Review History


All reviews of published articles are made public. This includes manuscript files, peer review comments, author rebuttals and revised materials. Note: This was optional for articles submitted before 13 February 2023.

Peer reviewers are encouraged (but not required) to provide their names to the authors when submitting their peer review. If they agree to provide their name, then their personal profile page will reflect a public acknowledgment that they performed a review (even if the article is rejected). If the article is accepted, then reviewers who provided their name will be associated with the article itself.

View examples of open peer review.

Summary

  • The initial submission of this article was received on March 31st, 2020 and was peer-reviewed by 2 reviewers and the Academic Editor.
  • The Academic Editor made their initial decision on April 25th, 2020.
  • The first revision was submitted on July 17th, 2020 and was reviewed by 2 reviewers and the Academic Editor.
  • A further revision was submitted on October 7th, 2020 and was reviewed by 4 reviewers and the Academic Editor.
  • A further revision was submitted on February 23rd, 2021 and was reviewed by 1 reviewer and the Academic Editor.
  • A further revision was submitted on July 29th, 2021 and was reviewed by the Academic Editor.
  • The article was Accepted by the Academic Editor on August 19th, 2021.

Version 0.5 (accepted)

· Aug 19, 2021 · Academic Editor

Accept

Thank you for addressing the suggested editing. The manuscript in its current form reads much smoother; however, I did note a few more areas to be addressed. The sequences are still a bit troublesome to extract from the website in a ready fashion; however, information to get to them does seem available. Regardless, I believe the manuscript is now in a form to move forward after the long run of edits. Please consider this as my approval for the manuscript to move forward for acceptance. Congratulations. The suggested edits are listed below:

Example of annotation:
LINE NO.: / PREVIOUS FORM / SUGGESTED FORM / [ADDITIONAL NOTES, NONE [.]]
LINE 26: / differentially / differential / [.]
LINE 65: / is worthwhile (Pazuki / is a worthwhile investigation (Pazuki / [.]
LINE 96: / chips in / chip technology in / [.]
LINE 113: / and haploid induction / and the haploid induction / [.]
LINE 138: / and them rinsed / and then rinsed / [.]
LINE 174: / USA) digestion was then / USA); digestion was then / [add punctuation.]
LINE 189: / using by / by using / [.]
LINE 191: / to use that to base / to use this for base / [.]
LINE 209: / Over represented / Over-represented / [.]
LINE 278: / reveal significantly genes / reveal significantly expressed genes / [.]

[# PeerJ Staff Note - this decision was reviewed and approved by Julin Maloof, a PeerJ Section Editor covering this Section #]

Version 0.4

· Apr 1, 2021 · Academic Editor

Major Revisions

The manuscript has recently been transferred to my desk. I may need to have some additional reviews submitted, but before that I would advise some of the following modifications be done before review. In general, at this point, the manuscript reads well; however, critical data needed to support your claims is not available. For instance, the reference genome when searched at: http://cmb.bnu.edu.cn/Cucumis_ sativus_V20 was not available. Since your raw data was uploaded to NCBI you might elect to also have NCBI host an assembly for the genome. Otherwise, supplemental sequence data will need to be provided to provide the readership the ability to download gene sequences individually for validation. A connection would need to be made between classified gene IDs, their annotations, and their sequence data; it can be provided as supplement or coordinates provided within an available reference genome sequence. There is binning of important DEGs based on GO: classification, but there are no sample representative available. Supplemental files of important groups, like the seven groups represented in Fig. 8, would allow comparison of diversity of genes within similar bins (and this would also be a good discussion point). The justifications for the need to improve ovarian culture is there, but the ability for readers to validate findings is not. The manuscript is still considered as requiring major revision. Below are some edits to consider:

Example of annotation:
LINE NO.: / PREVIOUS FORM / SUGGESTED FORM / [ADDITIONAL NOTES, NONE [.]]
LINE 52: / Eevaluating / Evaluation / [.]
LINE 60: / In vitro / *In vitro* / [ *italics*]
LINE 66: / In vitro / *In vitro* / [.]
LINE 72: / in vitro / *in vitro* / [.]
LINE 73: / in vitro / *in vitro* / [.]
LINE 81: / in vitro / *in vitro* / [.]
LINE 96: / in vitro / *in vitro* / [.]
LINE 97: / in vitro / *in vitro* / [.]
LINE 98: / in vitro / *in vitro* / [.]
LINE 105: / collecting of extensive / collecting extensive / [.]
LINE 115: / in vitro / *in vitro* / [.]
LINE 116: / regeneration was / regeneration / [.]
LINE 117: / divided into / into / [.]
LINE 118: / Via / *Via* / [.]
LINE 138: / sterilized the / sterilized with / [.]
LINE 195: / analyzed of the / analyzed the / [.]
LINE 195: / raw were data / raw data was / [.]
LINE 196: / Using / Using the / [.]
LINE 217: / USA). according / USA) according / [.]
LINE 218: / primer 5.0 / Primer 5.0 / [.]
LINE 229: / in vitro / *in vitro* / [.]
LINE 234: / were direct dead / were presumed dead / [?detected as?; ?found to be?]
LINE 246: / Enlarged of microspores / Enlarged microspores / [.]
LINE 268: / points used / points and used / [.]
LINE 272: / Bam alignment / BAM alignment / [ *.bam ]
LINE 274: / pre Million / per Million / [.]
LINE 281: / upregulated genes / up-regulated genes / [.]
LINE 281: / . / . / [use ";" to de-lineate values with "," in them; otherwise difficult to determine.]
LINE 282: / downregulated genes / down-regulated genes / [.]
LINE 282: / . / . / [use ";" to de-lineate values with "," in them; otherwise difficult to determine.]
LINE 295: / upregulation / up-regulation / [.]
LINE 295: / downregulation / down-regulation / [.]
LINE 327: / upregulated after / up-regulated after / [.]
LINE 328: / upregulated DEGs / up-regulated DEGs / [.]
LINE 334: / upregulated genes / up-regulated genes / [.]
LINE 336: / their upregulated / their up-regulated / [.]
LINE 338: / were upregulated / were up-regulated / [.]
LINE 339: / downregulated in / down-regulated in / [.]
LINE 348: / were upregulated / were up-regulated / [.]
LINE 349: / upregulated under / up-regulated under / [.]
LINE 353: / upregulated after / up-regulated after / [.]
LINE 356: / upregulated in / up-regulated in / [.]
LINE 359: / upregulated. / up-regulated. / [.]
LINE 359: / upregulated in / up-regulated in / [.]
LINE 365: / upregulated expression / up-regulated expression / [.]
LINE 374: / . / . / [use ";" to de-lineate values with "," in them; otherwise difficult to determine.]
LINE 431: / upregulated belonged / up-regulated belonged / [.]
LINE 432: / upregulation ratio / up-regulation ratio / [.]
LINE 433: / upregulated gene / up-regulated gene / [.]
LINE 455: / upregulated in / up-regulated in / [.]
LINE 456: / upregulated / up-regulated / [.]
LINE 456: / downregulated genes / down-regulated genes / [.]
LINE 460: / upregulated expression / up-regulated expression / [.]
LINE 469: / in vitro / *in vitro* / [.]
LINE 472: / pretreatment / pre-treatment / [.]
LINE 479: / upregulated genes / up-regulated genes / [.]
LINE 512: / upregulated / up-regulated / [.]
LINE 514: / other many / many other / [.]
LINE 522: / undifferentiated / un-differentiated / [.]
LINE 534: / ovules were sequencing / ovules yielded sequencing / [.]

·

Basic reporting

-

Experimental design

-

Validity of the findings

-

Additional comments

All my comments have been addressed. I have really appreciated the effort to improve the English style and the general structure of the manuscript.

Version 0.3

· Nov 16, 2020 · Academic Editor

Major Revisions

This manuscript has ben reviewed by several people, and while the consensus is that the manuscript has merit, some (reviewers 3 and 5) still say that major revisions are needed, including a major revision on the English. Please address all these concerns in full when submitting a revised version.

[# PeerJ Staff Note: Please ensure that all review comments are addressed in a rebuttal letter and any edits or clarifications mentioned in the letter are also inserted into the revised manuscript where appropriate.  It is a common mistake to address reviewer questions in the rebuttal letter but not in the revised manuscript. If a reviewer raised a question then your readers will probably have the same question so you should ensure that the manuscript can stand alone without the rebuttal letter.  Directions on how to prepare a rebuttal letter can be found at: https://peerj.com/benefits/academic-rebuttal-letters/ #]

[# PeerJ Staff Note: The review process has identified that the English language must be improved. PeerJ can provide language editing services - please contact us at copyediting@peerj.com for pricing (be sure to provide your manuscript number and title) #]

·

Basic reporting

All comments are now adressed.

Experimental design

No comments.

Validity of the findings

No comments.

Additional comments

As mentioned in the previous review, I have no scientific comments in this review. All have been previously addressed. I appreciate the effort made for the English style.

·

Basic reporting

• The style, punctuation and formatting still need to be improved. Indeed, I detected several minor errors, in lines: 164, 177, 213, 238.

• Sentences 97-101 would benefit to be rephrased

• Discussion is quite repetitive, and since it is based only on discussion of GO analysis and KEGG analyses I think it could be more concise. I suggest shortening without removing the articles suggested by previous reviewers which have instead helped reviewers helped in improving the discussion section.

• Conclusions section is too repetitive and they should not contain references to figures.

Experimental design

•This manuscript fits the aims and scope of PeerJ.
•The research question is well defined: obtaining a transcriptome landscape of early embryo development in cucumber ovary culture is of high interest for plant developmental biologist and for agricultural research.
•Some concerns about experimental procedures raised by previous reviewers still need to be addressed:

RNA quality control check results are not reported, please provide RNA integrity number (RIN) data if available or at least provide the values obtained from nanodrop analysis (260/280 and 260/230 ratios).

qPCR primer efficiency is still not reported.

It is still not clear in qPCR experiments the distinction between technical and biological replicates. Were the three technical replicates of qPCR related to only one biological replicate of the RNA-seq? Were the sample of RNA-seq and qPCR the same?

In qPCR experiments would be useful to show a gene whose expression level remains more or less the same along the different stages. This might be use in the future by other researchers as a possible housekeeping gene in the same developmental context.

Along results section, please repeat the value of fold change that has been considered for the selection of DEGs.

Validity of the findings

•Now that raw data has been provided, they will be of great value for other studies in the field of embryogenesis.
•The results obtained would increase their value if it were easier for the reader to extrapolate the results of the most interesting DEGs from the manuscript and from the figures. Here my suggestions:

Figure 3 is not very informative; I would prefer instead a table with a list of the principal DEGs between stage T0 and T1 which is for sure the more interesting time points of this study.

Results from 325 to 348 do not refer to any figure. I suppose that this part refers to Figure 9. This part should refer to a new figure or table that must be inserted in order between figure 5 and 6 and not at the end of the manuscript. This new figure or table should be, as already suggested by previous reviewer, a sort of matrix graph similar to that of figure 5 compiling the results on transcription factors.

In Fig. 5 change CTK with CKs (typical abbreviation for cytokinins)

Reviewer 4 ·

Basic reporting

In general I think the manuscript meets the basic reporting criteria. Grammar and spelling have been improved in successive revisions. There are however 3 points which I think must be addressed for the manuscript to be accepted: 1. the word papiloma in the methods section sounds incorrect as a previous reviewer pointed out. Do the authors refer to the papilla/papillae which form on the stigma of plant species-this needs to be checked. 2. I tested the link to access the data on the NIH website and it worked, however as a previous reviewer also commented the link is not included in the text of the manuscript or at least I could not find it. 3. the discussion section could be much more concise. The authors cite many other related studies in relation to their own data, I think such detail is unnecessary.

Experimental design

No comment

Validity of the findings

No comment

Additional comments

No comment

·

Basic reporting

The manuscript needs major English language revision.

Experimental design

The material analysed contains a number of different tissues/cells and non-embryogenic samples have not been included, making it difficult to assign gene expression profiles specifically to embryogenic egg cells/ovules

Validity of the findings

There are some small points to be addressed with respect to the experimental set-up, data analysis, presentation of the data and conclusions/discussion.

Additional comments

The manuscript by Deng et al presents a transcriptome analysis of different steps in haploid embryo induction (gynogenesis/parthenogenesis) from cucumber ovules. Although it is a descriptive/archival study i.e. it does not offer any insight into the mechanism behind haploid induction and development, it seems to have been fairly well performed and is still a good resource for researchers in the field. My main issues with the manuscript are the quality of the writing and the lengthy discussion.

After reading the Abstract I was quite intrigued and eager to read more as there is little known about the molecular regulation of gynogenesis systems. The abstract was well written, which seemed promising for the rest of the manuscript. Unfortunately, I was much less enthusiastic after reading the entire manuscript. Although the Abstract is well written, the other sections in the manuscript are not well written at all. The major problems are the phrasing and lack of attention to detail. The phrasing is often so awkward or the conclusions out of context that I even had difficulty understanding the background literature that they supplied for my own field of expertise (androgenesis). The manuscript needs extensive editing by a native speaker with a scientific background.
Examples:
“In Brassica napus, the expression of embryogenesis-related genes such as BBM, LEC1 (LEAFY COTYLEDON1 ) and LEC2 (LEAFY COTYLEDON2 ) were detected after culture
under heat stress at 32 °C for three days; after incubation at 24 °C, embryo-specific expressed genes, such as LEC1 and
LEC2 were detected (Malik et al., 2007).”
why is the distinction 32/24 relevant – do you mean during induction (32) and later, during outgrowth (24) ?
“ The BBM gene was the first key gene to be isolated in the process of spore cell division; and
was first expressed in zygotic embryogenesis and microspore embryogenesis (Pechan et al.,
1991; Boutilier et al., 2002). Khanday et al (2019) showed that BABYBOOM 1 (BBM 1) is a
member of the AP2 family of transcription factors”
“spore division” = microspore embryogenesis/haploid embryogenesis
“first expressed in zygotic embryogenesis and microspore embryogenesis” = doesn’t make sense
“showed that…is AP2 family” They did not show that it is an AP2 domain gene, it was already known. They showed that the gene is paternally-expressed (as did a group working in maize) and that egg cell expression induces parthenogenesis.

Some of the mistakes are the result of a non- critical eye and should be easy to fix. For example (there are many more):
Li et al.(2014), FAA( formalin
biological microscope ( = stereo microscope?),
heart chamber (= heat?),
magnetic beads with oligosaccharide (DT) ( = oligo dT?)
and embryo-like structures with integuments (do embryos have integuments?)
embryo maturation stage a nd shoot (= and)

Below are a number of specific questions/concerns

1. Material that was sequenced

Since the authors did not isolate egg cells for the transcriptome analysis, it is difficult to assign any of the changes that they observe specifically to egg cell parthenogenesis rather than effects in other sporophytic tissues. Likewise, what is the frequency of haploid embryo induction in this line? If a certain proportion of the ovules do not make embryos, then this will also confound interpretation of the transcriptome data.

I think these points should be made clearer throughout the manuscript, including the Discussion. For example, was the T0/T1 material used for sequencing from isolated ovules or from whole ovary slices? How does this affect interpretation of the expression patterns/gene functions?


2. Figure 1

The resolution in the images is very low. I am not sure if the resolution is low in the original figure or due to the pdf conversion process.

Showing the various treatments on the timeline e.g. heat stress would give a better overview of the culture procedure.

It would be helpful to provide more labels
a-f provide more labels for orientation purposes ovary, ovule, embryo. In f there is quite a bit of callus, a shoot and an embryo.
g-k, provide more labels in general. Specifically, in sections outline embryo sac, indicate ovule integuments/ovary, female gametophyte structures e.g. egg cell.
panel g- label ‘g’ extends beyond boundary of the image

What evidence is there that the egg cell enlarges and not one of the other female gametophyte cells?

What do non-responsive (non-embryogenic) egg cells/embryo sacs/ovules look like. A negative control would help convince me that the changes you observe are specific to the cells that undergo embryogenesis.


3. qRT-PCR

The authors indicate that they used the 2ddCt method ((Ct sample-Ct actin reference gene) –(Ct calibrator sample -Ct actin reference gene)). If the 2ddCt method was used, then the expression value for the sample used as a calibrator should be set to 1. I do not see this, so I suspect they used a different calculation.

The sentence “The results showed that the relative expression levels revealed by RNA-seq and qRT-PCR were closely correlated (Pearson's r = 0.53, 0.77, 0.57, 0.74, 0.79, 0.69, 0.81, 0.81, 0.86, 0.62, 0.81). The avlues are in the figure and do not need to be repeated here, where it is not even clear which value belongs to which gene. I also wouldn’t consider r=0.53 “closely correlated”.

Why were technical replicates used instead of biological replicates?

4. Figure 3
legend should be ‘upregulated’ and ‘down regulated’ not “upregulate” and “down regulate”

5. Figure 5
To improve the readability: move the sample legend to the top of the figure and show average expression values, not replicates.


6. Figures 4, 7, 8

Kegg pathway enrichment figures are not very informative– combine into one figure with 3 panels.

There are some pathways that are no applicable to plants e.g. ‘renin-angiotensin’, ‘lipolysis in adipocytes’. Were the pathways mapped to the correct organism?

7. Figure 9

What is meant by ‘specific-expressed’ gene?


8. Discussion:

The Discussion can be reduced by about 2/3. Given the experimental set-up it is quite speculative to discuss all the genes/pathways in detail. Just highlight similarities with other studies and the novel findings. Please also discuss the limitation of this study.


9. I would not use the term maturation. This is reserved for storage product accumulation phase. the term morphogenesis is more in line with the literature.

Version 0.2

· Aug 17, 2020 · Academic Editor

Major Revisions

It is clear that the article needs a major revision, and critically, a link to the raw data for the reviewers, as this is particularly clear from the comments of one of the reviewers. If the link is not present in a new, revised version of the article submitted (the authors decide not to include it), then I am sorry to say that the reviewers cannot do their job properly and, as a consequence, I am afraid that the article will have to be rejected.

[# PeerJ Staff Note: Please ensure that all review comments are addressed in a rebuttal letter and any edits or clarifications mentioned in the letter are also inserted into the revised manuscript where appropriate.  It is a common mistake to address reviewer questions in the rebuttal letter but not in the revised manuscript. If a reviewer raised a question then your readers will probably have the same question so you should ensure that the manuscript can stand alone without the rebuttal letter.  Directions on how to prepare a rebuttal letter can be found at: https://peerj.com/benefits/academic-rebuttal-letters/ #]

·

Basic reporting

The authors addressed all comments from the initial review. I only have uploaded an annotated pdf version of the text for the spelling or text structure.

Experimental design

All comments were addressed. The method part is now fully described.

Validity of the findings

no comment

Additional comments

The authors addressed all concerns raised by the review of the original manuscript. However, there are a series of grammar issues that I have annotated in an enclosed pdf. I do not need to see the text further for scientific issues if English is proofed properly.

·

Basic reporting

The English grammar, style, punctuation and formatting need to be improved. In fact, they seem worse than the previous version.

Literature references are ok.

Article structure ok. Raw data still not shared, as far as I can tell- I could not find the reference for the raw data accession in the manuscript. If it is there, it is difficult to find.
Since this is the second time they have not shared the raw data (I made this comment on the last revision), I would recommend rejecting the manuscript on this basis.

Article is self contained, the addressed my previous comment about an unpublished protocol, since the protocol is now published.

Experimental design

This is all ok, as I mentioned in my last review.

Validity of the findings

No underlying data for RNAseq provided, for the second time. As I mentioned above, I would recommend rejecting the article on this basis.

Additional comments

The response letter to the reviewers' criticisms lacks almost all pertinent detail. There was no point-by-point response to my comments. The only comment they addressed was the need to cite their new method (which they did, it is now a published paper), and a few sentences in the materials and methods. As far as I can tell, the other reviewer's comments were not addressed at all.

I could not find the accession number for the RNAseq data. This is the second submission of this manuscript without a link to the raw data. The most useful aspect of this article would be the availability of the raw data. On this basis, I recommend rejecting the article, because the authors are clearly trying to avoid releasing the raw data.

Version 0.1 (original submission)

· Apr 25, 2020 · Academic Editor

Major Revisions

Please read with care the commentaries of both reviewers of your manuscript, as they raise multiple points that merit attention.

[# PeerJ Staff Note: Please ensure that all review comments are addressed in a rebuttal letter and any edits or clarifications mentioned in the letter are also inserted into the revised manuscript where appropriate.  It is a common mistake to address reviewer questions in the rebuttal letter but not in the revised manuscript. If a reviewer raised a question then your readers will probably have the same question so you should ensure that the manuscript can stand alone without the rebuttal letter.  Directions on how to prepare a rebuttal letter can be found at: https://peerj.com/benefits/academic-rebuttal-letters/ #]

[# PeerJ Staff Note: It is PeerJ policy that additional references suggested during the peer-review process should only be included if the authors are in agreement that they are relevant and useful #]

·

Basic reporting

This manuscript entitled “Analysis of transcriptomic regulation mechanisms underlying cucumber (Cucumis sativus L.) embryogenesis during ovary culture” by Deng and co-authors, reports on the types of genes differentially expressed in key steps of plant regeneration from ovary culture in Cucumber.
The study is a transcriptomic analysis by RNAseq. The text is clear, and mostly in professional language. However, some sentences would benefit to be rephrased, such as in lines 71-74 and lines 99-100.
Conventional writing is sometimes missing as the names of proteins should be in upper cases. For example, this is the case in lines 88-89.
Background information is sufficiently included. Literature is mostly provided, maybe a bit old sometimes. In some cases, references are missing. For example, the relationship between microtubules and zygote development (line 429) could benefit from referring to:
Kimata Y, Higaki T, Kawashima T, et al. Cytoskeleton dynamics control the first asymmetric cell
division in Arabidopsis zygote. Proc Natl Acad Sci U S A. 2016;113(49):14157–14162. doi:10.1073/pnas.1613979113

The involved of WUS in shoot formation has been published in connection to cytokinin signalling (line 475). Some references (not exclusive):
Dai, X, Liu, Z, Qiao, M, Li, J, Li, S, Xiang, F ( 2017) ARR12 promotes de novo shoot regeneration in Arabidopsis thaliana via activation of WUSCHEL expression. J Integr Plant Biol 59: 747– 758
or
Meng, W. et al., 2017. Type-B ARABIDOPSIS RESPONSE REGULATORs Is Critical to the Specification of Shoot Stem Cell Niche by Dual Regulation of WUSCHEL. The Plant cell, 29(6), pp.1357–1372. Available at: http://www.plantcell.org/content/29/6/1357.long

It would be good if the authors may look into this connection in their study.

The article is properly structured with figures and tables of good quality. I could find the raw data of the RNAseq (I was looking for them to look at genes in auxin production). And I would miss a figure (something like a matrix graph as in Figure 5) compiling the results on transcription factors. Those are discussed in the discussion part but not in the results.
Conclusion of the article is overwise supported by the results.

Experimental design

This manuscript fits the Aims and Scope of the journal as original primary research in Biological Sciences.
The research question is well defined: what groups of genes are differentially expressed during the different steps of plant regeneration from ovary culture in cucumber. It aims at helping to improve the efficiency of the process by understanding the molecular mechanisms during each step. The gap in knowledge is here clearly established. However, the explanation in lines 73-74 why microspore-derived embryogenesis may not be a good method: is it true for cucumber?
However, the abstract would appreciate rephrasing. For example, you selected six key time points in three stages. A clarification would be required for the reader what these time points are. Also, the data are presented as a differential expression between successive time points. Then a very little emphasis is made in the abstract about the cytological analysis.
May I ask what is pre-embryo (abstract and introduction)?
This could be connected with my previous commented. Here no methods are provided for these results: what staining used, what microscope, live or fixed material, etc.
In the methods part, there are also missing details about
- amount of starting material for RNA isolation
- Method of RNA isolation (Trizol and then what?)
- RNAseq library preparation,
- Number of replicates for each sample
- RNA QC check,
- qPCR primer efficiency
- RNA for qPCR: from freshly collected samples or from the same RNA?
- qPCR: How many biological replicates? Technical replicates? Was it performed any statistical analysis?
The ovary culture method is presented as unpublished (line 123). If the policy of the journal is that a ready should be able to reproduce the presented data, the in vitro ovary culture method would need to be provided in the method part with sufficient details: renewal of the culture medium (timeframe?), any change in composition during each step (same medium from the ovary at T0 and germinating embryo with a shoot, in term of hormone composition for example?)

Validity of the findings

The validity of the results appears correct. However, I could not access the raw data to look for a specific pathway of interest, as I was missing them from the figures (see below). The results are interesting and will be to improve the culture of the ovary and plant regeneration.
RT-qPCR results are missing a solid statistical analysis, especially that they are compared to the results of RNAseq. The authors claim the RNAseq and qPCR results are consistent (line 239). Figure 2 (as it stands) lacks to demonstrate it for all the presented genes. Could it be an idea to use a double y-axis (one for DE in RNAseq and on for fold-change in qPCR)?
Concerning the results, please try to be accurate in referring to the genes in the text. For example,
- line 264: “cytokinin transfer protein” is, in fact, Histidine phosphotransferase protein;
- line 265: “cytokinin response proteins” are response regulators (RR)
- line 267: “ethylene-binding proteins” are EIN3 binding (F-box) proteins (EBF)
- line 268: “ethylene-responsive protein transcription factors” are ERF
And you mention nothing about auxin production (YUCCA, TAA1, others), auxin signalling (TIR1, ARF, Aux/IAA) or cytokinin production (LOG, CKX), despite the presence of a KEGG Zeatin biosynthesis enrichment.
In addition, transcription factors are a big part of the discussion, not they are not analyzed in the results. An analysis with a matrix graph would be welcome. It may also help to understand the process of plant regeneration.
Finally, in the discussion (around lines 400), I do not agree with the conclusion that “two genes related to hormone regulation that were highly expressed only at the stage of early embryo development (Csa6M147590.1 and Csa3M389850.1)”. From Figure 4, I could find that genes Csa1M600830.1, Csa1M006300.1, Csa3M822100.1, Csa1M0516890.1 and maybe others after analysis of the raw data, rather than the picture in the figure.
The authors do not over-interpret their data. The conclusions are solid and supported.

·

Basic reporting

The English is generally good, though uneven. Ironically, the least clear part of the manuscript is the abstract. In the pdf file I have uploaded, I have made numerous comments about how to make the abstract more clear, and some grammatical corrections.

The raw data is not shared, as far as I can tell. This is totally essential for publication. There is a link in the cover letter that goes nowhere.

The background and literature references are fine.

There isn't a hypothesis. It's a manuscript reporting a transcriptome analysis, with validation of 10 genes by qPCR. The microscopic analysis of induction of embryos from ovule culture is interesting.

Experimental design

The aim of the article is to study transcriptome changes associated with induction of embryo calli from ovules of Cucumber. The authors point out the importance of the technique for double haploid production from ovule culture in cucumber breeding. The point of the article is to determine the transcriptome changes associated with ovule culture that accompany this important technique (double haploid induction). Thus, the research question is meaningful, and the data are worth publishing.

The methods are well described, except for several glaring omissions, which absolutely need to be addressed before publication:

1) the raw data are not available (link in the cover letter does not work)
2) The authors do not state how many biological replicates were used for their RNAseq analysis. From Table S2 I think it is probably three biological reps. If this is so, this needs to be stated clearly in the text.
3) Regarding qPCR replicates: Looking at the excel file with the source data for the qRT-PCR, it looks like they have three technical replicates for each sample, and possibly biological replicates. In the legend for Figure 2, they say they did three 'independent experimental replicates'. What does this mean? They need to refer to technical and biological replicates, so it is clear what they did.
4) On line 155, the refer to a 'highly efficient in vitro ovary culture technology system of cucumber (unpublished)'. This is not acceptable. They need to put a description of this culture technology here, or publish it somewhere else and cite the publication.
5) On line 185, the authors use the abbreviation 'TDZ'. I supposed this is some kind of cytokinin (zeatin). Whatever it is, it must be the key factor for induction of embryogenesis from ovaries, and must be spelled out. Also the supplier of TDZ must be mentioned.
6) The authors should provide more detail in the methods on exactly which part of the tissues were used for the different RNAseq and qPCR samples. I.e. how much of the ovule? Was carpel tissue included also? Were the maturing embryos dissected from the undifferentiated callus tissue, etc.

Validity of the findings

Underlying data have not been provided. Once they are provided, the data will be of value to the field of embryogenesis, in particular for studies of inducing embryogenesis, and apomixis.

Regarding conclusions, many analysis of GO categories and KEGG analyses are provided. But this kind of analysis isn't that informative, even though it's the only kind of analysis that is possible with the data in its current state. I think that it would be good to make the article more concise.

Things that must be changed to be acceptable for publication. These are also listed as comments in the pdf file that I have uploaded.

1) Remove 'mechanism' from the title. There are no mechanisms here. An appropriate title would be something like 'Transcriptome analysis of ovary culture-induced embryogenesis in cucumber (Cucumis sativus L.)'
2) Line 46: state what the timpoints where
3) Line 61: delete 'shoot formation might be regulated by' too speculative
4) Line 180: define 'papilloma'

Additional comments

My only other suggestion would be that it would be interesting to put the authors' findings in the context of other papers focusing on asexual induction of embryos (tissue culture and apomixis). And especially to discuss the paper from the Sundaresan lab on induction of rice embryos using Baby Boom: Khanday I, Skinner D, Yang B, Mercier R, Sundaresan V (2019) A male-expressed rice embryogenic trigger redirected for asexual propagation through seeds. Nature 565(7737):91–95.

All text and materials provided via this peer-review history page are made available under a Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.