Hydroponic Optimization and Screening of Aluminum Tolerance on Finger Millet (Eleusine coracana (L.) Gaertn.) Accessions and Cultivars
, Kassahun Tesfaye 2,4, Cecilia Hammenhag 3, Rodomiro Ortiz 3 and Mulatu Geleta 3
Round 1
Reviewer 1 Report
The manuscript “Hydroponic Optimization and Screening of Al-Tolerance on Finger Millet accessions and cultivars” from Haftom Brhane et all. describes an experiment to test Al tolerance of finger millet and cultivars accessions. The experimental design is well explained and the results seem relevant.
Major remarks
In general
I miss the scientific context. The experimental conditions chosen are based on the response of plants to different concentrations of Al and a specific acidity, but it is not mentioned whether these conditions are comparable to free Al in natural soils. The experimental levels may be outside the relevant range in nature, which would make the results difficult to extrapolate to real life.
In addition, germinating seeds are tested and seedling length is measured. It is not known whether the sensitivity of seedlings to Al correlates with later plant performance.
The hydroponic system, although elegant, does not represent the effect in soil. Therefore, a conformation experiment consisting of growing selected tolerant and intolerant accessions in acidic soil would complete the story.
I also miss the discussion of the relevant agronomic conditions. Why is the soil acidic, is it because of NO emissions or because of soil erosion? Couldn't basic fertilizer be an alternative, cheaper and more sustainable solution? This should at least be discussed.
Specific:
It is not clear why 75μM and 100μM were chosen as concentration. Is 100μM a concentration that occurs in acidic soils in Etheopia? Same question for the acidity.
Some measurements from the pilot experiment seem not to be confirmed in the large experiment, e.g.:
Ec-215944 table 1 RLL is 36% and RSL is 133% while in table 2 it is 50% and 69%, respectively
Ec-216051 table 1 RLL is 20% and RSL is 133% while in table 2 it is 42% and 47%, respectively
Ec-216057 table 1 RLL is 13% and RSL is 170% while in table 2 it is 38% and 53%, respectively
The best lines Ec-215836, Ec-215845 and Ec-229722 performed better with 100 μM Al than without, what could this mean? And does experimental Al tolerance mean tolerance to all changes in acidic soils? I understand that the availability of other ions may decrease...The relevance of the experiment related to real soil conditions should be discussed.
Minor remarks:
P3, 2nd par. line 4: Al2(SO4)3.18H2O is the chemical formula for the provided salt form. In solution it is just Al2(SO4)3
Table 1: RRL% would suggest a percentage in the column, however at 0μM a different value is given, which is confusing. Taking into account the accuracy of the measurement, values may be given without digits after the point.
page 11: “RGI” is not explained
P3 line 8: “The pH of the nutrient adjusted at 4.3 by using 1M HCl or NaOH and renewed every day (24 hrs.) in….” unclear, change to: “The pH of the nutrient solution was adjusted to 4.3 using 1M HCl or NaOH, and the solution was renewed every day (24 hrs.) in order to refresh the detoxified solution ….”
P3 line 12: “recoded” should be “recorded” or “measured”
P3, 3rd par. line 1: “… and SL also measured from the cotyledon to the tip of the shoot using a ruler. ” must be “.. and SL also measured from the base of the cotyledon to the tip of the shoot using a ruler.
Author Response
RESPONSE TO REVIEWER-1’S COMMENTS
We appreciate the comments and carefully considered them during the revision of the manuscript and provided a point-by-point response.
COMMENT-1: The manuscript “Hydroponic Optimization and Screening of Al-Tolerance on Finger Millet accessions and cultivars” from Haftom Brhane et all. Describes an experiment to test Al tolerance of finger millet and cultivars accessions. The experimental design is well explained and the results seem relevant.
RESPONSE TO COMMENT-1: Thank you for the positive assessment of the manuscript, valuable comments and the advice.
COMMENT-2: I miss the scientific context. The experimental conditions chosen are based on the response of plants to different concentrations of Al and a specific acidity, but it is not mentioned whether these conditions are comparable to free Al in natural soils. The experimental levels may be outside the relevant range in nature, which would make the results difficult to extrapolate to real life.
RESPONSE TO COMMENT-2: Thank you for your suggesting to consider the level of acidity in nature, while using hydroponic nutrient solution. This research focused only on preliminary optimizing and screening of finger millet accessions to observe which genetic materials are tolerant and which are susceptible to what extent they are tolerant. Our work is continuous and we will evaluate selected finger millet accessions (from tolerant, intermediate, and susceptible) from this hydroponic under multi-site filed.
COMMENT-3: In addition, germinating seeds are tested and seedling length is measured. It is not known whether the sensitivity of seedlings to Al correlates with later plant performance.
RESPONSE TO COMMENT-3: We appreciate your suggestion to include correlation analysis to correlate the plant performance under early stage (root length and shoot length) with later performance such as grain yield, plant height etc. Our work begins from preliminary optimizing and screening up to multi-site field evaluation, genomic and transcriptomic analysis on selected accessions. Therefore, we will include correlation analysis and other analysis in our next manuscript which include data from multi-site field.
COMMENT-4: The hydroponic system, although elegant, does not represent the effect in soil. Therefore, a conformation experiment consisting of growing selected tolerant and intolerant accessions in acidic soil would complete the story.
RESPONSE TO COMMENT-4: The comment is highly appreciated. More discussion have been added (See line from 520 – 672, with track change).
COMMENT-5: I also miss the discussion of the relevant agronomic conditions. Why the soil is acidic, is it because of NO emissions or because of soil erosion? Couldn't basic fertilizer be an alternative, cheaper and more sustainable solution? This should at least be discussed.
RESPONSE TO COMMENT-5: Thank you for the comment. More discussion about this is now added with track changes (see page number 2, 2nd paragraph line from 10-16).
COMMENT-6. It is not clear why 75μM and 100μM were chosen as concentration. Is 100μM a concentration that occurs in acidic soils in Ethiopia? Same question for the acidity.
RESPONSE TO COMMENT-6: Thank you for the comment. We have published one paper (Brhane et al. 2018) on optimization and screening of Al-tolerance on finger millet. We used that research as a starting point to conduct this new project. In that manuscript we have reported 112.5 µM Al-concentration was the optimum (threshold) level. To confirm that we conducted rapid optimization experiment under control, 75, and 100 µM Al-concentrations and then we choose 100 µM as threshold.
COMMENT-7. Some measurements from the pilot experiment seem not to be confirmed in the large experiment, e.g.:
Ec-215944 table 1 RLL is 36% and RSL is 133% while in table 2 it is 50% and 69%, respectively
Ec-216051 table 1 RLL is 20% and RSL is 133% while in table 2 it is 42% and 47%, respectively
Ec-216057 table 1 RLL is 13% and RSL is 170% while in table 2 it is 38% and 53%, respectively
RESPONSE TO COMMENT-7: Thank you very much for spotting the difference of RRL and RSL values across the tables. This variation is due to there is variation in size and color among individuals in each accession. To minimize such big variation we select individuals having the same color, same seed size, and even after germination we used seedlings with similar performance. By taking this variation in to account we select one individual from each accessions for multi-site field evaluation, genomic and transcriptomic analysis.
COMMENT-8. The best lines Ec-215836, Ec-215845 and Ec-229722 performed better with 100 μM Al than without, what could this mean? And does experimental Al tolerance mean tolerance to all changes in acidic soils? I understand that the availability of other ions may decrease...The relevance of the experiment related to real soil conditions should be discussed.
RESPONSE TO COMMENT-8: after we decided the threshold level of Al-stress, we rapidly screened more accessions at 100 µM and control (without Al-stress) in parallel. Relative root length (RRL; root length under treatment/root length under control*100) was estimated and these accessions showed better performance than others. Therefore, it is not to mean that the selected best lines were performed better at 100 µM than without. Thank you also for the second question “does Al tolerance mean tolerance to all changes in acidic soils? There are different factors associated with soil acidity such as phosphorus deficiency, calcium deficiency, decreasing microbial diversity, difficulties in influx of macro-nutrients, manganese stress. We tried to control everything like we provide calcium, phosphorus and other nutrients in to the hydroponics, except we added more Al in to the solution to create Al-stress on plants.
COMMENT-9. P3, 2nd par. line 4: Al2(SO4)3.18H2O is the chemical formula for the provided salt form. In solution it is just Al2(SO4)3
RESPONSE TO COMMENT-9. Thank you for the comment. Now it is corrected as Al2 (SO4)3, see line P3, 2nd par. line 4, 8.
COMMENT-10. Table 1: RRL% would suggest a percentage in the column, however at 0μM a different value is given, which is confusing. Taking into account the accuracy of the measurement, values may be given without digits after the point.
RESPONSE TO COMMENT-10. Thank you for the comment. Now it is corrected see page 4 with track change.
COMMENT-11. Page 11: “RGI” is not explained
RESPONSE TO COMMENT-11. Thank you for the comment. Now it is corrected see page 11 with track change.
COMMENT-12. P3 line 8: “The pH of the nutrient adjusted at 4.3 by using 1M HCl or NaOH and renewed every day (24 hrs.) in….” unclear, change to: “The pH of the nutrient solution was adjusted to 4.3 using 1M HCl or NaOH, and the solution was renewed every day (24 hrs.) in order to refresh the detoxified solution ….”
RESPONSE TO COMMENT-12. Thank you for the comment. Now it is corrected following your suggestion see page 11 with track change. The pH of the nutrient was adjusted to 4.3 by using 1M HCl or NaOH and the solution was renewed every day (24 hrs.) in order to refresh the detoxified solution and ensure continuous exposure of the seedlings to Al-ions.
COMMENT-13. P3 line 12: “recoded” should be “recorded” or “measured”
RESPONSE TO COMMENT-13. Thank you for the comment. Now it is corrected with track change.
COMMENT-14. P3, 3rd par. line 1: “… and SL also measured from the cotyledon to the tip of the shoot using a ruler. ” must be “.. and SL also measured from the base of the cotyledon to the tip of the shoot using a ruler.
RESPONSE TO COMMENT-14. Thank you for the comment. Now it is corrected with track change as “SL also measured from the base of the cotyledon”.
We hope that the revision made based on your comments are satisfactory.
Reviewer 2 Report
The work presented by Haftom Brhane et al. focus on the results of rapid hydroponic screening methods for Al tolerance of different Finger millet cultivars and accessions. The topic of the manuscript is of a great importance, and all these results can help in new breeding programs and ongoing breeding works, because authors tested 328 finger millet accessions and 15 cultivars. All studies related to heavy metals are valuable not just for researchers, but decision makers, and can help prepare and implement an action plan.
Because of the absence of numbering of the lines, I cannot write my suggestions in detail in my review report. All my comments are in the attached pdf file.
General comments:
1. Extensive editing of English language required!
2. References must be numbered in order of appearance in the text (including citations in tables and legends) and listed individually at the end of the manuscript.
3. Several flaws were found in the manuscript; a lack of a clear explanations was also observed. Sometimes it was difficult to understand the sections, it is confusing, it contains very general statements.
4. Correction and supplement is needed in the Introduction section. There is not any description about the heavy metals, hydroponic methods, screening methods, etc.
5. I suggest a more precise definition of the research aim.
6. Please, insert some nice, good quality photos, it will improve the quality of the ms.
7. Tables must be corrected (see comments in the pdf file).
8. The ‘Discussion’ part is a summary of the result with some comparison with other studies. Please, supplement if possible.
9. Where is the Supplementary Table and Figure?
Comments for author File: 
Comments.pdf
Please, see my general comments!
Author Response
RESPONSE TO REVIEWER-2’S COMMENTS
We appreciate the comments and we carefully considered them during the revision of the manuscript and provided a point-by-point response.
COMMENT-1: Hydroponic Optimization and Screening of Al-Tolerance on Finger Millet accessions and cultivars. I suggest inserting the scientific name!....Finger Millet (Eleusine coracana (L.) Gaertn.) accessions
RESPONSE TO COMMENT-1: Thank you for the comment and we have inserted the scientific name of finger millet both in the tile and abstract with track change. The updated title is now “Hydroponic Optimization and Screening of Al-Tolerance on Finger Millet (Eleusine coracana (L.) Gaertn.) Accessions and Cultivars”
COMMENT-2: "...annual self-pollinating allotetraploid (xxx),...". re-write the sentence
RESPONSE TO COMMENT-2: Comment accepted with thanks, and comma added with track change.
COMMENT-3: cereal crop that belongs
RESPONSE TO COMMENT-3: Thank you for the comment. the sentence is now improved following your suggestion "...cereal crop belonging to...." with track change.
COMMENT-4: In Ethiopia, it is commonly grown throughout Tigray, in parts of Oromia (Wellega, IIluababora, and Hararghe), Amhara (Gonder and Goj[1]jam) and the Southern Nations and Nationalities and People’s region (Gamo-Gofa and Hossana) (Admassu et al., 2009; Kinfe et al., 2017).
RESPONSE TO COMMENT-4: Thank you for the comment. In Ethiopia, finger millet is produced by small scale farmers in Tigray, Wellega, IIluababora, Hararghe, Gonder, Gojjam, Gamo-Gofa and Hossana [5, 6]. We hope this addresses your concerns.
COMMENT-5: In Ethiopia, finger millet ranks sixth important cultivated cereal crop after teff, wheat, maize, barley, and sorghum (Seyoum et al., 2019). Do you mean: '...millet is the sixth most important cultivated...'?
RESPONSE TO COMMENT-5: Thank you for the comment. In Ethiopia, finger millet is the sixth most important cultivated cereal crop after teff, wheat, maize, barley, and sorghum [7].
COMMENT-6: , essential amino acids, gluten-free, and
RESPONSE TO COMMENT-6: Thank you for the comment. Now it is corrected as with track change.
COMMENT-7: Even though it is an important nutritional security and environment resilient crop, according to Ethiopian central statistics agency its current productivity is low i.e. 2.76 t ha-1 . This might be due to unavailability of improved cultivars, poor attitude towards the crop, drought, blast, soil salinity, soil acidity, and moisture stress are the most serious challenges (Lule et al., 2014; Semahegn et al., 2021). Re-write the sentence
RESPONSE TO COMMENT-7: Thank you for the comment. Now we re-write the sentence with track change.
COMMENT-8: This limitation can be reduced by developing finger millet cultivars which are more resistance or tolerance to acidic soils. Re-write this sentence
RESPONSE TO COMMENT-8: Thank you for the comment. This limitation can be reduced by developing finger millet cultivars which are more tolerant or resistant to acidic.
COMMENT-9: Table 1. Root length (RL) and shoot length (SL) of finger millet accessions and cultivars (Acc/Cul) grown under 0 µM, 75 µM, and 100 µM Al-concentrations in hydroponic nutrient solution. Relative root length (RRL) and relative shoot length (RSL) in percent estimated. The Table is confusing. Preparing two Tables from Table 1. Would make the tables more understandable. Table or Figure: with the SL and RL (cm) Table: with RRL and RSL (%) data In both tables, units and significant differences (*) between genotypes should be inserted.
RESPONSE TO COMMENT-9: thank you for the comments and we made two figures and one table from Table 1 following your kind suggestions.
COMMENT-10: This implies that a low Al concentration (less than 75 µM) was not strong enough to create stress condition on finger millet root and high Al concentration above 100 µM inhibits growth in all finger millet varieties without discrimination (Table 1). Re-write this sentence
RESPONSE TO COMMENT-10: This indicates low Al concentration was not strong enough to create stress condition on finger millet root and high Al concentration above 100 µM inhibits growth in all finger millet varieties without discrimination (Table 1).
COMMENT-11: Based on their RRL, the 328 accessions were classified into three tolerance groups. Accessions grouped as Al-tolerant were those that had RRL ≥ 80%, whereas intermediate were between 80% and 20% cm and susceptible were those that had below 20%. I suggest putting this information also to the Material and methods section.
RESPONSE TO COMMENT-11: based on your suggestion we moved this part to material and methods indicated with track change.
COMMENT-12: Corrections are needed in this Table: Units are missing, Structural corrections, There are 4 tables without Table captions (Which table related to the control results and which to the other (100 µM Al3+ concentration)?
RESPONSE TO COMMENT-12: Thank you for your comments. Now we added correction in to the table such as units of RRL and RSL as percent, we added caption in each table. The table generally indicates RRL and RSL of finger millet accessions evaluated under control and treatment (100 uM Al-concentration). The RRL was calculates as Root length under treatment/Root length under control*100, the same to relative shoot length.
COMMENT-13: The authors describe the results for the third time in this section. I suggest writing about future prospects/opportunities/solutions, benefits, and further plans related to this study.
RESPONSE TO COMMENT-9: Even though the level of soil acidity is not known in Ethiopia, results of the present study suggests that there are individual accessions that can tolerate acidic soils and some of the m are highly susceptible. To confirm their performance, the accessions should be tested under multi-site filed by considering control and treated environments. Furthermore, association studies also be considered to correlate field performance with genomic background. Transcriptomic analysis on the best tolerant and least susceptible should be tested by taking samples from different plant tissue (root, leaf, and stem) by taking at different time interval (0, 12hrs, 24hrs, 48, and 72hrs). Finally, Al-tolerant lines identified from this study will be used as input to the finger millet breeding program in relation to Al-tolerance in the country and beyond. If anyone is interested to study Al-tolerance on finger millet it is required to include wild types for comparative analysis.
COMMENT-14: References must be numbered in order of appearance in the text (including citations in tables and legends) and listed individually at the end of the manuscript.
RESPONSE TO COMMENT-14: references cited in order of appearance following the guidelines.
COMMENT-15: There is not any description about the heavy metals, hydroponic methods, screening methods, etc.
RESPONSE TO COMMENT-15: thank you for the comment. Now description of hydroponic and about heavey metal is added in the introduction with track change.
COMMENT -16: Please, insert some nice, good quality photos, it will improve the quality of the ms.
RESPONSE TO COMMENT-16: thank you for your comment to add photo. Now, good quality photo is added as figure 4.
COMMENT -17: Where is the Supplementary Table?
RESPONSE TO COMMENT-17: it was submitted separately, but here also attached.
We hope that the revision made based on your comments are satisfactory.
Reviewer 3 Report
Hydroponic Optimization and Screening of Al-Tolerance on Finger Millet accessions and cultivars
Manuscript ID: agronomy-2405662
Submitted to Agronomy Section Crop Breeding and Genetics
The manuscript “Hydroponic Optimization and Screening of Al-Tolerance on Finger Millet accessions and cultivars” presents a one-year study of Aluminium stress tolerance of a large group (328 accessions) of Eleusine coracana.
The authors previously studied the effect of Aluminium on 288 accessions and six improved national cultivars of finger millet and the results were published (Brhane et al. 2018) in the Journal of Agricultural Biotechnology and Sustainable Development, DOI: 10.5897/JABSD2017.0289). However, in the manuscript submitted for review, the authors did not refer to the results obtained previously in 2018.
The Abstract of the manuscript is adequate to the content.
The Introduction provides a characterization of the species studied, however, it is brief in providing knowledge on the toxic effects of Al on plants. Please expand this part of the text.
Furthermore, the authors do not explain why they undertake the experiment in hydroponics. Please provide an explanation in the text of the manuscript.
P1 L 30 Finger millet (Eleusine coracana L???)
Please insert the discoverer.
The methodology should be supplemented with some information: Why were these 15 specific varieties chosen? Under which conditions of temperature, lighting was the experiment conducted?
Figure 1 is missing.
A fundamental deficiency of the manuscript is the lack of repetition of the experiment. In my opinion, it would be better to study fewer objects but more thoroughly and according to the rules of mathematical statistics, i.e. using 2-3 repetitions of the experiment. No repetition makes it impossible to perform statistical analyses.
P 3 L 110-111 In the Materials and Methods chapter, the authors mention that 274 accessions were selected for further field, genomic and transcriptomic evaluations, but they do not present the results of these studies.
The authors should take care to explain the abbreviations used for the first time in each chapter, e.g. P 3 L 113-114 - RL, SL.
In my opinion, the root and stem length measurements were made on insufficient number of plants, i.e. 5 individuals per accession, which may influence the final result.
Shoot length (SL) of the accession and cultivars grown under control (0 μ M Al-concentration) varied from 0.46 cm (Urji) to 1.18 cm (Ec-215888 and Ec-215911). At 75 μ M Al-concentration RSL ranged from 63.46% (215897) to 175.86% (Urji), and at 100 μ M Al-con-centration RSL ranged from 41.69% (215888) to 195.22% (213314) (Table 1).
Where are the results given in cm? Please provide a source.
P 4 Table 1 Are the differences between the genotypes presented in Table 1 statistically significant? If so, indicate which ones?
P 5 Table 2
Please state if SS is the total sum of squares or the intra-group sum of squares. Why were only 5 plants measured per accession ?
P 5 L 163-165 ......At Al concentration above 100 μ M, the growth of roots of all the varieties were greatly hampered to the extent that there was nearly no difference among them……………………………
While the methodology states that the highest Al dose used was 100 μM.
P 5 L168-172 ……. Finger millet accessions (n=328) were rapidly screened after initially decided the op-
timum Al concentration (i.e. 100 mM). There were observable differences among individ-
uals within an accession such as variation in grain color and grain size and to take this
heterogeneity into account each accession was evaluated systematically by recording data
from similarly performing individuals…………..
These observations are not supported by the results (figures, tables). Furthermore, the authors refer to Table 3.2 (P5 L176) , which is not included in the text of the manuscript or in supplementary materials.
P5 L 174-175 Where did the RRL threshold values come from to determine the three tolerance groups? There is no reference to the literature. Explain it in the text.
In my opinion, the results for the control varieties in Table 3 are missing.
The discussion needs to be revised. Some parts of the text are not related to the results, e.g. Discussion P10 L13-15 and P10 L 31-35.
P11 L 45-49” ……………..and develop Al-tolerant genes as enhance their tolerance……..”
Genotypes develop genes? What do you mean by develop genes? Explain it in the manuscript.
P11 L 70-72 “Accessions identified as Al-tolerant under hydroponic experiment often showed improved agronomic performance compared to Al-susceptible (Fikiru et al., 2011; Hede et al., 2002;
Negusse et al., 2022)”.
Sometimes in the discussion the authors use incorrect citations, for example (Fikiru et al. 2011). The study material consisted of 10 improved lentil varieties. The authors do not mention anything about these varieties being AL-tolerant
Some conclusions are not supported by the results: “Lower Al-concentration below 75 μ M had no significant effect on the RL and SL of most finger millet cultivars and accessions, while the growth starts to decline with increasing Al-concentration”. In my opinion the lowest concentration used in the experiment was 75 μM.
In my opinion, it would be useful to enrich the manuscript with a table showing which part of Ethiopia the accessions come from and what is the acidity of the soils there? Furthermore, improvement of English language is necessary (Notes made on the initial PDF file). The text contains a number of language errors. It cannot be printed in its current form.
Improvement of English language is necessary (Notes are made on the initial PDF file). The text contains a number of language errors. It cannot be printed in its current form.
Author Response
RESPONSE TO REVIEWER-3’S COMMENTS
We appreciate the comments and we carefully considered them during the revision of the manuscript and provided a point-by-point response.
COMMENT-1: The authors previously studied the effect of Aluminium on 288 accessions and six improved national cultivars of finger millet and the results were published (Brhane et al. 2018) in the Journal of Agricultural Biotechnology and Sustainable Development, DOI: 10.5897/JABSD2017.0289). However, in the manuscript submitted for review, the authors did not refer to the results obtained previously in 2018.
RESPONSE TO COMMENT-1: Thank you for the comment and we have included our previous findings with track change on page 4 and page 13.
COMMENT-2: The Introduction provides a characterization of the species studied, however, it is brief in providing knowledge on the toxic effects of Al on plants. Please expand this part of the text.
RESPONSE TO COMMENT-2: Comment accepted with thanks, and we have included toxic effect of Al-stress on plant and its management practice in Ethiopia is now added following your recommendation. See on page 2 paragraph 2 line 9-14 and paragraph 3 line 1-7.
COMMENT-3: Furthermore, the authors do not explain why they undertake the experiment in hydroponics. Please provide an explanation in the text of the manuscript.
RESPONSE TO COMMENT-3: Thank you for the comment. Now we have explained why we used hydroponic to screen finger millet accessions against Al-stress with track change. See page 2, paragraph 4 line 1-10. We hope this addresses your concerns.
COMMENT-4: P1 L 30 Finger millet (Eleusine coracana L???)
RESPONSE TO COMMENT-4: Thank you for the comment. Corrected as Finger millet (Eleusine coracana (L) Gaertn.).
COMMENT-5: The methodology should be supplemented with some information: Why were these 15 specific varieties chosen? Under which conditions of temperature, lighting was the experiment conducted?
RESPONSE TO COMMENT-5: Thank you for the comment. Now how we selected the specific cultivars and accessions and their germinations conditions added following your comments. See from page 2 paragraph 5 line 6 up to page 3 from line 1 to 9.
COMMENT-6: Figure 1 is missing.
RESPONSE TO COMMENT-6: Thank you for the comment. Now it is added. See page 3.
COMMENT-7: P 3 L 110-111 In the Materials and Methods chapter, the authors mention that 274 accessions were selected for further field, genomic and transcriptomic evaluations, but they do not present the results of these studies.
RESPONSE TO COMMENT-7: Thank you for the comment. Now we remove that part since we diidn’t have data on filed, genomic and transcriptomic analysis.
COMMENT-8: The authors should take care to explain the abbreviations used for the first time in each chapter, e.g. P 3 L 113-114 - RL, SL.
RESPONSE TO COMMENT-8: Thank you for the comment. now RL, Sl as well as other abbreviations cross checked following your recommendations.
COMMENT-9: Shoot length (SL) of the accession and cultivars grown under control (0 μ M Al-concentration) varied from 0.46 cm (Urji) to 1.18 cm (Ec-215888 and Ec-215911). At 75 μ M Al-concentration RSL ranged from 63.46% (215897) to 175.86% (Urji), and at 100 μ M Al-con-centration RSL ranged from 41.69% (215888) to 195.22% (213314) (Table 1).
Where are the results given in cm? Please provide a source.
RESPONSE TO COMMENT-9: thank you for the comments. Now we split Table one in to two Figures and one table. The results given in cm are now shown in Figure 2 for the root length and Figure 3 for the shoot length measurement. Table 1 indicates RRL and RSL of the accessions.
COMMENT-10: Please state if SS is the total sum of squares or the intra-group sum of squares. Why were only 5 plants measured per accession ?
RESPONSE TO COMMENT-10: thank you for your comment. The abbreviation SS is now corrected and it represents total sum of squares.
COMMENT-11: Furthermore, the authors refer to Table 3.2 (P5 L176) , which is not included in the text of the manuscript or in supplementary materials.
RESPONSE TO COMMENT-11: thank you for spotting the error. Now it is corrected to Table 2.
COMMENT-12: P5 L 174-175 Where did the RRL threshold values come from to determine the three tolerance groups? There is no reference to the literature. Explain it in the text.
RESPONSE TO COMMENT-12: Thank you for your comments. There is clear standard to group accessions as tolerant, intermediate, susceptible based on their RRL or RL. We made this grouping by in-house way.
COMMENT-13: In my opinion, the results for the control varieties in Table 3 are missing.
RESPONSE TO COMMENT-9: Table 3 shows RRL and RSL of 328 finger millet accessions. RRL and RSL were analyzed as follows: Therefore, there is no separate column for the control values.
COMMENT-14: Some conclusions are not supported by the results: “Lower Al-concentration below 75 μ M had no significant effect on the RL and SL of most finger millet cultivars and accessions, while the growth starts to decline with increasing Al-concentration”. In my opinion the lowest concentration used in the experiment was 75 μM.
RESPONSE TO COMMENT-14: thank you for the comments. The conclusion part is now modified following your suggestion.
COMMENT-15: P11 L 70-72 “Accessions identified as Al-tolerant under hydroponic experiment often showed improved agronomic performance compared to Al-susceptible (Fikiru et al., 2011; Hede et al., 2002; Negusse et al., 2022)”.
RESPONSE TO COMMENT-15: thank you for the comment. it is to mean that the authors noted that Al-tolerant varieties showed better performance in RL, SL, and fresh weight than the susceptible.
We hope that the revision made based on your comments are satisfactory.
Round 2
Reviewer 1 Report
The authors provided in many cases good explanations. However, I am not satisfied with the answer provided in comment-7
COMMENT-7. Some measurements from the pilot experiment seem not to be confirmed in the large experiment, e.g.: (..)
RESPONSE TO COMMENT-7: (..). This variation is due to there is variation in size and color among individuals in each accession. To minimize such big variation we select individuals having the same color, same seed size, and even after germination we used seedlings with similar performance. By taking this variation in to account we select one individual from each accessions for multi-site field evaluation, genomic and transcriptomic analysis.
I don’t understand this; the results of the two experiments seem totally not related. Please explain.
Figure 2 and 3 show the same data as table 1, show either the table or the figure (in case of keeping the figure, move the table to supplemental files).
line 230: “least” should be “most”
line 231: Figure 5 does only show Ec-215836, the reference at that location in the sentence would suggest all the mentioned acc. there.
The full table 3 can become a supplemental file and be replaced by a small table with representative examples that are mentioned in the text.
The paper wants to make a point of Al-tolerance related to acidification of soil. However, the first sentence of the conclusion is "Even though the level of soil acidity is not known in Ethiopia (..) 74 of them are highly susceptible".
If soil acidity is not known, why is the research done? The conclusion mixes up tolerance to Al and acidity. I think it is not the same. Also, I do not understand why a transcriptomics experimental set-up needs to be mentioned in the conclusion.
The revised version seems to have additional errors, periods where they do not belong and other small errors.
Author Response
RESPONSE TO REVIEWER-1’S COMMENTS
We appreciate the comments and carefully considered them during the revision of the manuscript and provided a point-by-point response.
COMMENT-1. Some measurements from the pilot experiment seem not to be confirmed in the large experiment, e.g.:
Ec-215944 table 1 RLL is 36% and RSL is 133% while in table 2 it is 50% and 69%, respectively
Ec-216051 table 1 RLL is 20% and RSL is 133% while in table 2 it is 42% and 47%, respectively
Ec-216057 table 1 RLL is 13% and RSL is 170% while in table 2 it is 38% and 53%, respectively
RESPONSE TO COMMENT-1: In most of the accessions the individuals seeds are different. We observe variation in seed color, germination time, and even in their root length during hydroponic treatment. Therefore, the difference among the individuals within accessions obtained during the optimization and mass screening is expected. To minimize such big variation among individual seedling within an accession we select individuals having the same color, same seed size, and even after germination we used seedlings with similar performance. We hope this addresses your concerns.
COMMENT-2. Figure 2 and 3 show the same data as table 1, show either the table or the figure (in case of keeping the figure, move the table to supplemental files).
RESPONSE TO COMMENT-2: Thank you very much for the comment. We move Table 1 to supplementary Table as per your comment.
COMMENT-9. line 230: “least” should be “most”
RESPONSE TO COMMENT-9. Thank you for the comment. Now it is corrected as “most”.
COMMENT-10. The paper wants to make a point of Al-tolerance related to acidification of soil. However, the first sentence of the conclusion is "Even though the level of soil acidity is not known in Ethiopia (..) 74 of them are highly susceptible". If soil acidity is not known, why is the research done?
RESPONSE TO COMMENT-10. Thank you for the comment. We added information on the level of soil acidity in Ethiopia on the introduction part page 2 paragraph 2, line 3-4. At the same time we updated the conclusion part.
COMMENT-11. The conclusion mixes up tolerance to Al and acidity. I think it is not the same. Also, I do not understand why a transcriptomics experimental set-up needs to be mentioned in the conclusion.
RESPONSE TO COMMENT-11. Thank you for the comment. Now the conclusion section is improved based on your comment.
We hope that the revision made based on your comments are satisfactory.
Reviewer 2 Report
The new version of the manuscript has been substantially improved and all my comments have been taken into account by the authors. I consider that the work can be published in Agronomy journal.
Please, check the minor typing errors in the Discussion section, please (highlighted in yellow/green in the attached Pdf)
Comments for author File: Comments.pdf
Author Response
RESPONSE TO REVIEWER-2’S COMMENTS
The new version of the manuscript has been substantially improved and all my comments have been taken into account by the authors. I consider that the work can be published in Agronomy journal.
Thank you for accepting our responses to your comments.
Please, check the minor typing errors in the Discussion section, please (highlighted in yellow/green in the attached Pdf)
Thank you for spotting the minor typing errors and now we incorporated the corrections
Reviewer 3 Report
Hydroponic Optimization and Screening of Al-Tolerance on Finger Millet accessions and cultivars
Manuscript ID: agronomy-2405662
Submitted to Agronomy Section Crop Breeding and Genetics
I have read the revised version of the manuscript. The authors followed most of my comments, however, some of comments were omitted:
1) In my opinion, the root and stem length measurements were made on insufficient number of plants ( i.e. 5 individuals per accession), which may influence the final result. A fundamental deficiency of the manuscript is the lack of repetition of the experiment. No repetition makes it impossible to perform statistical analyses.
Figures 2 and 3 show root length and shoot length of finger millet accessions however, there is no information which accessions differ significantly. The authors were previously asked to supplement this information but they did not do it.
2) Some sentences in the text still need rewriting:
“The seedlings were allowed to grow for about 10 days weeks in hydroponics………………….”
What does it mean 10 days weeks?
3) The description of figure 4 needs to be improved and use a uniform nomenclature of accessions: “Figure 4. Examples of seedlings grown for 10 days in Al3+-containing nutrient solution. Accessions 215836 was scored as tolerant. Accession 215804 was scored as sensitive.”
Which spelling is correct? Ec – 215836 or 215836 ? Lack of consistency in labeling the accessions.
4) “It is advisable to use seedlings with similar vigor and this is achieved by selecting seedlings with similar-sized endosperm. similar initial root length and similar seed age to consider better performing individuals [25, 29]”
The authors do not explain how this fragment of the text relates to the obtained results? The reader does not know it.
5) “In this study. a hydroponic nutrient solution was employed to identify the threshold level of Al concentration in finger millet landraces and cultivars. Finger millet accessions and cultivars were evaluated at three Al-concentrations including the control (0. 75. and 100 μM). At low Al-concentration. it is difficult to properly discriminate finger millet accessions and cultivars in relation to their Al-tolerance. The reason could be due to that low Al-concentration (less than 75 μM) was not strong enough to create Al- stress condition at finger millet roots.”
How does one know it? Is it from the presented studies or from the literature? The methodology of the manuscript states that the lowest Al dose was 75 μM.
6) P14 L 44-46” ……………..and develop Al-tolerant genes as enhance their tolerance……..”
Genotypes develop genes? What do the authors mean by “develop genes”? Authors should explain it in the manuscript.
7) The last comment concerns the conclusions. The conclusions have been completely changed from those previously attached. In my opinion, this section of the text should include thoughts derived from the results obtained. At least in one or two sentences.
Author Response
RESPONSE TO REVIEWER-3’S COMMENTS
Comment-1: In my opinion, the root and stem length measurements were made on insufficient number of plants (i.e. 5 individuals per accession), which may influence the final result. A fundamental deficiency of the manuscript is the lack of repetition of the experiment. No repetition makes it impossible to perform statistical analyses.
Response to comment-1: Thank you for the comment. We understand your concern and to minimize the influence getting data from 5 individuals on the final output we first germinate 15 individual seeds and then 10 seedlings were selected based on their similar germination conditions. Afterwards, 10 seedlings were transferred in to two centrifuge tubes (5 seedlings each) then treated under hydroponic nutrient solution. Finally, we collect data from 5 individual seedlings per accession having similar growth rate. Measuring RL from 5 individuals per accessions and in total 328 *5=1640. Similarly measuring SL of 1640 seedling is tedious, time and energy taking. Therefore, we use this hydroponic experiment to preliminary screen the randomly collected accessions and used as input for further evaluation under multi-filed, genomic and transcriptomic analysis. It is continuous research project.
Figures 2 and 3 show root length and shoot length of finger millet accessions however, there is no information which accessions differ significantly. The authors were previously asked to supplement this information but they did not do it.
Yes, we have been asked this question in your round one comments. However, based on comments from other reviewers we construct Figure 1 and 2. As you can see the performance of each accessions from the figures there is big difference between accessions. To make statistically meaning full following your comment, we construct figure-4. Fig.4 indicates significant variation among accessions grown under the different Al-concentrations. We hope this addresses your concerns.
Comment-2: Some sentences in the text still need rewriting:
“The seedlings were allowed to grow for about 10 days weeks in hydroponics………………….”
What does it mean 10 days weeks?
Response to comment-2: thank you for spotting minor typing error and now it is corrected as follows ….. Then the seedlings were transferred to hydroponic nutrient solution and treated for about 10 days within the greenhouse adjusted to a temperature of 18°C and a humidity level of 65% at the Swedish University of Agricultural Sciences (SLU, Alnarp, Sweden).
Comment-3: The description of figure 4 needs to be improved and use a uniform nomenclature of accessions: “Figure 4. Examples of seedlings grown for 10 days in Al3+-containing nutrient solution. Accessions 215836 was scored as tolerant. Accession 215804 was scored as sensitive.”
Which spelling is correct? Ec – 215836 or 215836 ? Lack of consistency in labeling the accessions.
Response to comment-3: Thank you for spotting the error and now we removed the prefix Ec-.
Comment-4: “It is advisable to use seedlings with similar vigor and this is achieved by selecting seedlings with similar-sized endosperm. Similar initial root length and similar seed age to consider better performing individuals [25, 29]”
The authors do not explain how this fragment of the text relates to the obtained results? The reader does not know it.
Response to comment-4: thank you for the comment. Now, we added the explanation with track change on page 13, paragraph 2.
Comment-5: “In this study. a hydroponic nutrient solution was employed to identify the threshold level of Al concentration in finger millet landraces and cultivars. Finger millet accessions and cultivars were evaluated at three Al-concentrations including the control (0. 75. and 100 μM). At low Al-concentration. it is difficult to properly discriminate finger millet accessions and cultivars in relation to their Al-tolerance. The reason could be due to that low Al-concentration (less than 75 μM) was not strong enough to create Al- stress condition at finger millet roots.”
How does one know it? Is it from the presented studies or from the literature? The methodology of the manuscript states that the lowest Al dose was 75 μM.
Response to comment-5: thank you again. The two Al-concentrations (75 and 100 µM) were selected by considering the optimization protocol we have developed previously by Haftom et al., 2018. Haftom et al., reported the optimum Al-concentration was 112.5 uM Al-con.
Comment-6: 6) P14 L 44-46” ……………..and develop Al-tolerant genes as enhance their tolerance……..”
Response to comment-6: thank you for the comment. Now the sentence is modified as follows: Genotypes collected from acidic environment may accumulate mutation that adapt acidic environment and develop rapid Al-tolerance mechanisms by activating genes responsive for secretion of mucilage, organic acid anions when they exposed to phototoxic form of Al within minutes after exposure. Thus due to natural selection only the tolerant genotypes survive.
Comment-7: The last comment concerns the conclusions. The conclusions have been completely changed from those previously attached. In my opinion, this section of the text should include thoughts derived from the results obtained. At least in one or two sentences.
Response to comment-6: thank you for the comment. The conclusion is updated as per your comment.
“Results of the present study suggests that there are individual accessions that can tolerate acidic soils in a better way and some of them are highly susceptible. Lower Al-concentrations had no significant effect on the RL of most finger millet cultivars and accessions, while the growth starts to decline with increasing Al-concentration. At 100 µM Al-concentration, cultivars Tadesse, Padet, and Kumsa as well as accessions 212462, 215804, and 238323 were Al-susceptible. Thus, these cultivars should not be recommended in area where soil acidity is predominant. On the other hand, Urji, Bareda, and Axum cultivars as well as 215836, 215845 and 229722 accessions were relatively tolerant against Al and can be promoted in area where soil acidity is highly prevalent. To confirm their performance, the accessions should be tested under multi-site filed by considering control and treated environments. Further-more, association studies also be considered to correlate field performance with genomic background. Transcriptomic analysis on the best tolerant and least susceptible should be tested by taking samples from different plant tissue (root, leaf, and stem) by taking at different time interval (0, 12hrs, 24hrs, 48, and 72hrs). Finally, Al-tolerant lines identified from this study will be used as input to the finger millet breeding program in relation to Al-tolerance in the country and beyond. If anyone is interested to study Al-tolerance on finger millet it is required to include wild types for comparative analysis.”
We hope that the revision made based on your comments are satisfactory.
