The Development of a Transformation System for Four Local Rice Varieties and CRISPR/Cas9-Mediated Editing of the OsCCD7 Gene

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The manuscript entitled “Development of a transformation system for four local rice varieties and CRISPR/Cas9-mediated edition of OsCCD7 Gene” has been critically reviewed and found to be suitable for publication with the following minor modification.

1. Supplementary files were missing and therefore could not be reviewed properly.
2. Line 61-62: "in addition......respectively" may be deleted.
3. Line 91-97, designing of gRNA and construction of binary vector with CAS9 may be included with a brief detail. The target of the gRNAs to the CCD7 gene may be explained elaborately. 
4. Line 106, How is 60-70% humidity maintained in a closed and aseptic media condition?
5. Different media were used in the manuscript, like infection buffer, agrobacterium suspension, co-cultivation medium, recovery medium, differentiation medium, etc. However, there was no description of the composition of the media in the manuscript. These may be provided in the manuscript. What is a recovery culture condition?
6. Line 150: What is AS concentration?

Author Response

Dear Editor,

Thanks very much for your regarding to our manuscript entitled “Development of a transformation system for four local rice varieties and CRISPR/Cas9-mediated editing of OsCCD7 Gene”. These comments are very helpful for us to revise and improve the manuscript. We have revised the manuscript according to the reviewers' comments. Attached please find the revised version. The response to the reviewer’s comments and the main corrections in the manuscript are as flowing:

Reviewer 1

The manuscript entitled “Development of a transformation system for four local rice varieties and CRISPR/Cas9-mediated edition of OsCCD7 Gene” has been critically reviewed and found to be suitable for publication with the following minor modification.

Thank you for your constructive comments and suggestions on our paper titled “Development of a transformation system for four local rice varieties and CRISPR/Cas9-mediated editing of the OsCCD7 gene.” These comments and suggestions have been of great value and assistance in revising and improving our paper. We have carefully addressed all of your comments. Below are our responses to each comment. The revised version has been marked in red for easy reference. The main corrections in the paper and our responses to the reviewers are as follows:

Comment 1. Supplementary files were missing and therefore could not be reviewed properly.

Response: Thank you for your suggestion. We sincerely apologize for this oversight. The supplementary files have now been uploaded with the revised submission and are available for review. Thank you for highlighting this issue.

Comment 2. Line 61-62: "in addition......respectively" may be deleted.

Response: Thank you for your suggestion. We have deleted the phrase "in addition...respectively" from Line 61-62 in the revised manuscript.

Comment 3. Line 91-97, designing of gRNA and construction of binary vector with CAS9 may be included with a brief detail. The target of the gRNAs to the CCD7 gene may be explained elaborately.

Response: Thank you for your suggestion. We revised as “To download cDNA sequence of OsCCD7 gene of rice Nipponbare in NCBI database (https://www.ncbi.nlm.nih.gov/), and submit to CRISPR-P v2.0 database (http://crispr.hzau.edu.cn/cgi-bin/CRISPR2/SCORE) for target site prediction. The target sites proximal to the start codon (ATG) were selected to verify the location within exonic regions, referring to alignment between gDNA and cDNA sequences of OsCCD7 gene. The target regions were amplified from varieties WD68 and H128, and to confirm the identity sequences with Nipponbare genome, or else redesigning target sites. Based on GT bon scaffold (gRNA scaffold), two optimal targets were isolated and cloned into pBK1-Cas9-U3 vector by Golden Gate assembly (Eco31 I restriction sites), to get the fusion vector CRISPR/Cas9-ΔOsCCD7. The fusion vector was transformed into E. coli DH5ɑ to prepare plasmid stocks. The pBK1-Cas9-U3 vector was ordered from the Wuhan Boyuan Biotechnology Co., LTD.(#REC42-I, China), with vector map and construction procedures following the manufacturer's protocol. The gRNA sequences and target regions were listed in Table S1 and Fig. S1, respectively. ”

Comment 4. Line 106, How is 60-70% humidity maintained in a closed and aseptic media condition?

Response: Thank you for your question. Here is our explanation:By using breathable membrane sealed culture containers and culturing in a controlled environment incubator equipped with a humidity control system, humidity (60-70%) is maintained.Modifications were made in the text. (Line118-119)

Comment 5. Different media were used in the manuscript, like infection buffer, agrobacterium suspension, co-cultivation medium, recovery medium, differentiation medium, etc. However, there was no description of the composition of the media in the manuscript. These may be provided in the manuscript. What is a recovery culture condition?

Response: Thank you for your suggestion. I have added the culture medium components used in the paper to the manuscript. Added to Materials and Methods in 2.4.5.

Comment 6. Line 150: What is AS concentration?

Response: Thank you for your question. AS (acetosyringone) concentration refers to the amount (typically in μM or mg/L) of this phenolic compound used to induce Agrobacterium virulence genes during genetic transformation. In this study, different concentrations of AS were used, including 100 μM and 200 μM.

 

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

Development of a transformation system for four local rice varieties and\CRISPR/Cas9-mediated edition of OsCCD7 gene

The authors established a transformation system on four rice varieties popular in China using Agrobacterium to increase yield and grain quality. To achieve this, the authors first optimized transformation efficiency by testing the effects of various culture media on callus induction, differentiation, shoot regeneration and rooting. Further, the authors developed a

CRISPR/Cas9-mediated gene edition technique to improve the architecture and tiller development. This was achieved by editing the OsCCD7 gene which resulted in a significant increase in tiller number and dwarfism.

 

My comments:

 

Abstract

This section summarizes the methodology, results and conclusions. It is well detailed and provides a basis for efficient transformation of local rice varieties and germplasm innovation.

I have a few comments the authors may consider in revising their manuscript as follows:

 

Line 15: Change ‘mediums’ to ‘media’

Line 16: Place a comma after ‘differentiation’

Line 25: ‘300 mg/L Tmt’ what is the treatment? Is this an antibiotic?

 

Introduction

This section seems to be comprehensive and up to date. The quality of English in this section is much better than other sections. I don’t have a tool to check for similarity in sentence structure with published literature. There are a total of 10 citations in this section and the latest is 2024. I have not seen any 2025 reference. The quality of literature review appears to be fine and covers all the aspects of transformation and gene editing in rice. I have suggested adding three refences (2018-2019) that are listed at the end of this review. It is up to the authors to decide whether or not to cite these references in the introduction or Discussion section or both.

 

Methods & Methods

 

The Methods and Materials section seems to be logical and sufficient for performing the experiments pursued in this study. I recommend the authors follow my comments below which will improve the quality of their work before being accepted for publication:

 

Line 105: ‘Callus size, fresh weight, dry weight, and induction rate were recorded’. Explain how these growth parameters were measured?

Line 106: Explain ‘dark conditions’

Line 108: ‘The basic medium composes…’ Do you mean components? Or composition?

Rewrite the whole sentence.

Line 121: ’optimal density (OD600) of 0.6-0.8’. How many cells/mL? Please place cells/mL in parenthesis after OD.

Line 125: ‘Contamination rates’ delete ‘rates’

Lines 137 & 138: Explain how you performed the transformation of Agrobacterium with

pCambia2301-GUS.

Line 154: Add (v/v) after 70%

Line 170: Change ‘callus’ to ‘calli’

Line 171: Change ‘was’ to ‘were’

Line 171: Change ‘calluses’ to ‘calli’

Lines 172 & 173: Place a ‘period’ after ‘plasmid’ and start a new sentence: ‘The transformed calli were regenerated into transgenic seedlings’

Lines 186 & 187: Rephrase this sentence: ‘sent to sequence for mutation sequences detection’

Line 197: Change to: ‘Data were analyzed using SPSS 18.0.’

Line 201: Supplemental

 

Results

 

This section presents the results in detail, but the quality of English is poor and needs to be improved and revised. Read my comments below and make changes.

 

Remove the word ‘Different’ from the x axis in all the figures and Tables. Use the word ‘Treatment’ instead. 

 

Lines 219-220: Rephrase as: Values with different letters for each variety are significantly different at p<0.05 (n=15).

Line 230: Please add this sentence after ‘respectively’.: Further, we observed increases in callus size (36.52 mm²), fresh weight (230.20 mg) and dry weight (9.56 mg) in E33 variety following A17 treatment.

Line 236: Change ‘treatment’ to ‘treatments’

Line 238: Change to ‘under S1, S4, and S5 treatments’

Line 243: Change ‘Resistant’ to ‘Resistance’

Line 255: Change to ‘improvement’

Line 262: Change ‘Resistant’ to ‘Resistance’

Lines 281-283: ‘Differentiation rates were highest under M3 treatment in XG293, WD68, H128, and E33, with differentiation rates of 18.87%, 8.51%, 21.06%, and 11.93%, respectively (Fig. 4C)’. The Figure does not show this. The values are a lot lower than those presented here.

Line 288: In Figure 4, (C) is not labeled.

Lines 289-291: Rephrase as: Values with different letters for each variety are significantly different at p<0.05 (n=15).

Line 298: Delete this: ‘into regenerated seedlings’

Lines 313-315: Rephrase as: Values with different letters for each variety are significantly different at p<0.05 (n=15).

Lines 332-333: Rephrase as: Values with different letters for each variety are significantly different at p<0.05 (n=15).

Lines 341 & 352: Use singular words: …’concentration’… ‘duration’

Lines 342 & 343: Rephrase as: Values with different letters for each variety are significantly different at p<0.05 (n=15).

Lines 356, 357 & 377: Change ‘mediums’ to ‘media’

Line 360: Change ‘medium’ to ‘media’ before (Fig. 7).

Line 360: Add ‘often’ after ‘medium’: ‘Seedlings grown on M medium exhibited the lowest values’.  

Lines 367-369: ‘Compared with NA medium, MA medium significantly improved stem diameter, total root length, root surface area, and root volume, indicating superior rooting effects’. This is not true for all the varieties tested.

Line 372: I cannot see the data in Fig. S4 since it did not come with the main file.

Line 377: Delete ‘is’ and replace it with a comma: SD, stem diameter (cm); PH, plant height (cm); RFN, root fork number; …..  

Line 378: Change ‘numbers’ to ‘number’

Line 379: Change ‘cm2’ to ‘cm²

Line 380: Change ‘cm3’ to ‘cm³

Line 380: Change ‘numbers’ to ‘number’

Lines 383-384: Unlike WD68, XG293 hardly shows it is in the heading stage. Can you show a better picture?

Line 398: Change to ‘lanes’

Line 399: Change ‘plant’ to ‘negative control’

Line 403-406: Edited as: ‘The two editing sites in both varieties were sequenced, the target I showed no mutation in WD68 genome, whereas there were a base mutation and a base insertion upstream of the PAM site in H128.’

Line 408: Remove the second ‘in’

Lines 425-429:  Edited as: Phenotypic traits in osccd7 rice mutants: WD68-WT vs. WD68-Δosccd7 (60 days) (A); 120 days of growth (B); harvest time (C); Sword leaf morphology (D); leaf height (E); Tiller number (F); Stem diameter (G); Effective tiller number (H); Flag leaf length (I) and Flag leaf width (J). H128-WT vs. H128-Δosccd7 (60 days) (A); 120 days of growth (B); harvest time (C); Sword leaf morphology (D); Leaf height (E); Tiller number (F); Stem diameter (G); Effective tiller number (H); Flag leaf length (I) and Flag leaf width (J).

Line 430: Change to: Spike-related traits in WT vs. osccd7 mutants.

 

Discussion

 

In this section the authors compared their results with those previously published and made some solid conclusions. The references seem to be relevant and up to date. However, the quality of English like in ‘Results’ section needs to be improved.

 

I have a few comments as follows:

 

Line 457: What do you mean by ’generations’. Do you mean ‘studies’

Line 458: Remove ‘found’

 

Conclusions

In this section, the authors highlighted the main findings of their work and compared the transformation rates in the four rice varieties. Further, they were able to show that deletion of  Osccd7 gene caused an increase in tillering and shorter plant height.

 

References

I have identified the following references that are relevant to the work presented here but are absent in the list of references and are not cited in the Introduction or Discussion sections. I suggest that the authors consider adding these references.

CRISPR-Cas9-Mediated Genome Editing of Rice Towards Better Grain Quality (2018)

https://doi.org/10.1007/978-1-4939-8914-0_18

 

Applications of the CRISPR/Cas9 System for Rice Grain Quality Improvement: Perspectives and Opportunities (2019)

https://doi.org/10.3390/ijms20040888

 

 CRISPR/Cas9: Development and Application in Rice Breeding (2019)

https://doi.org/10.1016/j.rsci.2019.08.001

 

Comments on the Quality of English Language

The quality of English in some sections of the manuscript is poor and needs to be improved. I have indicated where revisions are needed.

Author Response

Dear Editor,

Thanks very much for your regarding to our manuscript entitled “Development of a transformation system for four local rice varieties and CRISPR/Cas9-mediated editing of OsCCD7 Gene”. These comments are very helpful for us to revise and improve the manuscript. We have revised the manuscript according to the reviewers' comments. Attached please find the revised version. The response to the reviewer’s comments and the main corrections in the manuscript are as flowing:

 

Abstract

This section summarizes the methodology, results and conclusions. It is well detailed and provides a basis for efficient transformation of local rice varieties and germplasm innovation.

I have a few comments the authors may consider in revising their manuscript as follows:

1. 1.Line 15:Change ‘mediums’ to ‘media’

Response: Thank you for your careful review. We apologize for our oversight. We have made corrections based on your comments.(line 15)

2. Line 16:Place a comma after ‘differentiation’

Response: Thank you for your careful review. We apologize for our oversight. We have made corrections based on your comments.(1ine 16)

3. Line 25:300 mg/L Tmt’ what is the treatment? Is this an antibiotic?

Response: Thank you for your question. 300 mg/L Tmt refers to 300 mg/L of Timentin, an antibiotic commonly used to treat bacterial infections.

 

Introduction

This section seems to be comprehensive and up to date. The quality of English in this section is much better than other sections. I don’t have a tool to check for similarity in sentence structure with published literature. There are a total of 10 citations in this section and the latest is 2024. I have not seen any 2025 reference. The quality of literature review appears to be fine and covers all the aspects of transformation and gene editing in rice. I have suggested adding three refences (2018-2019) that are listed at the end of this review. It is up to the authors to decide whether or not to cite these references in the introduction or Discussion section or both.

 

Methods & Methods

The Methods and Materials section seems to be logical and sufficient for performing the experiments pursued in this study. I recommend the authors follow my comments below which will improve the quality of their work before being accepted for publication:

4. Line 105: ‘Callus size, fresh weight, dry weight, and induction rate were recorded’. Explain how these growth parameters were measured?

Response: Thank you for your question. Callus size: Measure the diameter length and diameter width (mm) using a Vernier caliper. The size is calculated as diameter length × diameter width (mm²); Fresh weight: Weigh the fresh sample using an electronic balance (g); Dry weight: Weigh after drying at 60°C to constant weight (g); Induction rate (%) = (Numbers of induced callus / Numbers of cultured mature seeds) × 100.

5. Line 106:Explain ‘dark conditions’

Response: Thank you for your question. “Dark conditions” refers to an experimental setup in which the culture material is completely isolated from all light sources (wrapped in aluminum foil and placed in a light-proof container).

6. Line 108:‘The basic medium composes…’ Do you mean components? Or composition?Rewrite the whole sentence.

Response: We appreciate your correction. We have corrected “medium composes” to “medium components”. I have added the culture medium components used in the paper to the manuscript. Added to Materials and Methods in 2.4.5. Thank you for your careful reading.

7. Line 121:optimal density (OD600) of 0.6-0.8’. How many cells/mL? Please place cells/mL in parenthesis after OD.

Response: Thank you for your question. OD600=0.6-0.8 refers to the absorbance value range measured by a spectrophotometer at a wavelength of 600 nm. This range corresponds to a live bacterial count of approximately 1×10⁸ CFU/mL (E. coli). The relevant changes have been made in the article.(line 131-133)

8. Line 125:‘Contamination rates’ delete ‘rates’

Response: Thank you for your advice, we have deleted ‘rates’.

9.Lines 137 & 138: Explain how you performed the transformation of Agrobacterium with pCambia2301-GUS.

Response: Agree with comments, the sentence was rewritten with ‘The plasmid was extracted and transformed into Agrobacterium tumefaciens strain EHA105 with heat shock treatment at 42℃’.

10. Line 154:Add (v/v) after 70%

Response: Thank you for your careful review. We apologize for our oversight. We have made corrections based on your comments.

11. Line 170:Change ‘callus’ to ‘calli’

Response: Thank you for your careful review. We apologize for our oversight. We have made corrections based on your comments.

12. Line 171:Change ‘was’ to ‘were’

Response: Thank you for your careful review. We apologize for our oversight. We have made corrections based on your comments.

13. Line 171:Change ‘calluses’ to ‘calli’

Response: Thank you for your careful review. We apologize for our oversight. We have made corrections based on your comments.

14. Lines 172 & 173:Place a ‘period’ after ‘plasmid’ and start a new sentence: ‘The transformed calli were regenerated into transgenic seedlings’

Response: Thank you for your careful review. We apologize for our oversight. We have made corrections based on your comments.

15. Lines 186 & 187:Rephrase this sentence: ‘sent to sequence for mutation sequences detection’.

Response: We appreciate your correction. We have changed the original text from “ sent to sequence for mutation sequences detection” to “sent to sequence for mutation sequences detection”. Thank you for your careful reading.

16. Line 197:Change to: ‘Data were analyzed using SPSS 18.0.’

Response: We appreciate your correction. We have changed the original text from “ Data was analyzed in SPSS 18.0,” to “Data were analyzed using SPSS 18.0.”. Thank you for your careful reading.

17. Line 201:Supplemental

Response: We appreciate your suggestion. We have supplemented the content of the data analysis section in the manuscript.

 

Results

 

This section presents the results in detail, but the quality of English is poor and needs to be improved and revised. Read my comments below and make changes.

Response: We have made every effort to improve the manuscript and have made some revisions to it. These changes will not affect the content and structure of the paper. We have not listed the changes here, but they are marked in red in the revised document. We sincerely thank the editor/reviewer for their enthusiastic work and hope that the corrections will be approved. Secondly, we invited Professor  Zhaoshi Xu from the Chinese Academy of Agricultural Sciences to help polish our article. We hope that the revised manuscript will be accepted by you.

18. Remove the word ‘Different’ from the x axis in all the figures and Tables. Use the word ‘Treatment’ instead.

Response: Thank you for your suggestion. We have replaced "Different" with "Treatment" on all x-axes in the figures and tables as requested.

19. Lines 219-220:Rephrase as: Values with different letters for each variety are significantly different at p<0.05 (n=15).

Response: We appreciate your correction. We have changed the original text from “ Lower case letters indicate the significant difference among different treatments for the same variety (P<0.05) (n=15).” to “Values with different letters for each variety are significantly different at p<0.05 (n=15).”. Thank you for your careful reading.

20. Line 230:Please add this sentence after ‘respectively’.: Further, we observed increases in callus size (36.52 mm2), fresh weight (230.20 mg) and dry weight (9.56 mg) in E33 variety following A17 treatment.

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

21. Line 236:Change ‘treatment’ to ‘treatments’;

Response: Thank you for your suggestion. We have changed "treatment" to "treatments" in the relevant sentence.

22. Line 238:Change to ‘under S1, S4, and S5 treatments’;

Response: Thank you for your suggestion. We have changed "under treatment S1, S4, and S5 " to "under S1, S4, and S5 treatments" in the relevant sentence.

23. Line 243:Change ‘Resistant’ to ‘Resistance’;

Response: Thank you for your suggestion. We have changed "Resistant" to "Resistance" in the relevant sentence.

24. Line 255:Change to ‘improvement’;

Response: Thank you for your suggestion. We have changed "improvements" to "‘improvement" in the relevant sentence.

25. Line 262:Change ‘Resistant’ to ‘Resistance’

Response: Thank you for your suggestion. We have changed "Resistant" to "Resistance" in the relevant sentence.

26. Lines 281-283:‘Differentiation rates were highest under M3 treatment in XG293, WD68, H128, and E33, with differentiation rates of 18.87%, 8.51%, 21.06%, and 11.93%, respectively (Fig. 4C)’. The Figure does not show this. The values are a lot lower than those presented here.

Response: We sincerely apologize for this error. The correct differentiation rates under M3 treatment should indeed be 9.44%, 10.42%, 10.53%, and 5.97% for XG293, WD68, H128, and E33, respectively (Fig. 4C). We have carefully verified and corrected all data in the revised manuscript. Thank you for catching this important discrepancy.

27. Line 288:In Figure 4, (C) is not labeled.

Response: We sincerely apologize for this oversight. The label "(C)" has now been clearly added to Figure 4 in the revised manuscript. Thank you for pointing out this issue.

28. Lines 289-291:Rephrase as: Values with different letters for each variety are significantly different at p<0.05 (n=15);

Response: Thank you for your suggestion. We have revised the text to: “Values with different letters for each variety are significantly different at p<0.05 (n=15)”

29. Line 298:Delete this: ‘into regenerated seedlings’;

Response: Thank you for your suggestion. We have deleted the phrase 'into regenerated seedlings' as requested.

30. Lines 313-315:Rephrase as: Values with different letters for each variety are significantly different at p<0.05 (n=15);

Response: Thank you for your suggestion. We have revised the text to: “Values with different letters for each variety are significantly different at p<0.05 (n=15)”

31. Lines 332-333:Rephrase as: Values with different letters for each variety are significantly different at p<0.05 (n=15);

Response: Thank you for your suggestion. We have revised the text to: “Values with different letters for each variety are significantly different at p<0.05 (n=15)”

32. Lines 342 & 343:Rephrase as: Values with different letters for each variety are significantly different at p<0.05 (n=15);

Response: Thank you for your careful review. We sincerely apologize for these oversights and have now carefully addressed all similar instances throughout the manuscript based on your valuable comments. All corrections have been consistently applied in the revised version.

33. Lines 341 & 352:Use singular words: …’concentration’… ‘duration’

Response: Thank you for your suggestion. We have revised Lines 341 & 352 to use the singular forms "concentration" and "duration" respectively, as suggested.

34. Lines 356, 357 & 377:Change ‘mediums’ to ‘media’;

Response: We have changed "mediums" to "media" in Lines 356, 357, and 377 as suggested. Thank you for your careful review.

35. Line 360:Change ‘medium’ to ‘media’ before (Fig. 7);

Response: Thank you for your suggestion. We have changed "medium" to "media" in Line 360 before (Fig. 7) as suggested.

36. Line 360:Add ‘often’ after ‘medium’: ‘Seedlings grown on M medium exhibited the lowest values’.

Response: Thank you for your suggestion. We have added "often" as suggested, now reading: "Seedlings grown on M medium often exhibited the lowest values." (Line 360).

37. Lines 367-369:‘Compared with NA medium, MA medium significantly improved stem diameter, total root length, root surface area, and root volume, indicating superior rooting effects’. This is not true for all the varieties tested.

Response: We sincerely apologize for this oversight. We revised it as “Compared with NA medium, MA medium improved stem diameter, total root length, root surface area, and root volume, whereas reaching a significant level in root surface area, except of H128, indicating superior rooting effects.”

38. Line 372:I cannot see the data in  S4 since it did not come with the main file.

Response: We sincerely apologize for this oversight. The data for Fig. S4 have now been properly uploaded as supplementary material with the revised submission. Thank you for bringing this to our attention.

39. Line 377:Delete ‘is’ and replace it with a comma: SD, stem diameter (cm); PH, plant height (cm); RFN, root fork number; …..

Response: Thank you for your suggestion. We have revised the text as follows: "SD, stem diameter (cm); PH, plant height (cm); RFN, root fork number;..."

40. Line 378:Change ‘numbers’ to ‘number’;

Response: Thank you for your suggestion. We have changed "numbers" to "number" in the relevant sentence.

41. Line 379:Change ‘cm2’ to ‘cm²;

Response: Thank you for your suggestion. We have changed "cm2" to "cm²" in the relevant sentence.

42. Line 380:Change ‘cm3’ to ‘cm³;

Response: Thank you for your suggestion. We have changed "cm3" to "cm³" in the relevant sentence.

43. Line 380:Change ‘numbers’ to ‘number’；

Response: Thank you for your suggestion. We have changed "numbers" to "number" in the relevant sentence.

44. Line 398:Change to ‘lanes’

Response: Thank you for your suggestion. We have changed "lane" to "lanes" in the relevant sentence.

45. Line 399:Change ‘plant’ to ‘negative control’；

Response: Thank you for your careful review. We sincerely apologize for these oversights and have now carefully addressed all similar instances throughout the manuscript based on your valuable comments. All corrections have been consistently applied in the revised version.

46. Lines 383-384:Unlike WD68, XG293 hardly shows it is in the heading stage. Can you show a better picture?

Response: Thank you very much for your kind review. We do not have the better pictures, due to relatively poor regeneration seedlings in winter, we are very sorry for that.

47. Line 403-406:Edited as: ‘The two editing sites in both varieties were sequenced, the target I showed no mutation in WD68 genome, whereas there were a base mutation and a base insertion upstream of the PAM site in H128.’

Response: Thank you very much for your kind review. We have revised it.

48. Line 408:Remove the second ‘in’

Response: Thank you for your careful review. We apologize for our oversight. We have made corrections based on your comments.

49. Lines 425-429:Edited as: Phenotypic traits in osccd7 rice mutants: WD68-WT vs. WD68-Δosccd7 (60 days) (A); 120 days of growth (B); harvest time (C); Sword leaf morphology (D); leaf height (E); Tiller number (F); Stem diameter (G); Effective tiller number (H); Flag leaf length (I) and Flag leaf width (J). H128-WT vs. H128-Δosccd7 (60 days) (A); 120 days of growth (B); harvest time (C); Sword leaf morphology (D); Leaf height (E); Tiller number (F); Stem diameter (G); Effective tiller number (H); Flag leaf length (I) and Flag leaf width (J).

Response: Thank you very much for your kind review. We have revised it.

50. Line 430:Change to: Spike-related traits in WT vs.osccd7 

Response: Thank you for your careful review. We apologize for our oversight. We have made corrections based on your comments.

Discussion

In this section the authors compared their results with those previously published and made some solid conclusions. The references seem to be relevant and up to date. However, the quality of English like in ‘Results’ section needs to be improved.

Response: We have made every effort to improve the manuscript and have made some revisions to it. These changes will not affect the content and structure of the paper. We have not listed the changes here, but they are marked in red in the revised document. We sincerely thank the editor/reviewer for their enthusiastic work and hope that the corrections will be approved. Secondly, we invited Professor Xu Zhaoshi from the Chinese Academy of Agricultural Sciences, who has returned from studying in the United States, to help polish our article. We hope that the revised manuscript will be accepted by you.

51. Line 457:What do you mean by ’generations’. Do you mean ‘studies’

Response: Thank you for your comment. We have revised the term "generations" to "studies" throughout the manuscript to improve clarity.

52. Line 458:Remove ‘found’

Response: Thank you for your careful review. We apologize for our oversight. We have made corrections based on your comments.

Conclusions

In this section, the authors highlighted the main findings of their work and compared the transformation rates in the four rice varieties. Further, they were able to show that deletion of Osccd7 gene caused an increase in tillering and shorter plant height.

Response: Thank you very much for your strong support for our work.

References

53. I have verifiedthe following references that are relevant to the work but absent in the list of references, and not cited in the Introduction or Discussion sections. I suggest that the authors consider adding these references.

CRISPR-Cas9-Mediated Genome Editing of Rice Towards Better Grain Quality (2018)

https://doi.org/10.1007/978-1-4939-8914-0_18

Applications of the CRISPR/Cas9 System for Rice Grain Quality Improvement: Perspectives and Opportunities (2019)

https://doi.org/10.3390/ijms20040888

CRISPR/Cas9: Development and Application in Rice Breeding (2019)

https://doi.org/10.1016/j.rsci.2019.08.001

Response: Thank you for your suggestion. We have added the references that you provided in the introduction section. According to reference 1 “With the development of the Asian economy, rice breeding faces the challenge of regionalized demand for high-yielding, high-quality varieties, which traditional breeding methods struggle to meet”. According to reference 2 “CRISPR-Cas9 genome editing technology, with its high efficiency and precision, has become a key tool for improving rice quality and agronomic traits, such as stress tolerance and yield”. According to reference 3 “This technology has successfully edited multiple quality-related genes and advanced functional genomics research, providing a new approach for developing high-quality rice varieties that adapt to environmental and market demands”.

 

Author Response File: Author Response.pdf

Reviewer 3 Report

Comments and Suggestions for Authors

This work addresses a topic of importance to science and agriculture, particularly for the region where the studied varieties are grown. However, there are aspects that could be improved that would enhance the quality of this work. The introduction provides elements of the materials and methods or the results that should be highlighted in those sections and not in the introduction. In the materials and methods section, it is necessary to declare the experimental designs, the replicates of each experiment, and some elements related to data processing. In addition, it is necessary to present some data on the treatments presented in supplements that were not submitted as files for review. Perhaps it was necessary to transfer this information to supplements due to the very heavy workload. Therefore, this reviewer considers that the work is extremely large and could be divided into two for publication. This would facilitate improving the presentation of the results, the layout of the figures, and a better discussion of the results. In the results section, the authors sometimes provide unnecessary materials and methods because they have already been explained. Due to the amount of information, the discussion is brief. Some results and possible implications need to be explained. Overall, the work is publishable, but it could be further leveraged if divided into two papers that could be published as a series.

Comments for author File: Comments.pdf

Author Response

Dear Editor,

Thanks very much for your regarding to our manuscript entitled “Development of a transformation system for four local rice varieties and CRISPR/Cas9-mediated editing of OsCCD7 Gene”. These comments are very helpful for us to revise and improve the manuscript. We have revised the manuscript according to the reviewers' comments. Attached please find the revised version. The response to the reviewer’s comments and the main corrections in the manuscript are as flowing:

1. Line63:“CCD7 (Carotenoid Cleavage Dioxygenase 7)”, make a full stop here

Response: Thank you for your suggestion. We have revised it as “CCD7 (Carotenoid Cleavage Dioxygenase 7), a key enzyme in the biosynthesis of strigolactones (SLs), is responsible for catalyzing the carotenoids cleavage to generate SLs precursor molecules”

2. Line70-75:This information does not clearly state the objective of the research; rather, it attempts to provide information that should be clarified in detail in the materials and methods. It is suggested that the research objective be clearly stated, and should coincide with that of the abstract.

Response: Thank you for your suggestion. We have revised it as “This study aims to establish an efficient genetic transformation system for local rice varieties and utilize the CRISPR/Cas9-mediated gene editing tool to create novel germplasms with improved traits. Four local rice varieties were selected as materials to optimize hormone types and concentrations in induction media, reduce callus browning during subculture, and minimize contamination after Agrobacterium infection, thereby establishing an optimized induction culture system. The study also adjusted callus infection durations and Acetosyringone (AS) concentrations of infection buffer, transformed GUS gene as a marker, assessed GUS-expressing calli activity, and optimized rooting medium for regenerated seedlings.”

3. Line77-79:This sentence seems like a summary of the results that should not go in the introduction.

Response: Thank you for your suggestion. We have revised it as “A OsCCD7 gene, regulating on plant architecture of rice, was used to create the novel germplasms by the CRISPR/Cas9 tool, based on the efficient transformation system as above.”

4. Line79-81:This prayer is part of the materials and methods

Response: Thank you for your suggestion. We have revised it as “A OsCCD7 gene, regulating on plant architecture of rice, was used to create the novel germplasms by the CRISPR/Cas9 tool, based on the efficient transformation system as above.”

5. Line81-83:and this last sentence is part of the conclusions

Response: Thank you for your suggestion. We have removed the sentences.

6. Line88-89:“higher plant”, “low tiller numbers”, “stable yield”, “strong stress resistance”, “short growth period”, How tall is the height……….? Avoid using ambiguous terms.

Response: Thank you for your suggestion. We have replaced all vague terms with specific quantitative data. We have also presented this information in a table for easy reference (Table 1.).

7. Line96:“induced into”, It is better to use the term: cloned into, inserted into or ligated into. Induction typically refers to activating the expression of a gene. This should occur after this process, not during transformation.

Response: Thank you for your valuable suggestion. We have revised the text to use the correct terminology: " The target sites were isolated and cloned into the CRISPR/Cas9 vector "

8. Line97:I was unable to review the table because it was not provided.

Response: We sincerely apologize for this oversight. The missing supplement tables will be uploaded along with the revised manuscript for your review.

9. Line107:I was unable to review the table because it was not provided. Furthermore, it is important to consider that this information is too relevant to be included in supplements. It is suggested that it be included in the article content.

Response: Thank you for your advice. We sincerely apologize for that. We have re-uploaded the supplementary tables for your reviews. As the reagent formulation in tissue culture contains numerous components, presenting them in the main text would occupy considerable space. We hope to display them in the supplementary materials. If this is not acceptable, please let us know immediately, and we will promptly include them in the main text.

10. Line112:Same as the previous comment.

Response: Thank you for your advice. We hope to display them in the supplementary materials as above. If this is not acceptable, please let us know immediately, and we will promptly include them in the main text.

11. Line117:It is suggested that relevant information be placed in the manuscript and not in supplements.

Response: Thank you for your careful review. Many appendix charts merely served to supplement and clarify the classification criteria and growth status of regenerated seedlings. And also, this manuscripts was quite lengthy, so we hope to provide supplementary proof in the attachment. If not, please let me know, I will place the attached charts in the main text. Thank you!

12. Line196-201:It is important to explain the processing of the data in more detail. For example, there are several data points that are percentages; these must be tested for normality and homogeneity of variance, and then transformed if they do not meet these assumptions. The probability of the analysis (p-value) must be stated. The other data must also be tested for normality and homogeneity of variance. The experimental designs and repetitions must be stated in each case.

Response: Thank you for your feedback. We have already conducted normality tests and homogeneity of variance tests on the data points prior to data processing. The probability values (p-values) were analyzed using SPSS (IBM SPSS Version 21.0) software to perform Student’s t-test on the data. All experiments were conducted using a completely randomized design (CRD), with each replicate including 15 technical replicates (n=15).

13. Line205-206:This information is redundant. It's already included in the materials and methods. This session corresponds to the results.

Response: Thank you for your suggestion. We have removed the redundant information from the Results section to maintain focus on data presentation, as these methodological details are already fully described in the Materials and Methods section .

14. Line217-220:The figure, especially the graphics, do not have good resolution. It is suggested to display them separately to improve viewing.

Response: Thank you for your suggestion. We have optimized the resolution of all charts.

15. Line269-270：These are materials and methods

Response: Thank you for your suggestion. We have removed the redundant information from the Results section to maintain focus on data presentation.

16. Line287:Figure4, What do the bars in the columns indicate? Standard error, confidence interval, or standard deviation?

Response: Thank you for your question. The error bars in Figure 4 represent the standard error (SE) from 15 biological replicates, which is clearly stated in the revised legend.

17. Line352:Figure6, What do the bars at each point indicate? Standard error, confidence interval, or standard deviation? Why aren't bars used in all figures?

Response: Thank you for your question. The error bars in Figure 6 represent the standard error (SE) from 15 biological replicates, which is clearly stated in the revised legend.

18. Line377:Figure7, What do the bars at each point indicate? Standard error, confidence interval, or standard deviation? Why aren't bars used in all figures?

Response: Thank you for your question. The error bars in Figure 7 represent the standard error (SE) from 15 biological replicates, which is clearly stated in the revised legend.

19. Line453-462:The discussion is brief. It is necessary to discuss why the synergistic combinations achieved optimal callus induction.

Response: Thank you for your question. Based on your suggestion, we have added a discussion on synergistic mechanisms in section 4.1 of the discussion section.

20. Line501-504:These are the results. We would like more discussion of the biological processes in which they are involved.

Response: Thank you for your constructive suggestion. We have expanded the discussion section (Section 4.4) to analyze the relationship between these results and key biological processes, and cited relevant references from recent literature.

21. Line534-549:An important solution is provided by detecting mutant lines with lower yields. Clearly, these can be used as parents to improve the other traits that were positive after transformation. It would be useful to see a discussion of the other results of work using this approach. However, in this section, it is important to highlight the advantages of dwarfing and increased tillering as components of yield.

Response: Thank you very much for your strong support for our work.

 

Author Response File: Author Response.pdf

Reviewer 4 Report

Comments and Suggestions for Authors

The authors have standardized transformation system for four local rice varieties of Anhui province in China by studying various parameters like callus induction efficiency, anti-browning tendency of media, and agrobacterium transformation efficiency using GUS reporter. Further, the standardized method has been used for transformation of japonica rice WD68 and indica rice H128 with CRISPR/Cas9 construct targeting CCD7 gene. The research is important in order to expand the transformation amenability in local rice varieties which are largely grown in that area.

The methodology adopted by the authors is appropriate but inadequately described. For example, the authors should provide the sequence of gRNA and the target regions of the gene with the help of map for better understanding.

Further, there is no supplementary file attached with the manuscript and thus, I am unable to assess the primer details or other details of treatments for regeneration, antibrowning and transformation.

Authors are suggested to use expanded forms in abstract and at the first instance. E.g. tementin in place of tmt.

Authors have not provided any information about edits in plants. For confirmation of editing of CCD7 gene, they have carried out sequencing, but the results are not provided.

It is better to provide the phenotypic information on Plant architecture-related traits and Spike-related traits between WT and osccd7 mutants in a tabulated form rather than describing it in detail in results.

Modify the title by replacing the word “edition” by “editing”

Use the word editing instead of edition throughout the manuscript.

Comments on the Quality of English Language

Language need to be improved.

Author Response

Dear Editor,

Thanks very much for your regarding to our manuscript entitled “Development of a transformation system for four local rice varieties and CRISPR/Cas9-mediated editing of OsCCD7 Gene”. These comments are very helpful for us to revise and improve the manuscript. We have revised the manuscript according to the reviewers' comments. Attached please find the revised version. The response to the reviewer’s comments and the main corrections in the manuscript are as flowing:

Reviewer 4

The authors have standardized transformation system for four local rice varieties of Anhui province in China by studying various parameters like callus induction efficiency, anti-browning tendency of media, and agrobacterium transformation efficiency using GUS reporter. Further, the standardized method has been used for transformation of japonica rice WD68 and indica rice H128 with CRISPR/Cas9 construct targeting CCD7 gene. The research is important in order to expand the transformation amenability in local rice varieties which are largely grown in that area.

Thank you for your constructive comments and suggestions on our paper titled “Development of a transformation system for four local rice varieties and CRISPR/Cas9-mediated editing of the OsCCD7 gene.” These comments and suggestions have been of great value and assistance in revising and improving our paper. We have carefully addressed all of your comments. Below are our responses to each comment. The revised version has been marked in red for easy reference. The main corrections in the paper and our responses to the reviewers are as follows:

 

Comment 1. The methodology adopted by the authors is appropriate but inadequately described. For example, the authors should provide the sequence of gRNA and the target regions of the gene with the help of map for better understanding.

Response: Thank you for your suggestion. We revised as “To download cDNA sequence of OsCCD7 gene of rice Nipponbare in NCBI database (https://www.ncbi.nlm.nih.gov/), and submit to CRISPR-P v2.0 database (http://crispr.hzau.edu.cn/cgi-bin/CRISPR2/SCORE) for target site prediction. The target sites proximal to the start codon (ATG) were selected to verify the location within exonic regions, referring to alignment between gDNA and cDNA sequences of OsCCD7 gene. The target regions were amplified from varieties WD68 and H128, and to confirm the identity sequences with Nipponbare genome, or else redesigning target sites. Based on GT bon scaffold (gRNA scaffold), two optimal targets were isolated and cloned into pBK1-Cas9-U3 vector by Golden Gate assembly (Eco31 I restriction sites), to get the fusion vector CRISPR/Cas9-ΔOsCCD7. The fusion vector was transformed into E. coli DH5ɑ to prepare plasmid stocks. The pBK1-Cas9-U3 vector was ordered from the Wuhan Boyuan Biotechnology Co., LTD.(#REC42-I, China), with vector map and construction procedures following the manufacturer's protocol. The gRNA sequences and target regions were listed in Table S1 and Fig. S1, respectively. ”

Comment 2. Further, there is no supplementary file attached with the manuscript and thus, I am unable to assess the primer details or other details of treatments for regeneration, antibrowning and transformation.

Response: Thank you for bringing this to our attention. We sincerely apologize for this oversight. The supplementary document containing all primer details and regeneration, anti-browning, and transformation protocols has now been correctly uploaded to the attachments along with the revised submission.

Comment 3. Authors are suggested to use expanded forms in abstract and at the first instance. E.g. tementin in place of tmt.

Response: Thank you for your suggestion. We have replaced all abbreviations (including "tmt" to "Timentin") with their full forms at first mention in both the abstract and main text, with abbreviations subsequently used where appropriate.

Comment 4. Authors have not provided any information about edits in plants. For confirmation of editing of CCD7 gene, they have carried out sequencing, but the results are not provided.

Response: Thank you for your advice. We apologize for the oversight. The confirmation of the CCD7 gene editing sequencing results has been included in the supplement (Fig. S5). The supplement information will be uploaded along with the revised manuscript.

Comment 5. It is better to provide the phenotypic information on Plant architecture-related traits and Spike-related traits between WT and osccd7 mutants in a tabulated form rather than describing it in detail in results.

Response: Thank you for your advice. We apologize for this oversight. The phenotypic information between the wild-type and osccd7 mutant has been presented in box plots, bar charts, and violin plots, respectively.

Comment 6. Modify the title by replacing the word “edition” by “editing”

Response: Thank you for your careful review. We apologize for our oversight. We have made corrections based on your comments.

Comment 7. Use the word editing instead of edition throughout the manuscript.

Response: Thank you for your careful review. We apologize for our oversight. We have made corrections based on your comments.

 

Author Response File: Author Response.pdf