Multiple Transcriptomic Networks Regulate the Callus Development Process in Panax ginseng

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The manuscript “Multiple Transcriptomic Networks Regulate the Callus Development Process in Panax ginseng” offers relevant insights into transcriptomic changes during callus induction and growth, based on a well-structured combination of time-course and gamma irradiation treatments. While the study employs appropriate methods and presents the data clearly, several aspects require clarification.

(1) In section 4.1, the heading states that ARR12 and RR1 networks are “positively correlated with callus induction,” whereas the data and conclusions suggest that their roles are inhibitory. This discrepancy should be clarified—perhaps by explaining that although these modules are active during the induction phase, they function as repressors.

(2) The number of biological replicates used in the gamma irradiation experiment is not specified, which affects the transparency of the experimental design.

(3) The description of network analysis tools such as WGCNA lacks detail and should be expanded to include key parameters and citations.

(4) Figures, especially Figures 7 to 9, would benefit from clearer labeling and consistent module color coding across panels. The discussion could also acknowledge the need for functional validation of key regulators.

Author Response

Please see the attachment.

Author Response File: Author Response.docx

Reviewer 2 Report

Comments and Suggestions for Authors

Dear Authors,

This work represents the first attempt at transcriptomic analysis of ginseng callus tissues and the identification of genes associated with the regulation of its developmental processes. Overall, the logic and structure of the paper support the authors' main conclusions and findings. However, this paper requires major revision before publication in Agronomy. My comments are provided below.

Abstract
The abstract is written to the point and reflects the main idea of the study. However, in it and throughout the text, do not forget to mark the Latin names of species (Panax ginseng and others in italics). Please check this throughout the text. Also, I do not understand the meaning of the expression "Intensive network analysis". It would be better to reformulate it into something more precise and scientific. Regarding terminology, there is also a question: What exactly do you mean by "regulatory network" - are they in fact gene regulatory networks, or is it something else?

Keywords. I advise you to expand the list of keywords and not duplicate them with the title of the article. For example, "RNA-seq, plant development, gamma-ray, callus, gene regulatory network", etc.

The introduction is currently very short and requires expansion. In particular, the topic of the influence of gamma radiation on callus formation and approaches to cell induction and callus cultivation for P. ginseng are not covered. Also, the topic of ginseng development transcriptomes is not covered. All this will help readers to better understand the goal-setting and novelty of your research. The last paragraph also needs to be expanded and clarified, describing more precisely the main idea and methodology in your work.
Line 35-36 "but the cell biomass was inversely proportional to the cell biomass" - please correct this statement

Materials and methods need significant improvements before publication. 
2.1. Line 65-66: "Prior to the experiment, seeds were dehisced and cold treated according to previously described methods." - It is not clear why the seeds were exposed to cold. Can you explain this in the text? Also, please explain shortly the conditions of cold treatment used.
The same remark about the use of gamma radiation. Why was it used? If I understand correctly, it is reducing plant growth and development processes (doi.org/10.1038/s41598-022-14949-6). 
What are the principles used to select somatic embryos (size, stage)?
2.2. Line 80: "samples were collected according to the irradiation dose at 60 days" - which dose was used here?
How many RNA libraries were sequenced in total? How many treatment and control groups were entered? Specify the length of the reads (it is probably 150 and not 151).
Sections 2.3 and 2.4 are called the same "DEG identification and functional annotation". Either clarify the names or merge them into one section.
2.3. "T2T level genome was applied for the reference sequences" - The abbreviation T2T is not clear here, and it would be better to provide a link to the reference genome that you used along with a link to the article. 
Also, you used a rather strict threshold for identifying DEGs (FC > 4). I advise you to make a separate supplementary material with a full table of DEGs, including a softer threshold (for example, log2(FC) > 0.5).
2.4. Line "GeneMania from cytoscape" - do you mean Cytoscape plugin of GeneMania? 
I have a question about the methodology on this point. Why are you using co-expression data from the Arabidopsis database (lines 115-116) when you already have more accurate co-expression data calculated using the WGCNA approach? What parameters/functions were used when working with GENIE3?

The results section requires significant revisions. 
3.1. Am I correct in understanding that differentially expressed genes were identified only for the 15-day transcriptomes? This section needs some clarification:
1. How many DEGs were identified in each group?
2. Full DEG tables should be provided in the supplementary materials.
3. The legends in Figure 2 are unclear - do graphs B and C show processes only for 15 days in comparison with the other groups or not?
4. Figures 1-3 need to be improved in resolution before publication, because many legends are currently presented in poor and insufficient quality. This is especially true for Figure 3.
3.2. Section needs serious revision. I don't like the way you presented the gene network. In the form of separate patches, I suggest making it a single visualization in Cytoscape, marking different types of connections in different colors (for example, GeneMania associations in red, Genie associations in black, etc. Also, on this network you can mark how each gene is upregulated/downregulated on average. For example, in the 15-day group, think about it. The second point concerns the text. I advise you to combine the results section with the discussion and discuss here the functions of the identified genes in the gene network according to the literature. This will greatly improve the reader's experience.
3.3. In Figure 5 C, one of your control points (g0Gy) is significantly out of alignment with the principal components (bottom right). Did you take its clustering into account in any way? Did you remove it as an outlier?
Figure 6 is presented in low resolution, and, again, it is not clear for which groups of genes GO, GSEA analysis was performed. Part of the letter D is hidden behind the figure, please fix it.
3.4. All my comments on section 3.2 apply to this section as well. It needs to be improved significantly.
3.5. The objects in Figure 8 are very small, the identifiers on the gene networks are not informative and overlap each other, and the hitmaps are impossible to read. I advise you to enlarge this figure or split it into two that will be readable. Please revise it.
Figure 9 overall improves the reading experience, thanks to including it. Again, I suggest merging the results with the discussion so that readers can have a clear reading experience of your manuscript.

The discussion section provides some interpretation of the results obtained, and I encourage authors to check whether all relevant genes/pathways are covered in this section and combine it with the results.
The conclusion section is written briefly and to the point.
In the supplementary materials, the statement that you provide gene networks, however, they were not loaded in the first revision of the manuscript. Please be more careful with loading supplements into the system in the future.

Author Response

Please see the attachment.

Author Response File: Author Response.docx

Round 2

Reviewer 2 Report

Comments and Suggestions for Authors

Dear Authors,

The new version of the manuscript has been significantly improved. I recommend it for publication in Agronomy.