Survival and State of Garlic Explants of Two Lithuanian Cultivars after Cryopreservation
Round 1
Reviewer 1 Report (Previous Reviewer 3)
The authors did their best, added photos and mostly corrected what was requested. I think the manuscript is improved enough for publication.
Author Response
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Best regards.
Authors.
Author Response File: 
Author Response.pdf
Reviewer 2 Report (Previous Reviewer 1)
The authors have improved the text. Nonetheless, some points still require correction before publishing.
- The style of the Abstract requires improvement.
- The choice of keywords is incorrect. I suggest: Allium sativum L., PVS3, survival, vitrification
- Change 'amount/number' in the text and Figures to 'share' when referring to percentage values.
- Figure 1 lacks scale bars. The quality of the figures is low.
- The symbol of degrees is incorrect (it should be 'o', not '0').
- In the graphs, 'a' should indicate the highest values, not the lowest.
- The authors mentioned the survival rate of the explants but what about the recovery?
- Please provide information on the producers of crucial chemicals (PGRs, agar, etc.) used in this study (name, city, state, country).
- What was the number of explants used in each treatment? The number of replication and repetitions is unclear.
- How many explants were placed in each cryovial/Petri dish?
- The authors should consider if they are referring to 'variety' or 'cultivar' (this is not the same).
- The Conclusions are chaotic and unprecise. This part should be rewritten.
Author Response
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Best regards.
Authors.
Author Response File: Author Response.pdf
Reviewer 3 Report (New Reviewer)
Dear Authors,
I think the structure of the study is quite inadequate. I think that the study should be reconsidered, from the method used to the results. In this context;
# What is new in the method, the protocol implemented for these two different local varieties mentioned? What is different if the method used for cryopreservation of these varieties was taken from one reference? These points should be emphasized in the study. or if there is an optimization for these types, what are the results.
# How many parameters were tried in the applications? What was the criterion that was positive for the explant in the results? Has acclimatization work been done? If not, why? It is a powerful support study in germplasm conservation studies.
# What is the "surviving" criterion? Explant germination after optimized incubation time? or shoot growth after incubation? This should be clearly stated in the study. In cryopreservation studies, if "surviving" is mentioned, it refers to viability, and we usually determine this by a method such as the tetrazolium (TZ) test after liquid nitrogen thawing. The TZ test alone is not sufficient for a cryopreservation study. Regeneration of explants must also be evaluated. Because the explant may survive after liquid nitrogen, but it may stay for a long time without any regeneration and then die. In this context, regeneration is more important than vitality.
# Figure 1 has a very low resolution. Nothing is clear from this figure used. The results are given in the form of graphs and standard errors do not appear in these graphs. It would have made more sense if it was given as a table instead.
Kind Regards
Comments for author File: Comments.pdf
Author Response
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Best regards,
Authors
Author Response File: Author Response.pdf
Reviewer 4 Report (New Reviewer)
In their work “Survival and state of garlic explants of two Lithuanian cultivars after cryopreservation”, the authors investigated the survival rate and state of explants after cryopreservation of two cultivars of Lithuanian garlic (‘Žiemiai’ and ‘Dangiai’). Their results confirm that the efficiency of garlic cryopreservation depends on the genotype, the type of explant, and the dehydration conditions. The success of cryopreservation depends on many factors, including genotypes, and specific conditions. Although cryopreservation of garlic has been widely studied, a specific protocol for cryopreservation for the cultivars ‘Žiemiai’ and ‘Dangiai’ is not present in the available literature. For this reason, I think this study warrants publication in “Horticulturae”, with minor revisions. My recommendations are:
- Please add error bars in Figures 2, 3, and 4;
- Please include the principal coordinate analysis in section 2.3;
- Please improve the English language quality
Author Response
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Best regards,
Authors
Author Response File: Author Response.pdf
Round 2
Reviewer 2 Report (Previous Reviewer 1)
The authors made all the necessary changes. The manuscript is now suitable for publication.
Reviewer 3 Report (New Reviewer)
Dear authors,
You have performed all suggested corrections. In this context, your article has become suitable for publication in the journal.
Kind regards
This manuscript is a resubmission of an earlier submission. The following is a list of the peer review reports and author responses from that submission.
Round 1
Reviewer 1 Report
The manuscript fits the criteria of communication but some improvements are needed as listed below:
- The Abstractly is badly written and incomplete. The background and methodology of the study is missing.
- Keywords should be arranged alphabetically.
- Line 23: corrected the reference style.
- Moderate grammar and punctuation corrections are needed, e.g. in lines 68, 74, 89, 108, 130-132, 168,
- Please provide information on the producers of key chemicals (name, city, state, and country).
- Line 81: change “frozen” to “stored”.
- “We consider surviving ex-98 plants to be those with 2-4 points damage” – why not 2-5?
- According to the authors, the control was “non-frozen” (its should be non-LN-treated), but was the control treated with PVS3 or not? Please explain clearly in the methodology what was the control.
- the Discussion section could be more profound.
- Line 220 unclear: do you mean the mean share?
- Some recent references on garlic cryopreservation are missing, e.g. https://doi.org/10.5586/asbp.9132
Author Response
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Author Response File: 
Author Response.docx
Reviewer 2 Report
1) In abstract section, the author wrote that “These results confirm that the efficiency of garlic cryopreservation depends on the combination of factors.” Therefore, it is necessary to clear indicate the specific combination factors.
2) In introduction section, it needs to be added more literature studies related to the cryopreservation features of Lithuanian garlic cultivars.
3) The literature review is too general. Moreover, the literature gap is not discussed.
4) In a current form, a novelty of the conducted research is unclear.
5) In present article, it needs to add the main research contributions of the present research compared with the existing research.
6) “The assessment of the state of explants showed that the amount of dark green explants in control (not frozen) experiment reached 92% for cultivar ‘Žiemiai’ and 82% for cultivar ‘Dangiai’. Some of the explants showed the intensive prolongation of the leaf bases and rooting. The average amount of survived explants (state 2-4 points) of both cultivars reached 72,3% after cryopreservation. Cultivar ‘Žiemiai’ showed a higher capability for survival.”
Hence, it is necessary to provide the more reasonable and adequate explanation for the above research findings.
7) “A comparison of Lithuanian cultivars proved that a creamy white cultivar ‘Žiemiai’ showed a higher survival rate after cryopreservation compared with light purple ‘Dangiai’.”
Similarly, it is necessary to provide the more reasonable and adequate explanation for the above research findings.
8) “A comparison of the effect of explant type showed that the survival rate of stem domes sampled from bulbils was significantly higher than that of stem domes sampled from cloves.”
Hence, it is necessary to provide the more reasonable and adequate explanation for the above research findings.
9) “Significant differences in survival rate and the state of cryoprotected explants were observed under three different PVS3 exposure durations.”
Therefore, it is necessary to provide the more reasonable and adequate explanation for the above research findings.
10) The abstract and conclusion sections need to be improved.
11) The results are mainly presented by figures. It is necessary to provide the sufficient explanation of the research results. This is also the weakest aspect of the study.
12) The author needs to discuss all the research results in more detail and give more reasonable and sufficient explanation for all the research findings.
13) Results and discussion should be modified accordingly to the aim of the article.
14) The limitations of the study are not considered.
Author Response
Thank you for the review. Please see an attachment for the answer.
Author Response File: Author Response.docx
Reviewer 3 Report
Manuscript brings interesting information however, the main objection to this work is that it is actually unfinished. You estimate the survival rate, but you don't show the data on how many of these surviving explants are actually regenerated. The research should have continued. The results you show are, in my opinion, rather subjective assessments of the condition (state) of the explants and cannot be independently published as a paper in a journal such as Horticulturae.
The English language must be proofread
The Abstract should be supplemented with basic data on the research methodology
Row 23: I believe you can get the latest FAO data on garlic production
In Materials and methods “not etiolated, medium-green colour” was designated with Point 4,
In Results you say for Point 4 “etiolated dark-green colour explants “ (Row 122)
93: When did you count the survived explants? After how many days of cultivation? Should be specified.
98: “We consider surviving explants to be those with 2-4 points damage.” And what about explants with Point 5? Are they not surviving?
132-133: There is something wrong with this sentence – when –while??
140-141: “The average survival rate of stem domes from bulbils reached up to 79,5% and up to 70% from cloves in the control experiment.” Average for two genotypes or what?
Do results listed under 3.1. and 3.2. come out from data of all dehydration treatments (1.5, 2 and 3 hours). That is completely unclear.
166: Influence of ? dehydration condition…
169-170: “survival rate for tested cultivars of both types of explants reached 79% in the control variant and 65,9% after cryopreservation while PVS3 was used for 1,5 h.” - in which order, for which cultivars?
175: dark green (Point 4) ??
Why didn't you show photos of explants in different states (Point 1-5)?
Somewhere in the results you mentioned that you had contamination, especially with explants obtained from cloves. Why did you not report the percentage of contamination? How could you assess, and did you assess the state of contaminated explants?
Author Response
Thank you for the review. Please see an attachment for the answer.
Author Response File: Author Response.docx
Round 2
Reviewer 2 Report
1) The abstract need to be improved with more quantitative values of results obtained.
2) In present article, it needs to add the differences and main research contributions of the present research compared with the existing research.
3) The conclusion section needs to be improved.
Author Response
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Author Response File: Author Response.pdf
Reviewer 3 Report
Dear authors,
although you have significantly improved parts of the work according to the suggestions, a lot of things remained unclear.
186-187: „We used explants 2 cm in diameter and 3 cm in length“ ??? In materials and methods you say 2 mm in diammeter and 3 mm in length
143-144: „The total amount of survived explants of this cultivar reached 88.5%...“ How? 39+28+9.8 =76.8%
Figure 1: Shouldn't the total percentages of individual categories (state of explants) for each genotype be 100% in total? It's not for you. For example for the cultivar 'Žiemiai' the number of survivors is 39+28+9.8 =76.8% + 13% non-survivors (in Point 1)= 90%. Where did the other explants up to 100% disappear?
Then I realized that in Materials and methods you say that you had a 5 point scaling, and you specify 6 points (from 0-5). For 0 you say they are dead, brown no green. It means that explants scored with 0 and 1 are not alive. And you didn't show those rated with 0 on the graphs at all. That's why the percentages don't match.
118-121: „Explants were characterized by five points rating scale: 0 - dead, brown no green; 1 - etiolated, brown, pale; 2 yellowish brown colour; 3 - etiolated, medium-green colour; 4 - not etiolated, medium green colour; 5 - not etiolated, dark green colour, prolongation of the leaf bases“
Therefore, I still consider these explant evaluations (points) to be very subjective and vague. If you could show us with a photo what kind of explants you rated with 0, 1, 2, 3, 4, and 5, it would be much better. This way, I still stand by the assessment that this research is not complete, despite the fact that you categorize the work as communication.
Author Response
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