Droplet-Vitrification Protocol for Cryopreservation of Ginger (Zingiber officinale) Shoot Tips
and Jie Li 1,*
Round 1
Reviewer 1 Report
Comments and Suggestions for AuthorsThis manuscript investigates the cryopreservation of ginger shoot tips using the droplet-vitrification method. The protocol was optimized for one genotype and its applicability was tested in three other genotypes. The authors investigated different parameters within the cryoprocedure as well as media for shoot tip regrowth. In addition, histological and ultrastructural analyses were performed to study the different parameters used within the cryoprocedure. The manuscript is interesting and well developed. Below I have suggested many points that would make the document even more valuable, most of which relate to the need for additional information on materials and methods to make it easier for others to use the proposed procedure. I have also suggested rewording some sentences.
Please find the detailed report below:
Title: Use a hyphen for droplet-vitrification
Include figures and tables as soon as they are mentioned in the text to make them easier to read.
L 13: I assume that the term "widely applicable" means a lot more than 4 genotypes. So please consider deleting "widely applicable".
L 35: Use words that are not already in the title.
L 46-48: Consider rephrasing to “The conservation of plant genetic resources is essential for the future efforts of breeding programs. As a result, numerous genebanks have been established around the world to conserve and utilize germplasm resources.”
L 48: liquid nitrogen (LN)
L 50-51: Consider rephrasing to “Once cryopreserved, the sample maintains its viability upon thawing, allowing for indefinite storage.”
L 54: Consider adding additional references where the morphological anatomy of cryopreserved plants was similar to that of controls - for example, the following reference (https://doi.org/10.3390/horticulturae9070841) on pineapple cryopreservation, where the authors found that no differences were observed in any of the indicators evaluated when plantlets obtained from cryopreserved shoot tips were compared with controls.
L 54-57: Consider deleting this sentence - once the number may not reflect current numbers as it increases over the years and the paper referenced is about 5 years old.
L 58: Consider rephrasing to “To date, studies on the cryopreservation of ginger shoot tips are relatively scarce.”
L 59: G Yamuna et al. also worked on vitrification - I assume it was the best protocol (80% regrowth) compared to encapsulation-dehydration and encapsulation-vitrification - so sentence needs rewording.
L 62: I suggest adding a sentence introducing the droplet-vitrification procedure, adding its original reference, and then the sentence that you compared it (L 62-64) to the method already used for this plant species.
L 65: However, to the best of our knowledge,
L 66-67: Consider rephrasing to “Therefore, efforts to develop a droplet-vitrification protocol for this plant species would further increase the cryopreservability of ginger genetic resources.” Consider adding a sentence on the potential use of the developed protocol for disease eradication (cryotherapy) in addition to conservation efforts – add references on cryotherapy – some for your consideration: https://doi.org/10.1111/ppa.13565 https://doi.org/10.1007/s11240-020-01846-x
L 68-70: Consider rephrasing to “The aim of this study was to establish an efficient and widely applicable droplet-vitrification protocol for in vitro cryopreservation of shoot tips from four Chinese ginger genotypes.”
L 70-71: observations, as well as evaluation of genetic stability by ISSR analysis
*Describe ISSR at first mention
L 83: Describe MS at first mention
L 84: Complete information on the culture medium is required – sucrose, pH, agar? – Please add this information in detail for others to replicate the procedure.
L 85: “Sub-85 culture was conducted every five weeks” – How? Using the same medium? Please describe in detail
L 86-87: “Shoot induction from nodal segments of five-week-old stock cultures was established in our previous study” – please describe the procedure in detail then reference your previous study - This is not clear in the figures - please describe it in more detail.
L 93: from XXX-week old induced shoots.
L 93: Delete “According to our preliminary studies”
L 94: “added for 1-4 days”? it is not clear - is this a variation in the duration of treatment - also include full information on the preculture medium - was it liquid? solid? pH? Autoclaved or filter-sterilized? Be sure to provide detailed information.
L 95: “had been treated with a loading” ?? You mentioned in the previous sentence that they were precultured, not loaded on loading solution. Sentence needs checking
L 98: Describe PVS2 at first mention
L 98: Add all treatments in this interval (0-25 min), because the current way the reader does not know if it was assessed in a 5 or 10 min interval.
L 100: PVS2 - Autoclaved or filter-sterilized? Add this to the text.
L 105: How about the pH of the unloading solution?
L 106: Delete “According to our preliminary studies”
L 106-113: Describe abbreviations – complete information on culture media is needed… sucrose, pH…
L 111: Only for SRM4, cryopreserved shoot tips were incubated in the dark for 3 days and then transferred to light conditions, or was this also done for the other media - it is not clear…. Revise this.
L 122: Preliminary studies, or are you assessing this in this manuscript - if so, grammar needs review.
L 155: How many subcultures were performed on the recovered shoots and controls prior to ISSR analysis? Add this to the text.
L 157: Describe CTAB at first mention
L 157: Briefly describe the protocol, then add the reference
L 159: Include primer sequences either in the text as a table or in the Supplementary Material.
L 160-170: Describe abbreviations
L 171: same comment as above
L 173: How many subcultures were performed on the recovered shoots and controls prior to FCM analysis? Add this to the text.
L 203-205: How long was the incubation at this concentration? This is required in this section.
L 223-224: Consider rephrasing to “As the exposure time increased, the regrowth rate continued to improve, reaching a maximum of 33.33% at 20 min”.
L 224-226: Consider rephrasing to “Conversely, the regrowth rate of the treated control shoot tips decreased from 100% without PVS2 exposure to 36.67% when the PVS2 exposure time was 25 min.”
L 233: Add: What was the difference among the SRM1-3 and the SRM4?
L 241: PVS2 alone (PVS2 - AsA)
L 242-244: In comparison to what?
L 247: Parentheses are not needed in this sentence - revise it.
L 252: 54.17%, respectively,
L 255: So that means 3 months without any subculture.??
L 256-259: it (acclimatization) was not included in the Materials and Methods. Please add a Materials and Methods topic describing this.
L 260: Be sure to re-describe what positive and negative controls mean, otherwise the reader will have to read the Materials and Methods again to get it.
L 297: add the age to these plants….
L 298: on survival and regrowth rates
Table 1 – add a line below –LN/+LN.
Table 4 – You could add the statistical test to Table 4
L 376: Be sure to re-describe what positive and negative controls mean, otherwise the reader will have to read the Materials and Methods again to get it.
Table 5 – Add lines for the separation of references, also below the first line.
L 403-405: Consider rephrasing to “These previous studies used only a single genotype, which may limit the ability to assess the effects of cryopreservation procedures across multiple genotypes.”
L 405: Before talking about droplet-vitrification, add a sentence emphasizing that in the present study, the protocol was optimized for one genotype and tested in additional 3 genotypes to evaluate its applicability. After that, authors can add a sentence to explain why droplet-vitrification was chosen over other methods.
L 409: droplet-vitrification
L 413: 54.17%, respectively
L 415: “In these studies” ?? What studies?? – You can add something like “In the previous studies on cryopreserving Z. officinale….
L 416-418: This information is not clear in the Materials and Methods - please describe it in detail in the Materials and Methods.
L 417: served as source of material for shoot tip isolation
L 422: and most importantly, shoot tip uniformity (https://doi.org/10.3390/plants10102190)
L 425: “Samples”?? why not say shoot tip?
L 442: " PVS are crucial for cryopreservation techniques based on vitrification” – poor sentence – Please explore this further.
* This is the first time PVS has been added to the text - you have added PVS2 before - so description for PVS is needed.
L 469-471: poor sentence – Please explore this further.
L 471-472: “Notably, the recovery medium containing only GA3 significantly mitigated the risk of genetic variation.” Which medium? This is not clear - refer to the abbreviation used.
L 474-475: poor sentence – Please explore this further and add references to support this sentence
L 479: Antioxidants were also used in reference number 35.
L 484: antioxidants have also been added to the preculture or loading steps – reference number 35.
L 503: clonal cryopreservation
Comments on the Quality of English LanguageIn order to make the text more direct, I have suggested some sentence improvement/rephrasing.
Author Response
Comments 1: Title: Use a hyphen for droplet-vitrification. Include figures and tables as soon as they are mentioned in the text to make them easier to read.
Response 1: Special thanks to you for pointing this out. We agree with this comment. Except for the title, the other parts of the manuscript were all expressed as ‘droplet-vitrification’. Based on your comment, we have revised the title and marked it in red. Mention exactly where in the revised manuscript this change can be found in L2.
Comments 2: L 13: I assume that the term "widely applicable" means a lot more than 4 genotypes. So please consider deleting "widely applicable".
Response 2: We sincerely thank the reviewer for pointing this out. We agree with this comment. Other reviewers suggested that the study may have been conducted on a single genotype rather than all four. Because all experiments and analyses were conducted only on Z. officinale ‘Yunnan Xiaohuangjiang’. There are no cryopreservation results, preliminary applications (e.g., ascorbic acid, sucrose concentrations), or ISSR analyses presented for the other three genotypes. Although the manuscript mentions four genotypes, no statistical results are provided for the others. Based on your comment, we kept one genotype and have deleted ‘widely applicable’. Mention exactly where in the revised manuscript this change can be found in L13.
Comments 3: L 35: Use words that are not already in the title.
Response 3: Special thanks to you for pointing this out. We agree with this comment. Based on your comment, we have deleted ‘Zingiber officinale’, ‘shoot tips’ and ‘droplet-vitrification’. Mention exactly where in the revised manuscript this change can be found in L39-40.
Comments 4: L 46-48: Consider rephrasing to “The conservation of plant genetic resources is essential for the future efforts of breeding programs. As a result, numerous genebanks have been established around the world to conserve and utilize germplasm resources.”
Response 4: We appreciate the reviewer's feedback regarding the deficiencies in our writing. We agree with this comment. Based on your comment, we have revised the sentence and marked it in red. Mention exactly where in the revised manuscript this change can be found in L52-54.
Comments 5: L 48: liquid nitrogen (LN)
Response 5: We sincerely thank the reviewer for pointing this out. We agree with this comment. Based on your comment, we have revised it and marked it in red. Mention exactly where in the revised manuscript this change can be found in L55.
Comments 6: L 50-51: Consider rephrasing to “Once cryopreserved, the sample maintains its viability upon thawing, allowing for indefinite storage.”
Response 6: We sincerely appreciate the reviewer for identifying the shortcomings in our writing. We agree with this comment. Based on your comment, we have revised the sentence and marked it in red. Mention exactly where in the revised manuscript this change can be found in L56-57.
Comments 7: L 54: Consider adding additional references where the morphological anatomy of cryopreserved plants was similar to that of controls - for example, the following reference (https://doi.org/10.3390/horticulturae9070841) on pineapple cryopreservation, where the authors found that no differences were observed in any of the indicators evaluated when plantlets obtained from cryopreserved shoot tips were compared with controls.
Response 7: We thank the reviewer for highlighting the missing information in our manuscript. We agree with this comment. Based on your comment, we have added the references and marked them in red. Mention exactly where in the revised manuscript this change can be found in L60.
Comments 8: L 54-57: Consider deleting this sentence - once the number may not reflect current numbers as it increases over the years and the paper referenced is about 5 years old.
Response 8: We sincerely thank the reviewer for pointing this out. We agree with this comment. Based on your comment, we have deleted the reference.
Comments 9: L 58: Consider rephrasing to “To date, studies on the cryopreservation of ginger shoot tips are relatively scarce.”
Response 9: We sincerely appreciate the reviewer for identifying the shortcomings in our writing. We agree with this comment. Based on your comment, we have rephrased the reference and marked them in red. Mention exactly where in the revised manuscript this change can be found in L61.
Comments 10: L 59: G Yamuna et al. also worked on vitrification - I assume it was the best protocol (80% regrowth) compared to encapsulation-dehydration and encapsulation-vitrification - so sentence needs rewording.
Response 10: We sincerely thank the reviewer for pointing this out. We agree with this comment. Based on your comment, we have reworded the reference and marked them in red. Mention exactly where in the revised manuscript this change can be found in L63.
Comments 11: L 62: I suggest adding a sentence introducing the droplet-vitrification procedure, adding its original reference, and then the sentence that you compared it (L 62-64) to the method already used for this plant species.
Response 11: We extend our gratitude to the reviewer for the insightful comment. We agree with it. Based on your comment, we have added the sentences introducing the droplet-vitrification procedure and marked them in red. Mention exactly where in the revised manuscript this change can be found in L69-77.
Comments 12: L 65: However, to the best of our knowledge,
Response 12: We sincerely appreciate the reviewer for identifying the shortcomings in our writing. We agree with this comment. Based on your comment, we have corrected and marked them in red. Mention exactly where in the revised manuscript this change can be found in L85-86.
Comments 13: L 66-67: Consider rephrasing to “Therefore, efforts to develop a droplet-vitrification protocol for this plant species would further increase the cryopreservability of ginger genetic resources.” Consider adding a sentence on the potential use of the developed protocol for disease eradication (cryotherapy) in addition to conservation efforts – add references on cryotherapy – some for your consideration: https://doi.org/10.1111/ppa.13565 https://doi.org/10.1007/s11240-020-01846-x
Response 13: We sincerely thank the reviewer for pointing this out. We agree with this comment. Based on your comment, we have rephrased the sentences, added a sentence on the potential use of the developed protocol for disease eradication (cryotherapy) in addition to conservation efforts and marked them in red. Mention exactly where in the revised manuscript this change can be found in L87-90.
Comments 14: L 68-70: Consider rephrasing to “The aim of this study was to establish an efficient and widely applicable droplet-vitrification protocol for in vitro cryopreservation of shoot tips from four Chinese ginger genotypes.”
Response 14: We appreciate the reviewer for the comment. We agree with it. Other reviewers suggested that the study may have been conducted on a single genotype rather than all four. Based on your comments, we have rephrased the sentence and marked them in red. Mention exactly where in the revised manuscript this change can be found in L91-93.
Comments 15: L 70-71: observations, as well as evaluation of genetic stability by ISSR analysis
*Describe ISSR at first mention
Response 15: We sincerely appreciate the reviewer for identifying the shortcomings in our writing. We agree with this comment. Based on your comment, we have corrected and marked them in red. Mention exactly where in the revised manuscript this change can be found in L93-94.
Comments 16: L 83: Describe MS at first mention
Response 16: We sincerely thank the reviewer for pointing this out. We agree with this comment. Based on your comment, we have corrected and marked them in red. Mention exactly where in the revised manuscript this change can be found in L103.
Comments 17: L 84: Complete information on the culture medium is required – sucrose, pH, agar? – Please add this information in detail for others to replicate the procedure.
Response 17: We sincerely thank the reviewer for pointing this out. We agree with this comment. Based on your comment, we have added the information and marked them in red. Mention exactly where in the revised manuscript this change can be found in L105-106.
Comments 18: L 85: “Sub-85 culture was conducted every five weeks” – How? Using the same medium? Please describe in detail
Response 18: We extend our gratitude to the reviewer for the insightful comment. We agree with it. Based on your comment, we have added the information and marked them in red. Mention exactly where in the revised manuscript this change can be found in L108-109.
Comments 19: L 86-87: “Shoot induction from nodal segments of five-week-old stock cultures was established in our previous study” – please describe the procedure in detail then reference your previous study - This is not clear in the figures - please describe it in more detail.
Response 19: We sincerely thank the reviewer for pointing this out. We agree with this comment. Based on your comment, we have added the information and marked them in red. Mention exactly where in the revised manuscript this change can be found in L109-113.
Comments 20: L 93: from XXX-week old induced shoots.
Response 20: We appreciate the reviewer for the comment. We agree with it. Based on your comment, we have added and marked it in red. Mention exactly where in the revised manuscript this change can be found in L117.
Comments 21: L 93: Delete “According to our preliminary studies”
Response 21: We sincerely thank the reviewer for pointing this out. We agree with this comment. Based on your comment, we have deleted it. Mention exactly where in the revised manuscript this change can be found in L115.
Comments 22: L 94: “added for 1-4 days”? it is not clear - is this a variation in the duration of treatment - also include full information on the preculture medium - was it liquid? solid? pH? Autoclaved or filter-sterilized? Be sure to provide detailed information.
Response 22: We sincerely thank the reviewer for pointing this out. We agree with this comment. Based on your comment, we have added the information and marked them in red. Mention exactly where in the revised manuscript this change can be found in L117-118.
Comments 23: L 95: “had been treated with a loading” ?? You mentioned in the previous sentence that they were precultured, not loaded on loading solution. Sentence needs checking
Response 23: We thank the reviewer for pointing out the potentially misleading sentences in our manuscript. We agree with this comment. Based on your comment, we have rephrased and marked it in red. Mention exactly where in the revised manuscript this change can be found in L119-120.
Comments 24: L 98: Describe PVS2 at first mention
Response 24: We thank the reviewer for highlighting the missing information in our manuscript. We agree with this comment. Based on your comment, we have added and marked it in red. Mention exactly where in the revised manuscript this change can be found in L124.
Comments 25: L 98: Add all treatments in this interval (0-25 min), because the current way the reader does not know if it was assessed in a 5 or 10 min interval.
Response 25: We thank the reviewer for highlighting the missing information in our manuscript. We agree with this comment. Based on your comment, we have added and marked it in red. Mention exactly where in the revised manuscript this change can be found in L124.
Comments 26: L 100: PVS2 - Autoclaved or filter-sterilized? Add this to the text.
Response 26: We thank the reviewer for pointing out the missing information in our manuscript. We agree with this comment. Based on your comment, we have added and marked it in red. Mention exactly where in the revised manuscript this change can be found in L126.
Comments 27: L 105: How about the pH of the unloading solution?
Response 27: We thank the reviewer for highlighting the missing information in our manuscript. We agree with this comment. Based on your comment, we have added and marked it in red. Mention exactly where in the revised manuscript this change can be found in L134-135.
Comments 28: L 106: Delete “According to our preliminary studies”
Response 28: We sincerely thank the reviewer for pointing this out. We agree with this comment. Based on your comment, we have deleted it. Mention exactly where in the revised manuscript this change can be found in L135.
Comments 29: L 106-113: Describe abbreviations – complete information on culture media is needed… sucrose, pH…
Response 29: We thank the reviewer for highlighting the missing information in our manuscript. We agree with this comment. Based on your comment, we have added and marked it in red. Mention exactly where in the revised manuscript this change can be found in L136-145.
Comments 30: L 111: Only for SRM4, cryopreserved shoot tips were incubated in the dark for 3 days and then transferred to light conditions, or was this also done for the other media - it is not clear…. Revise this.
Response 30: We thank the reviewer for pointing out the potentially misleading sentences in our manuscript. We agree with this comment. Based on your comment, we have rephrased and marked it in red. Mention exactly where in the revised manuscript this change can be found in L147.
Comments 31: L 122: Preliminary studies, or are you assessing this in this manuscript - if so, grammar needs review.
Response 31: We appreciate the reviewer for the comment. We agree with it. Based on your comment, we have deleted and marked it in red. Mention exactly where in the revised manuscript this change can be found in L159.
Comments 32: L 155: How many subcultures were performed on the recovered shoots and controls prior to ISSR analysis? Add this to the text.
Response 32: We thank the reviewer for highlighting the missing information in our manuscript. We agree with this comment. Based on your comment, we have added and marked it in red. Mention exactly where in the revised manuscript this change can be found in L185-186.
Comments 33: L 157: Describe CTAB at first mention
Response 33: We thank the reviewer for highlighting the missing information in our manuscript. We agree with this comment. Based on your comment, we have added and marked it in red. Mention exactly where in the revised manuscript this change can be found in L189.
Comments 34: L 157: Briefly describe the protocol, then add the reference
Response 34: We appreciate the reviewer for the comment. We agree with it. The reference was added before in the manuscript (Porebski, S., Bailey L.G., Baum B.R. Modification of a CTAB DNA extraction protocol for plant containing high polysaccharides and polyphenol component. Plant Mol. Biol. Rep. 1997, 15, 8-15.). Based on your comment, we have described the protocol, and marked it red. Mention exactly where in the revised manuscript this change can be found in L189-213.
Comments 35: L 159: Include primer sequences either in the text as a table or in the Supplementary Material.
Response 35: We appreciate the reviewer for the comment. We agree with this comment. Based on your comment, we have added and marked it in red. Mention exactly where in the revised manuscript this change can be found in L215-218.
Comments 36: L 160-170: Describe abbreviations
Response 36: We thank the reviewer for highlighting the missing information in our manuscript. We agree with this comment. Based on your comment, we have added and marked it in red. Mention exactly where in the revised manuscript this change can be found in L188-202.
Comments 37: L 171: same comment as above
Response 37: We thank the reviewer for highlighting the missing information in our manuscript. We agree with this comment. Based on your comment, we have added and marked it in red. Mention exactly where in the revised manuscript this change can be found in L227-228.
Comments 38: L 173: How many subcultures were performed on the recovered shoots and controls prior to FCM analysis? Add this to the text.
Response 38: We extend our gratitude to the reviewer for the insightful comment. We agree with it. Based on your comment, we have added and marked it in red. Mention exactly where in the revised manuscript this change can be found in L231-233.
Comments 39: L 203-205: How long was the incubation at this concentration? This is required in this section.
Response 39: Thanks again for the reviewer's comments. We sincerely apologize for not clearly explaining the details in the manuscript. We agree with this comment. Based on your comment, we have added and marked it in red. Mention exactly where in the revised manuscript this change can be found in L274.
Comments 40: L 223-224: Consider rephrasing to “As the exposure time increased, the regrowth rate continued to improve, reaching a maximum of 33.33% at 20 min”.
Response 40: We appreciate the reviewer for the comment. We agree with it. Based on your comment, we have rephrased and marked it in red. Mention exactly where in the revised manuscript this change can be found in L294-295.
Comments 41: L 224-226: Consider rephrasing to “Conversely, the regrowth rate of the treated control shoot tips decreased from 100% without PVS2 exposure to 36.67% when the PVS2 exposure time was 25 min.”
Response 41: Thanks again for the reviewer's comments. We agree with it. Based on your comment, we have rephrased and marked it in red. Mention exactly where in the revised manuscript this change can be found in L294-297.
Comments 42: L 233: Add: What was the difference among the SRM1-3 and the SRM4?
Response 42: We thank the reviewer for highlighting the missing information in our manuscript. We agree with this comment. Based on your comment, we have added and marked them in red. Mention exactly where in the revised manuscript this change can be found in L305-309.
Comments 43: L 241: PVS2 alone (PVS2 - AsA)
Response 43: We appreciate the reviewer for the comment. We agree with it. Based on your comment, we have corrected and marked it in red. Mention exactly where in the revised manuscript this change can be found in L315.
Comments 44: L 242-244: In comparison to what?
Response 44: Thanks again for the reviewer's comments. We sincerely apologize for not clearly explaining the details in the manuscript. We agree with this comment. Based on your comment, we have rephrased and marked them in red. Mention exactly where in the revised manuscript this change can be found in L315-320.
Comments 45: L 247: Parentheses are not needed in this sentence - revise it.
Response 45: Thanks again for the reviewer's comments. Because we kept one genotype and deleted other three genotypes, so the sentence has been deleted.
Comments 46: L 252: 54.17%, respectively,
Response 46: Thanks again for the reviewer's comments. Because we kept one genotype and deleted other three genotypes, so the sentence has been deleted.
Comments 47: L 255: So that means 3 months without any subculture.??
Response 47: We sincerely thank the reviewer for pointing this out. We sincerely apologize for not clearly explaining the details in the manuscript. After 8 weeks of recovery culture, the cryopreserved shoot tips could grow into plants about 1 cm in length. Then, the regenerated plantlets were transferred to subculture media for another eight-week period to achieve a vigorous and robust growth state for transplanting. Based on your comment, we have rephrased and marked them in red. Mention exactly where in the revised manuscript this change can be found in L323-325.
Comments 48: L 256-259: it (acclimatization) was not included in the Materials and Methods. Please add a Materials and Methods topic describing this.
Response 48: We extend our gratitude to the reviewer for the insightful comment. We sincerely apologize for not clearly explaining the details in the manuscript. We agree with this comment. Based on your comment, we have rephrased and marked them in red. Mention exactly where in the revised manuscript this change can be found in L242-248.
Comments 49: L 260: Be sure to re-describe what positive and negative controls mean, otherwise the reader will have to read the Materials and Methods again to get it.
Response 49: Thanks again for the reviewer's comments. We agree with it. Based on your comment, we have added the information and marked it in red. Mention exactly where in the revised manuscript this change can be found in L330-333.
Comments 50: L 297: add the age to these plants….
Response 50: Thanks again for the reviewer's comments. We sincerely apologize for not clearly explaining the details in the manuscript. We agree with this comment. Based on your comment, we have added and marked it in red. Mention exactly where in the revised manuscript this change can be found in L368.
Comments 51: L 298: on survival and regrowth rates
Response 51: We sincerely thank the reviewer for pointing this out. We agree with this comment. Based on your comment, we have corrected and marked it in red. Mention exactly where in the revised manuscript this change can be found in L370.
Comments 52: Table 1 – add a line below –LN/+LN.
Response 52: Thanks again for the reviewer's comments. We’re sorry that we didn’t understand your meaning. Table 1 was already clear and may not need to be revised.
Comments 53: Table 4 – You could add the statistical test to Table 4
Response 53: Thanks again for the reviewer's comments. Because we kept one genotype and deleted other three genotypes, so the table has been deleted.
Comments 54: L 376: Be sure to re-describe what positive and negative controls mean, otherwise the reader will have to read the Materials and Methods again to get it.
Response 54: We sincerely thank the reviewer for pointing this out. We agree with this comment. Based on your comment, we have added the information and marked it in red. Mention exactly where in the revised manuscript this change can be found in L459-460.
Comments 55: Table 5 – Add lines for the separation of references, also below the first line.
Response 55: We sincerely thank the reviewer for pointing this out. We agree with this comment. Based on your comment, we have added lines. The original Table 4 has been deleted, and the original Table 5 has become the current Table 4. Mention exactly where in the revised manuscript this change can be found in table 4.
Comments 56: L 403-405: Consider rephrasing to “These previous studies used only a single genotype, which may limit the ability to assess the effects of cryopreservation procedures across multiple genotypes.”
Response 56: Thanks again for the reviewer's comments. Because we kept one genotype and deleted other three genotypes, so this sentence has been deleted.
Comments 57: L 405: Before talking about droplet-vitrification, add a sentence emphasizing that in the present study, the protocol was optimized for one genotype and tested in additional 3 genotypes to evaluate its applicability. After that, authors can add a sentence to explain why droplet-vitrification was chosen over other methods.
Response 57: We sincerely thank the reviewer for pointing this out. We kept one genotype and deleted other three genotypes. A sentence emphasizing that in the present study, the protocol was optimized for one genotype and tested in additional 3 genotypes to evaluate its applicability was not needed. In our manuscript, the advantages of droplet vitrification have been presented. However, there have been no reports on applying droplet vitrification in ginger. This explained why we chose droplet vitrification over other methods. Based on your comment, we have added a sentence to make the reason more convincing and marked it in red. Mention exactly where in the revised manuscript this change can be found in L495-498
Comments 58: L 409: droplet-vitrification
Response 58: We sincerely thank the reviewer for pointing this out. We agree with this comment. Based on your comment, we have corrected and marked it in red. Mention exactly where in the revised manuscript this change can be found in L501.
Comments 59: L 413: 54.17%, respectively
Response 59: Thanks again for the reviewer's comments. Because we kept one genotype and deleted other three genotypes, so this sentence has been deleted.
Comments 60: L 415: “In these studies” ?? What studies?? – You can add something like “In the previous studies on cryopreserving Z. officinale….
Response 60: Thanks again for the reviewer's comments. We sincerely apologize for not clearly explaining the details in the manuscript. We agree with this comment. Based on your comment, we have added and marked it in red. Mention exactly where in the revised manuscript this change can be found in L507.
Comments 61: L 416-418: This information is not clear in the Materials and Methods - please describe it in detail in the Materials and Methods.
Response 61: Thanks again for the reviewer's comments. We sincerely apologize for not clearly explaining the details in the manuscript. We agree with this comment. Based on your comment, we have added and marked it in red. Mention exactly where in the revised manuscript this change can be found in L109-113.
Comments 62: L 417: served as source of material for shoot tip isolation
Response 62: Thanks again for the reviewer's comments. We agree with it. Based on your comment, we have corrected and marked it in red. Mention exactly where in the revised manuscript this change can be found in L510.
Comments 63: L 422: and most importantly, shoot tip uniformity (https://doi.org/10.3390/plants10102190)
Response 63: Thanks again for the reviewer's comments. We agree with it. Based on your comment, we have corrected, added the reference and marked it in red. Mention exactly where in the revised manuscript this change can be found in L518.
Comments 64: L 425: “Samples”?? why not say shoot tip?
Response 64: Thanks again for the reviewer's comments. We agree with it. Based on your comment, we have corrected and marked it in red. Mention exactly where in the revised manuscript this change can be found in L518.
Comments 65: L 442: " PVS are crucial for cryopreservation techniques based on vitrification” – poor sentence – Please explore this further.
* This is the first time PVS has been added to the text - you have added PVS2 before - so description for PVS is needed.
Response 65: We sincerely thank the reviewer for pointing this out. We agree with this comment. Based on your comment, we have corrected and marked it in red. PVS first appeared in the text at L123-124. Mention exactly where in the revised manuscript this change can be found in L541-543.
Comments 66: L 469-471: poor sentence – Please explore this further.
Response 66: We extend our gratitude to the reviewer for the insightful comment. We agree with this comment. Based on your comment, we have corrected and marked it in red. Mention exactly where in the revised manuscript this change can be found in L568-572.
Comments 67: L 471-472: “Notably, the recovery medium containing only GA3 significantly mitigated the risk of genetic variation.” Which medium? This is not clear - refer to the abbreviation used.
Response 66: Thanks again for the reviewer's comments. We agree with it. Based on your comment, we have corrected and marked it in red. Mention exactly where in the revised manuscript this change can be found in L572.
Comments 68: L 474-475: poor sentence – Please explore this further and add references to support this sentence
Response 68: Thanks again for the reviewer's comment. We agree with it. Based on your comment, we have added and marked it in red. Mention exactly where in the revised manuscript this change can be found in L576-577.
Comments 69: L 479: Antioxidants were also used in reference number 35.
Response 69: Special thanks to you for your good comment. We agree with it. Based on your comment, we have added and marked it in red. Mention exactly where in the revised manuscript this change can be found in L581.
Comments 70: L 484: antioxidants have also been added to the preculture or loading steps – reference number 35.
Response 70: We thank the reviewer for thoroughly examining our manuscript and pointing this out. We agree with this comment. Based on your comment, we have added and marked it in red. Mention exactly where in the revised manuscript this change can be found in L586-587.
Comments 71: L 503: clonal cryopreservation
Response 71: We appreciate the reviewer for the comment. We agree with it. Based on your comment, we have added and marked it in red. Mention exactly where in the revised manuscript this change can be found in L609.
Author Response File: 
Author Response.pdf
Reviewer 2 Report
Comments and Suggestions for AuthorsDear Authors,
The manuscript presents an engaging and relevant study on developing a droplet vitrification protocol for cryopreservation of shoot apices of Zingiber officinale. The topic is essential for the conservation of plant genetic resources, and the design of the experiment appears robust, supported by comprehensive histological, ultrastructural, and molecular analyses. The manuscript is generally well-written, but there are areas where clarity, depth of discussion, and methodological details could be improved.
The introduction provides a good background, but developing the rationale for choosing droplet vitrification over other cryopreservation techniques is beneficial. What are the advantages and disadvantages? It would also be worth considering expanding the specific challenges of ginger cryopreservation that this study seeks to address.
The description of the cryopreservation protocol is detailed, but information on the criteria for selecting the four ginger genotypes is missing. Please also better explain the statistical methods used to analyze the data, including post hoc tests after ANOVA analysis.
The presentation of the results is clear, but more detailed statistical data (e.g., p-values) would increase the overall credibility of the results. Consider moving some of the results to the supplement - images of agarose gels should be considered.
The discussion effectively interprets the results but could be strengthened by a more critical analysis of the study's limitations and suggestions for future research. The discussion of survival and regrowth rates could be strengthened by comparing them with previous studies on other plant species. Please consider discussing the practical implications of the protocol for large-scale genetic conservation programs.
Best regards,
R.
Comments on the Quality of English LanguageThe manuscript is generally well written. However, there are grammatical errors and awkward phrasing. To improve readability, thorough linguistic editing is recommended.
Author Response
Comments 1: The introduction provides a good background, but developing the rationale for choosing droplet vitrification over other cryopreservation techniques is beneficial. What are the advantages and disadvantages? It would also be worth considering expanding the specific challenges of ginger cryopreservation that this study seeks to address.
Response 1: We extend our gratitude to the reviewer for the insightful comment. We agree with this comment. Based on your comment, we have added some sentences and made them in red. Mention exactly where in the revised manuscript this change can be found in L77-85, L87-90.
Comments 2: The description of the cryopreservation protocol is detailed, but information on the criteria for selecting the four ginger genotypes is missing. Please also better explain the statistical methods used to analyze the data, including post hoc tests after ANOVA analysis.
Response 2: We sincerely thank the reviewer for pointing this out. Other reviewers suggested that the study may have been conducted on a single genotype rather than all four. Because all experiments and analyses were conducted only on Z. officinale ‘Yunnan Xiaohuangjiang’. So we kept one genotype. Explain the statistical methods used to analyze the data, including post hoc tests after ANOVA analysis has been revised combined with the following comment. Based on your comment, we have added some information for selecting the genotype and made them in red. Mention exactly where in the revised manuscript this change can be found in L98-102, L257-258.
Comments 3: The presentation of the results is clear, but more detailed statistical data (e.g., p-values) would increase the overall credibility of the results. Consider moving some of the results to the supplement - images of agarose gels should be considered.
Response 3: Special thanks to you for pointing this out. Statistical data (e.g., p-values) was described in tables and figures. However, the section on materials and methods lacked a detailed description of statistical data (e.g., p-values). We agree with this comment. Based on your comment, we have added and marked it in red. Mention exactly where in the revised manuscript this change can be found in L257-258. We think that a selection of photographic images depicting agarose should be incorporated and presented within the manuscript. Following the recommendation put forward by one of the reviewers, the photographs of the remaining types of agarose gels have been strategically placed in the supplement, and this task has been duly accomplished.
Comments 4: The discussion effectively interprets the results but could be strengthened by a more critical analysis of the study's limitations and suggestions for future research. The discussion of survival and regrowth rates could be strengthened by comparing them with previous studies on other plant species. Please consider discussing the practical implications of the protocol for large-scale genetic conservation programs.
Response 4: We extend our gratitude to the reviewer for the insightful comment. We agree with this comment. Based on your comment, we have revised the sentence and marked it in red. Mention exactly where in the revised manuscript this change can be found in L587-592, L622-631.
Author Response File: Author Response.pdf
Reviewer 3 Report
Comments and Suggestions for AuthorsThe manuscript titled "Droplet Vitrification for Cryopreservation of Zingiber officinale Shoot-Tips" presents long-term cryopreservation studies conducted on four different genotypes. The introduction, materials and methods, and trial plan are well-structured and appropriately written. The study is interesting, and valuable results have been obtained. However, there are important issues in some sections of the manuscript that need to be addressed. The authors should consider these points and make the necessary major revisions to improve the manuscript.
- In the abstract, the four Zingiber officinale types are referred to as "varieties," while in the materials and methods section, they are described as "genotypes." To maintain consistency, the authors should use one term throughout the manuscript.
- The manuscript states that four different genotypes were used as plant materials and that cryopreservation, histological analysis, and ISSR analyses were performed. However, when examining the results and discussion sections, it appears that all experiments and analyses were conducted only on Z. officinale ‘Yunnan Xiaohuangjiang’. There are no cryopreservation results, preliminary applications (e.g., ascorbic acid, sucrose concentrations), or ISSR analyses presented for the other three genotypes. Although the manuscript mentions four genotypes, no statistical results are provided for the others. This suggests that the study may have been conducted on a single genotype rather than all four.
- The authors should clarify whether cryopreservation applications were performed on all four genotypes or only on Z. officinale ‘Yunnan Xiaohuangjiang’. If the study includes all four genotypes, statistical analyses comparing their responses should be included. If only one genotype was studied, the manuscript should be revised accordingly to avoid misleading references to multiple genotypes. If analyses on all four genotypes are added, the study could provide valuable insights into genotype-specific effects, which would enhance its originality and scientific contribution.
Author Response
Comments 1: In the abstract, the four Zingiber officinale types are referred to as "varieties," while in the materials and methods section, they are described as "genotypes." To maintain consistency, the authors should use one term throughout the manuscript.
Response 1: We sincerely thank the reviewer for pointing this out. Your second comment was that the study may have been conducted on a single genotype rather than all four. We agree with this comment. So ‘varieties’ has been deleted.
Comments 2: The manuscript states that four different genotypes were used as plant materials and that cryopreservation, histological analysis, and ISSR analyses were performed. However, when examining the results and discussion sections, it appears that all experiments and analyses were conducted only on Z. officinale ‘Yunnan Xiaohuangjiang’. There are no cryopreservation results, preliminary applications (e.g., ascorbic acid, sucrose concentrations), or ISSR analyses presented for the other three genotypes. Although the manuscript mentions four genotypes, no statistical results are provided for the others. This suggests that the study may have been conducted on a single genotype rather than all four.
Response 2: We extend our gratitude to the reviewer for the insightful comment. We agree with this comment. Based on your comment, we kept one genotype Z. officinale ‘Yunnan Xiaohuangjiang’. we have revised the relevant parts of the manuscript.
Comments 3: The authors should clarify whether cryopreservation applications were performed on all four genotypes or only on Z. officinale ‘Yunnan Xiaohuangjiang’. If the study includes all four genotypes, statistical analyses comparing their responses should be included. If only one genotype was studied, the manuscript should be revised accordingly to avoid misleading references to multiple genotypes. If analyses on all four genotypes are added, the study could provide valuable insights into genotype-specific effects, which would enhance its originality and scientific contribution.
Response 3: Special thanks to you for pointing this out. We agree with this comment. Based on your comment, we kept one genotype to avoid the misleading information. We have made revisions to the relevant parts of the manuscript.
Author Response File: Author Response.pdf
Reviewer 4 Report
Comments and Suggestions for AuthorsTitle
Drop vitrification for cryopreservation of shoot tips of Zingiber officinale 2
Add common name
Abstract
In general, the abstract lacks justification and is more like a conclusion. Because it does not explain which treatments were evaluated (materials and methods), the justification of the study is not clear. No perspective of the results obtained is mentioned.
Keywords
Do not repeat the words stated in the title
Introduction
Lines 40-41 too general an idea.
Lines 48-52 ideas should be supported with citations.
Lines 65-67 perhaps the cryopreservation methods developed in this species were already efficient, justify why do drop vitrification in ginger?
Doing a study for the first time in a certain species does not justify its publication.
The introduction does not justify why this cryopreservation technique was used in this species, and it does not mention the advantages of all the techniques used in this study (use of AsA and glutathione (GSH), ISSR, flow cytometry, etc.).
Materials and methods
Line 70 Why only the genotypes from China?
Line 75 “exceptional”? Avoid subjective words.
Lines 75-76 explain in the introduction why these genotypes were used.
Line 76-77 Why only this variety?
Line 86-87 Why 5 weeks?
Line 154 Why ISSR? There are new and more efficient molecular markers.
Line 183 How many apices were used for each experiment and treatment?
Materials and methods are scarce, routine, do not show the evaluation of different treatments in some sections or there is no mention of how the variables are evaluated.
Results
The results are meritorious and provide valuable information, however, most of them are only from one cultivar of ginger and in materials and methods more cultivars are mentioned (only table 4).
Discussion
Line 403-404 it seems to me that the same thing happened in this study, only one genotype was used.
The discussion begins on line 410.
Lines 429-441 do not discuss results, they are only mentioned in list order.
In general, the discussion needs to be improved, only previous studies where there is equality of experimental conditions can be discussed. The results obtained with similar works need to be discussed in depth.
Conclusion
The conclusion is well elaborated, a perspective of the observed results (utilization) is missing.
Author Response
Comments 1: Title Drop vitrification for cryopreservation of shoot tips of Zingiber officinale Add common name
Response 1: We sincerely thank the reviewer for pointing this out. We agree with this comment. Based on your comment, we have added the common name and marked it in red. Mention exactly where in the revised manuscript this change can be found in L2.
Comments 2: In general, the abstract lacks justification and is more like a conclusion. Because it does not explain which treatments were evaluated (Materials and methods), the justification of the study is not clear. No perspective of the results obtained is mentioned.
Response 2: We extend our gratitude to the reviewer for the insightful comment. We agree with it. Based on your comment, we have rewritten the abstract and made them in red. Mention exactly where in the revised manuscript this change can be found in L13-38.
Comments 3: Keywords Do not repeat the words stated in the title
Response 3: Special thanks to you for pointing this out. We agree with this comment. Based on your comment, we have deleted ‘Zingiber officinale’, ‘shoot tips’ and ‘droplet-vitrification’. Mention exactly where in the revised manuscript this change can be found in L39-40.
Comments 4: Introduction Lines 40-41 too general an idea.
Response 4: We appreciate the reviewer's feedback regarding the deficiencies in our writing. We agree with this comment. Based on your comment, we have revised the sentence and marked it in red. Mention exactly where in the revised manuscript this change can be found in L45-49.
Comments 5: Lines 48-52 ideas should be supported with citations.
Response 5: Special thanks to you for pointing this out. Lines 48-52 ideas were cited with several references in the manuscript. However, the sentence ‘At extremely low temperatures, the metabolic and biochemical reactions within the cells stop’ lacked a reference. We agree with this comment. Based on your comment, we have added a reference and made it in red. Mention exactly where in the revised manuscript this change can be found in L58.
Comments 6: Lines 65-67 perhaps the cryopreservation methods developed in this species were already efficient, justify why do drop vitrification in ginger?
Response 6: We thank the reviewer for highlighting the missing information in our manuscript. We agree with this comment. Based on your comment, we have added the references and marked them in red. Mention exactly where in the revised manuscript this change can be found in L65-69.
Comments 7: The introduction does not justify why this cryopreservation technique was used in this species, and it does not mention the advantages of all the techniques used in this study (use of AsA and glutathione (GSH), ISSR, flow cytometry, etc.).
Response 7: We sincerely thank the reviewer for pointing this out. We agree with this comment. Based on your comment, we have added some sentences to explain that why this cryopreservation technique was used in this species. Mention exactly where in the revised manuscript this change can be found in L65-69. The advantages of all the techniques used in this study (use of AsA and glutathione (GSH), ISSR, flow cytometry, etc.) were described in discussion. Some studies also adopt this writing style. Such as: Wang, L.Y., Li, Y.D., Sun, H.Y., Liu, H.G., Tang, X.D., Wang, Q.C., Zhang, Z.D. An efficient droplet-vitrification cryopreservation for valuable blueberry germplasm. Sci. Hortic. 2017, 219,60-69.
Comments 8: Materials and methods Line 70 Why only the genotypes from China?
Response 8: Thank you for the reviewer's question. This study was a project funded by the Chinese government, which focused on the cryopreservation study of several genotypes of Chinese ginger. Therefore, we selected several representative genotypes in China. Reviewer 3 pointed out that the manuscript states that four different genotypes were used as plant Materials and that cryopreservation, histological analysis, and ISSR analyses were performed. However, when examining the results and discussion sections, it appears that all experiments and analyses were conducted only on Z. officinale ‘Yunnan Xiaohuangjiang’. There are no cryopreservation results, preliminary applications (e.g., ascorbic acid, sucrose concentrations), or ISSR analyses presented for the other three genotypes. Although the manuscript mentions four genotypes, no statistical results are provided for the others. This suggests that the study may have been conducted on a single genotype rather than all four. Based on the reviewer 3’s comment, we kept one genotype and have revised the relevant parts of the manuscript.
Comments 9: Line 75 “exceptional”? Avoid subjective words.
Response 9: We sincerely thank the reviewer for pointing this out. We agree with this comment. Based on your comment, we have selected the word. Mention exactly where in the revised manuscript this change can be found in L98.
Comments 10: Lines 75-76 explain in the introduction why these genotypes were used.
Response 10: We sincerely thank the reviewer for pointing this out. We agree with this comment. Based on your comments, we have described the importance of the genotype in Materials and methods. Mention exactly where in the revised manuscript this change can be found in L98-102. In some references on cryopreservation, the rationale for genotype selection is typically placed in the "Materials and Methods" section, and we have adopted this manner of presentation. The representative references include:
Wang, R.R., Gao, X.X., Chen, L., Huo, L.Q., Li, M.F., Wang, Q.C. Shoot recovery and genetic integrity of Chrysanthemum morifolium shoot tips following cryopreservation by droplet-vitrification. Sci. Hortic. 2014,176, 330-339.
Bi, W.L., Hao, X.Y. Cui, Z.H., Volk, Z.H., Wang, Q.C. Droplet-vitrification cryopreservation of in vitro-grown shoot tips of grapevine (Vitis spp.). In vitro Cell. Dev.-PL. 2018, 54, 590-599.
Comments 11: Line 76-77 Why only this variety?
Response 11: Thank you for the reviewer's question. In some references on cryopreservation, one genotype was used to optimize key parameters affecting shoot tip cryopreservation. Additional genotypes were subsequently used for testing the procedure. So we selected ‘Yunnan Xiaohuangjiang’ to optimize key parameters. Based on the reviewer 3’s comment, we kept one genotype and have revised the relevant parts of the manuscript. The representative references include:
Wang, R.R., Gao, X.X., Chen, L., Huo, L.Q., Li, M.F., Wang, Q.C. Shoot recovery and genetic integrity of Chrysanthemum morifolium shoot tips following cryopreservation by droplet-vitrification. Sci. Hortic. 2014,176, 330-339.
Bi, W.L., Hao, X.Y. Cui, Z.H., Volk, Z.H., Wang, Q.C. Droplet-vitrification cryopreservation of in vitro-grown shoot tips of grapevine (Vitis spp.). In vitro Cell. Dev.-PL. 2018, 54, 590-599.
Zhang, J.M., Han, L., Lu, X.X., Volk, G.M., Xin, X., Yin, G.K., He, J.J., Wang, L., Chen, X.L. Cryopreservation of Jerusalem artichoke cultivars using an improved droplet-vitrification method. Plant Cell Tiss. Org. 2017,128, 577-587.
Comments 12: Line 86-87 Why 5 weeks?
Response 12: Thank you for the reviewer's question. Shoot induction from nodal segments of five-week-old stock cultures was according to Wang et al. (2021).
Wang, R.R, Liu, C.C., Wang, L.J., Yuan, M.Y., Li, J., Tang, Z.K. An improved micropropagation via nodal segments of Zingiber officinale. Eur. J Hortic. Sci. 2021, 86, 21-28.
Comments 13: Line 154 Why ISSR? There are new and more efficient molecular markers.
Response 13: Thank you for the reviewer's question. In recent reports on cryopreservation, the ISSR technique was still used to identify genetic stability. Therefore, we employed the ISSR technique in the present study and combined it with FCM to ensure the accuracy of the results. Some reports include:
Ma, X.Y, Blystad, D.R., Wang, Q.C., Tong, L., Stensbl, Y., Zhang, D., Hamborg, Z. Establishment of an efficient and wide-spectrum droplet-vitrification cryopreservation for raspberry (Rubus idaeus L.). germplasm and assessment of genetic integrity and vegetative growth in the regenerants. Plant Cell Tiss. Org. 2024,159, 58.
Malhotra, E.V., Mali, S.C., Sharma, S., Bansal, S. A droplet vitrification cryopreservation protocol for conservation of hops (Humulus lupulus) genetic resources. Cryobiology 2024,115, 104887.
Comments 14: Line 183 How many apices were used for each experiment and treatment?
Response 14: Thank you for the reviewer's question. For cryopreservation experiments, each treatment included three replicates, with a minimum of ten samples per replicate, and all experiments were performed twice. We described in L254-255. For histological and ultrastructural observation, each treatment include two replicates, twenty samples were utilized. We described in L258. For ISSR and FCM, thirty plantlets regenerated from cryopreserved shoot tips, as well as thirty plantlets from in vitro stock cultures, were randomly picked from a collective population consisting of three hundred cryo-derived plants and three hundred in vitro culture-derived plants. We described in L260-262.
Comments 15: Results The results are meritorious and provide valuable information, however, most of them are only from one cultivar of ginger and in Materials and methods more cultivars are mentioned (only table 4).
Response 15: We want to express our gratitude to the reviewer for the insightful comments. We agree with this comment. Based on the both your and reviewer 3’s comment, we kept one genotype and have revised the relevant parts of the manuscript.
Comments 16: Discussion Line 403-404 it seems to me that the same thing happened in this study, only one genotype was used.
Response 16: Thanks again for the reviewer's comments. We agree with this comment. Based on the both your and reviewer 3’s comment, we kept one genotype and have revised the relevant parts of the manuscript.
Comments 17: Lines 429-441 do not discuss results, they are only mentioned in list order.
Response 17: Thanks again for the reviewer's comments. We presented the concentrations and durations of sucrose preculture in these references to demonstrate the sentence ‘the concentration of sugars or sugar alcohols and the preculture duration must be optimized, which can be genotype-dependent’. Based on your comment, we have added some descriptions to make the discussion more in-depth and made them in red. Mention exactly where in the revised manuscript this change can be found in L520-526.
Comments 18: Conclusion The conclusion is well elaborated, a perspective of the observed results (utilization) is missing.
Response 18: We thank the reviewer for highlighting critical information initially absent from the manuscript. Since the conclusion in many reports is relatively brief, we have specifically written a paragraph on the prospects and placed it in the discussion section. We hope this approach is appropriate. Mention exactly where in the revised manuscript this change can be found in L622-631.
Author Response File: Author Response.pdf
Round 2
Reviewer 1 Report
Comments and Suggestions for AuthorsThe authors have done a great job in revising the document. I just add some minor comments below that need clarification.
L 80: droplet-vitrification
L 109-110: Add how long nodal segments were cultured on axillary induction medium before isolating shoot tips?
On line 116 you added "shoot tips were excised from the five-week-old induced shoots." - so it is a bit confusing because on lines 109-100 you say that nodal segments were cultured to promote axillary growth and then shoot tips were isolated. Please review this information and clarify how many days the nodal segments were cultured on axillary induction media prior to shoot tip isolation.
L 130: filled with LN
L 137-144: Instead of repeating "Before autoclaving at 121℃ for 20 min, the pH of the medium was adjusted to 5.8" for each recovery medium, you can say just once that the pH of all recovery media was adjusted to 5.8 before autoclaving at 121℃ for 20 min.
L 362: 3-4 LPs, excised for nodal segments grown for xxxx days (add for how long?), used for cryopreservation
Author Response
Dear Reviewer #2,
On behalf of my co-authors, we thank you again for giving us your positive and constructive comments and suggestions on our manuscript.
We have studied your comments carefully and have made revision by "making the changes in red" in the paper. We have tried our best to revise our manuscript according to the comments. Attached please find the revised version, which we would like to submit for your kind consideration.
We would like to express our great appreciation to you for comments on our paper. Looking forward to hearing from you.
Best regards.
Responds to the reviewer’s comments:
Comments 1: L80: droplet-vitrification
Response 1: We want to express our gratitude to the reviewer for identifying the errors in our manuscript. We agree with this comment. Based on your comment, we have corrected and made them in red. Mention exactly where in the revised manuscript this change can be found in L80.
Comments 2: L109-110: Add how long nodal segments were cultured on axillary induction medium before isolating shoot tips?
Response 2: We appreciate the reviewer for the comment. We agree with it. Based on your comment, we have added the information and made them in red. Mention exactly where in the revised manuscript this change can be found in L109.
Comments 3: On line 116 you added "shoot tips were excised from the five-week-old induced shoots." - so it is a bit confusing because on lines 109-100 you say that nodal segments were cultured to promote axillary growth and then shoot tips were isolated. Please review this information and clarify how many days the nodal segments were cultured on axillary induction media prior to shoot tip isolation.
Response 3: Special thanks to you for pointing this out. We agree with this comment. Based on your comment, we have added the information and marked it in red. Mention exactly where in the revised manuscript this change can be found in L116.
Comments 4: L 130: filled with LN.
Response 4: Thanks again for the reviewer's comments. We agree with this comment. Based on your comment, we have corrected and marked it in red. Mention exactly where in the revised manuscript this change can be found in L130.
Comments 5: L 137-144: Instead of repeating "Before autoclaving at 121℃ for 20 min, the pH of the medium was adjusted to 5.8" for each recovery medium, you can say just once that the pH of all recovery media was adjusted to 5.8 before autoclaving at 121℃ for 20 min.
Response 5: We want to express our gratitude to the reviewer for the insightful comments. We agree with this comment. Based on your comment, we have corrected and marked it in red. Mention exactly where in the revised manuscript this change can be found in L137-142.
Comments 6: L 362: 3-4 LPs, excised for nodal segments grown for xxxx days (add for how long?), used for cryopreservation.
Response 6: We thank the reviewer for highlighting the missing information in our manuscript. We agree with this comment. Based on your comment, we have added the information and marked it in red. Mention exactly where in the revised manuscript this change can be found in L360.
