Establishment of Genetic Transformation System of Non-Embryogenic Callus in Rosa rugosa

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

This manuscript presents a valuable technical advance, a transformation system for R. rugosa non-embryogenic callus. The work is sound and addresses a relevant gap, but requires major revisions to solidify its claims and methodological rigor before publication.

1. The introduction should more clearly state the specific applications for which this non-regenerative system is ideal.
2. The assertion that this provides a platform for "functional gene validation in Rosaceae species" is overstated. Narrow this to reflect that the system was validated for a metabolic gene in a non-regenerative context, and broader applicability remains to be shown.
3. Clarify which solvent was used for the final data in Figure 5C and justify the choice, ideally by presenting solvent comparison data in a supplement.
4. Quantifying "total carotenoids" solely by A450 is insufficiently specific. HPLC analysis is needed to confirm specific carotenoid composition, or conclusions must be rephrased to reflect an increase in "total yellow pigment (A450)."
5. A negative control (empty vector) is required to distinguish the effects of the transformation process itself from those of RrPSY1 overexpression.
6. Explicitly state what "n" represents (biological vs. technical replicates) and report the number of independent transformation events analyzed.
7. Report on the transformation efficiency to allow comparison with other systems.
8. Clarify the figure legend to accurately describe what is shown in panels A, B, and C.
9. Include a positive control (plasmid) on the PCR gel to confirm primer functionality and expected band size.
10. The speculation on differential kanamycin sensitivity (35 vs. 75 mg/L) should be either substantiated with a deeper discussion e.g., on metabolic uptake or removed.
11. Resolve the discrepancy between the stated optimal kanamycin concentration (35 mg/L in results) and the "Screening culture" medium (30 mg/L in Table 1).
12. A thorough proofreading is required to correct typos e.g., "Drafr," "RvPSY1," "proliferation" and minor grammatical errors.

I hope by addressing these points is essential to validate the study conclusions. I am looking forward to seeing the revised version of the manuscript.

 

 

This manuscript presents a valuable technical advance, a transformation system for R. rugosa non-embryogenic callus. The work is sound and addresses a relevant gap, but requires major revisions to solidify its claims and methodological rigor before publication.

1. The introduction should more clearly state the specific applications for which this non-regenerative system is ideal.
2. The assertion that this provides a platform for "functional gene validation in Rosaceae species" is overstated. Narrow this to reflect that the system was validated for a metabolic gene in a non-regenerative context, and broader applicability remains to be shown.
3. Clarify which solvent was used for the final data in Figure 5C and justify the choice, ideally by presenting solvent comparison data in a supplement.
4. Quantifying "total carotenoids" solely by A450 is insufficiently specific. HPLC analysis is needed to confirm specific carotenoid composition, or conclusions must be rephrased to reflect an increase in "total yellow pigment (A450)."
5. A negative control (empty vector) is required to distinguish the effects of the transformation process itself from those of RrPSY1 overexpression.
6. Explicitly state what "n" represents (biological vs. technical replicates) and report the number of independent transformation events analyzed.
7. Report on the transformation efficiency to allow comparison with other systems.
8. Clarify the figure legend to accurately describe what is shown in panels A, B, and C.
9. Include a positive control (plasmid) on the PCR gel to confirm primer functionality and expected band size.
10. The speculation on differential kanamycin sensitivity (35 vs. 75 mg/L) should be either substantiated with a deeper discussion e.g., on metabolic uptake or removed.
11. Resolve the discrepancy between the stated optimal kanamycin concentration (35 mg/L in results) and the "Screening culture" medium (30 mg/L in Table 1).
12. A thorough proofreading is required to correct typos (e.g., "Drafr," "RvPSY1," "proliferation") and minor grammatical errors.

I hope by addressing these points is essential to validate the study conclusions. I am looking forward to seeing the revised version of the manuscript.

Author Response

Comments 1: The introduction should more clearly state the specific applications for which this non-regenerative system is ideal.

response 1: We sincerely appreciate and fully agree with the reviewer's constructive suggestions. The Introduction section has been thoroughly restructured and rewritten. The specific applications of non-regenerative system isdiscribed in lines 48–63 of the revised manuscript.

Comments 2: The assertion that this provides a platform for "functional gene validation in Rosaceae species" is overstated. Narrow this to reflect that the system was validated for a metabolic gene in a non-regenerative context, and broader applicability remains to be shown.

response 2: Thanks for your comments. Based on your suggestions, we rewritten this sentence and its related sentences to make the description more accurate in the revised version, respectively in lines 24-26, 77-78, and 279-281.

Comments 3: Clarify which solvent was used for the final data in Figure 5C and justify the choice, ideally by presenting solvent comparison data in a supplement.

response 3:Thanks for your comments. We sincerely apologize for writing error in our original manuscript. In fact, we conducted the experiment using water-saturated n-butanol as the solvent. We have revised the detailed methods in the revised version (revised Line 110-119).

Comments 4: Quantifying "total carotenoids" solely by A450 is insufficiently specific. HPLC analysis is needed to confirm specific carotenoid composition, or conclusions must be rephrased to reflect an increase in "total yellow pigment (A450)."

response 4: We sincerely thank the reviewer for highlighting this significant oversight in our original analysis.  We changed the vertical coordinate " carotenoids content" in Figure 5C to"total yellow pigment (A450)", and the corresponding sentences have also been modified (revised Line 22, 209).

Comments 5: A negative control (empty vector) is required to distinguish the effects of the transformation process itself from those of RrPSY1 overexpression.

response 5: Thanks for your comments. In Figure 5, 35S:GFP served as the negative control (empty plasmid), as clarified in the revised manuscript (Line 101). Although GFP fluorescence and corresponding GFP bands were detectable in callus transformed with 35S:GFP, no significant yellow pigment accumulation was observed. Therefore, further quantitative analysis was not performed.

Comments 6: Explicitly state what "n" represents (biological vs. technical replicates) and report the number of independent transformation events analyzed.

response 6: Thanks for your comments. Following your suggestion, we made modifications to the relevant figure legends (Fig2-5) to make the description more accurate in the revised version.

Comments 7: Report on the transformation efficiency to allow comparison with other systems.

response 7: Thanks for your comments. According to statistics, 6.96% of the35S:RrPSY1-GFP transgenic calli showed GFP fluorescence. However, we have to admit that this data cannot fully represent the transformation efficiency, as no further verification has been conducted. We added a clarification in the revised manuscript (Line 205).

Comments 8: Clarify the figure legend to accurately describe what is shown in panels A, B, and C.

response 8: Thanks for your comments. We checked and revised all the legend descriptions to ensure that these descriptions can clearly depict the content shown in the figures.

Comments 9: Include a positive control (plasmid) on the PCR gel to confirm primer functionality and expected band size.

response 9: Thanks for your comments. In Figure 5, 35S:GFP served as the control (empty plasmid). A band of the expected size (720 bp) for the 35S:GFP control was detected using GFP-specific primers, which is consistent with the expected band size.

Comments 10: The speculation on differential kanamycin sensitivity (35 vs. 75 mg/L) should be either substantiated with a deeper discussion e.g., on metabolic uptake or removed.

response 10: Thanks for your constructive suggestions. We have added new descriptions to discuss this question (revised Line 251-257).

Comments 11: Resolve the discrepancy between the stated optimal kanamycin concentration (35 mg/L in results) and the "Screening culture" medium (30 mg/L in Table 1).

response 11: Thanks for your comments. We sincerely apologize for the mistakes in our writing. “30 mg/L” in Table 1 was revised to “35 mg/L”.

Comments 12: A thorough proofreading is required to correct typos (e.g., "Drafr," "RvPSY1," "proliferation") and minor grammatical errors.

response 12: Thanks for your comments, we are very sorry for the mistakes in spelling caused by our carelessness. In the revised version, we re-checked the text throughout, and we tried our best to improve the manuscript and made some changes.

 

Reviewer 2 Report

Comments and Suggestions for Authors

 

Review evaluation results for the Manuscript ID: cimb-3913427 entitled as “Establishment of genetic transformation system of non-embryogenic callus in Rosa.”

Points and questions to be addressed: -

1. My first comment is that the manuscript should be carefully edited. Because it looks that the manuscript was written with less attention for spells, fonts, repetition of references multiple times with fonts inconsistency.
2. The materials and methods were not clearly explained. For instance, media protocols were not well defined (summarized in table 1) with missing differentiation medium protocols.
3. There are no clues how embryonic and non-embryonic experiments have been executed.
4. How do you differentiate the embryonic vs non-embryonic calli? How much you are very sure whether the calli be embryonic or non-embryonic based on calli color and visual observation as it is very subjective?

Some typo and font errors

5. The abbreviations given in abstract “CAT and PRO” should be defined for the first time.
6. The word “within” line#28, I think should be “within” and “R. rugosa” line#33 should be in italic and “mediums” line#77 should be media.
7. References at line#296 and line#342 as well as at line#314 and line#325 among the repeated once.

Thank you

Author Response

Comments 1: My first comment is that the manuscript should be carefully edited. Because it looks that the manuscript was written with less attention for spells, fonts, repetition of references multiple times with fonts inconsistency.

response 1: Thanks for your comments. We sincerely apologize for all the writing issues that appeared in our manuscript. In the revised version, we re-checked the text throughout, and we tried our best to improve the manuscript and made changes.

Comments 2: The materials and methods were not clearly explained. For instance, media protocols were not well defined (summarized in table 1) with missing differentiation medium protocols.

response 2: Thanks for your comments. Firstly, we added the differentiation medium protocol to Table 1. Then, we made some revisions to the Materials and Methods section to make the expression clearer.

Comments 3: There are no clues how embryonic and non-embryonic experiments have been executed.

response 3:Thanks for your comments. We have rewritten and revised the "3.1 Comparison of embryogenic and non-embryogenic callus" section, providing a clearer and more detailed description of the experimental content and results.

Comments 4: How do you differentiate the embryonic vs non-embryonic calli? How much you are very sure whether the calli be embryonic or non-embryonic based on calli color and visual observation as it is very subjective?

response 4: Thanks for your comments. In this study, the two callus types were distinguished based on distinct morphological criteria. Specifically, embryogenic calli exhibited a compact, granular structure, whereas non-embryogenic calli appeared soft and watery. These morphological characteristics are well-established and consistent with those documented in the reference (doi.org/10.3390/ijms241914625; doi.org/10.3390/plants12152869). Furthermore, the calli are from long-term preserved lines in our laboratory, we have definitively confirmed that only the embryogenic callus possesses regenerative capacity. Thus, we are confident in the identity of the materials used.

Comments 5: The abbreviations given in abstract “CAT and PRO” should be defined for the first time.

response 5: Thanks for your comments. Upon its first introduction in the abstract (revised Line 16-17), we written in full name. Then we checked the entire text to ensure that the abbreviation was used elsewhere.

Comments 6: The word “within” line#28, I think should be “within” and “R. rugosa” line#33 should be in italic and “mediums” line#77 should be media.

response 6: We sincerely thanks for your careful review of our manuscript. We have made thorough revisions to address these issues. In the revised version: Line 28 corresponds to revised Line 64: ‘‘wuthin” was revised to “within’’. All “R. rugosa’’ in the text have been italicized. the singular form of " medium " is represented by " medium ", and the plural form is changed to " media ".

Comments 7: References at line#296 and line#342 as well as at line#314 and line#325 among the repeated once.

response 7: Thanks for your comments. In the revised version, we reorganized and formatted all the documents to eliminate any duplicate citations and ensure their accuracy.

 

Reviewer 3 Report

Comments and Suggestions for Authors

The work "Establishment of a Genetic Transformation System of Non-embryogenic Callus in Rosa rugosa" by Xinyun Liu et al. is well-executed, with careful recording of the various antibiotic concentrations used. While the practical part is reasonably well-executed, the theoretical justification is poorly described.
The authors did not explain why, out of the many physiological parameters, they chose to detect catalase activity and proline content. The connection with regeneration processes (embryogenesis) is completely unexamined and unexplained. This should be substantiated in a separate paragraph in the Introduction.
The Introduction should also discuss the relationship between non-embryogenic and embryogenic callus. Is it possible to obtain embryogenic callus from non-embryogenic callus, and what factors might influence this? References to relevant publications, not limited to the Rosa genus, should be provided.
End of the Introduction section. The authors describe their existing achievements. This should be moved to the Discussion or Conclusion. And at the end of the Introduction section, it's important to state what the authors intended to achieve in their work. For example: "The aim of our study was...."
Abstract. Lines 16-17. The sentence is incorrect. The same applies to Lines 141 and 223. This should be corrected, as it is correctly written in Line 126.
Results section. Subsection 3.3. This should rather be moved to the Materials and Methods section.
It's unclear how the authors eliminated live Agrobacterium from the callus after transformation. This could have produced false positives.
Overall, the manuscript should be corrected.

Author Response

Comments 1: The authors did not explain why, out of the many physiological parameters, they chose to detect catalase activity and proline content. The connection with regeneration processes (embryogenesis) is completely unexamined and unexplained. This should be substantiated in a separate paragraph in the Introduction.

response 1: Thanks for your comments. We sincerely appreciate you bringing this issue to our attention. In the revised version, we rewrote the introduction and added relative description of the differences between embryogenic and non-embryogenic callus (revise line 39-47). Moreover, in the result section of 3.1 (revised line146-147), we added sentence to further explain the role of detecting these two physiological indicators.

Comments 2: The Introduction should also discuss the relationship between non-embryogenic and embryogenic callus. Is it possible to obtain embryogenic callus from non-embryogenic callus, and what factors might influence this? References to relevant publications, not limited to the Rosa genus, should be provided.

response 2: Thanks for your comments. Based on our rewriting of the introduction, the main relationship and differences between the two types of callus are described in the first and second paragraphs of the introduction in the revised version. To our knowledge, no literature has reported the conversion of non-embryogenic callus into embryogenic callus. However, as previously described, embryogenic callus can transition into non-embryogenic callus during prolonged subculture.

In indirect somatic embryogenesis, the callus is an intermediate product produced on the explant. Depending on subsequent culture conditions (e.g., hormone ratios, types, and light condition), the intermediate callus differentiates into either embryogenic or non-embryogenic types. The materials in this study were the two well-defined callus types obtained post-induction. Consequently, the non-embryogenic callus in our experiments does not equate to the intermediate callus material..

Comments 3: End of the Introduction section. The authors describe their existing achievements. This should be moved to the Discussion or Conclusion. And at the end of the Introduction section, it's important to state what the authors intended to achieve in their work. For example: "The aim of our study was....".

response 3:We appreciate the reviewer's constructive suggestion. we have revised the concluding section of the introduction to better emphasize our research objectives in the revised version.

Comments 4: Abstract. Lines 16-17. The sentence is incorrect. The same applies to Lines 141 and 223. This should be corrected, as it is correctly written in Line 126.

response 4: Thanks for your comments. We sincerely appreciate you bringing this issue to our attention. We have corrected all the incorrect descriptions. All “CAT and PRO” have been changed to “CAT activity and PRO content” (revised Line 16-17,145,148,162, 236, and 256).

Comments 5: Results section. Subsection 3.3. This should rather be moved to the Materials and Methods section.

response 5: Thanks for your comments. We have moved this part to “2.3. Transformation procedure” of "Materials and Methods" section.

Comments 6: It's unclear how the authors eliminated live Agrobacterium from the callus after transformation. This could have produced false positives.

response 6: We sincerely appreciate the reviewers' comments and we also agree that there might be residual Agrobacterium in the transformed callus. In our study, positive transformants were carefully identified using a combination of GFP fluorescence, PCR, and yellow pigment quantification. Among them, the GFP fluorescence and PCR detection may yield false positives due to the residual presence of Agrobacterium, whereas pigment production is a definitive marker of functional plant gene expression. Therefore, in order to ensure the rigor of the conclusion, we emphasized that this transformation system is more suitable for the functional verification of secondary metabolism-related genes. For the validation of other types of genes, more stringent experimental designs would indeed be required, which represents a key direction for our future work.

Comments 7: Overall, the manuscript should be corrected.

response 7: Thanks for your comments. To improve the quality of the manuscript, we have thoroughly revised the manuscript. Special attention has been paid to refining sentence tenses, structural organization, and overall expression. We believe these revisions have significantly improved the fluency and readability of the article, thereby enhancing the clarity of the scientific narrative.

 

 

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

Authors made efforts to revise the manuscript. I have no more suggestions.

Reviewer 3 Report

Comments and Suggestions for Authors

After the corrections were made, the quality of the manuscript was significantly improved. The work can be recommended for publication.