Authors
The comments and suggestions in the review were not adequately addressed.
Comments on the Quality of English Language
The quality of newly added paragraphs is considerably lower than the rest of the text
Submission Date
Answer: Thank you for your suggestions and help! which will help improve the quality of your paper.The manuscript has been polished by experts from Beijing Liwen Editing Company, with emphasis on new paragraphs, and the full text has been further polished and corrected.
Author Response File: Author Response.pdf
