A Highly Efficient Protocol for Multiple In Vitro Somatic Shoot Organogenesis from the Hypocotyl- and Cotyledon-Derived Callus Tissues of Russian Cabbage Genotypes

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The manuscript “A Highly Efficient Protocol for Multiple In Vitro Somatic Shoot Organogenesis from the Hypocotyl- and Cotyledon-Derived Callus Tissues of White Cabbage Genotypes” investigates the shoot organogenesis system using two types of explants from three Brassica rapa genotypes. However, this manuscript has significant shortcomings in terms of novelty, depth of data, logical articulation, and presentation format. Specifically, the research content is rather conventional and fails to fully express its outstanding points; The research content is rather thin. It is suggested that experimental data be supplemented. The abstract fails to independently and clearly convey the core value of the paper. The discussion section is too scattered, and the main points of the article are not clearly presented. The elaboration on key issues is not strong enough; The technical flowchart fails to clearly and intuitively highlight the core content and lacks readability; The bar chart in figures 1 and 3 lacks statistical significance test marks; The illustration in figure 2 is not strong and lacks a scale; In the reference list, the format of the journal name is not uniform. Therefore, this manuscript fails to meet the academic standards of this journal. Furthermore, the manuscript has the following problems:

1. Novelty: The study appears rather conventional and fails to clearly articulate its innovative aspects and outstanding contributions compared to existing literature. 
2. Depth of Data: The experimental data presented seem insufficient to robustly support the conclusions. It is recommended to add experimental data.
3. Abstract: The core issues, methods, key findings, and main conclusions of the study are not clearly summarized (For example, lines 16-19 are not clear and can be ambiguous).
4. Technical flow chart: The current flow chart fails to clearly and intuitively outline the core steps and technical key points of the experiment, and is not readable. It is suggested to redesign it.
5. Figure 1 and Figure 3: The bar chart lacks a statistical significance test annotation; please add the necessary statistical analysis annotation.
6. Figure 2: The picture is not illustrative, and the key picture lacks a scale, so readers cannot judge the actual size. Please add the scale.
7. Discussion: This section is out of focus and fails to closely focus on the core findings ( For example, lines 334-395 do not highlight the argument, do not conduct in-depth analysis, and do not have new insights ). Recommend a new discussion.
8. References: The format of journal names in the reference list is not uniform ( For example, lines 436-437 are inconsistent with the format of lines 438-439 ). Please unify the format according to the requirements of journals.

Author Response

Dear Reviewer,

On behalf of ourselves and the co-authors, we thank you for your valuable comments.

The manuscript “A Highly Efficient Protocol for Multiple In Vitro Somatic Shoot Organogenesis from the Hypocotyl- and Cotyledon-Derived Callus Tissues of White Cabbage Genotypes” investigates the shoot organogenesis system using two types of explants from three Brassica rapa genotypes. However, this manuscript has significant shortcomings in terms of novelty, depth of data, logical articulation, and presentation format. Specifically, the research content is rather conventional and fails to fully express its outstanding points; The research content is rather thin. It is suggested that experimental data be supplemented. The abstract fails to independently and clearly convey the core value of the paper. The discussion section is too scattered, and the main points of the article are not clearly presented. The elaboration on key issues is not strong enough; The technical flowchart fails to clearly and intuitively highlight the core content and lacks readability; The bar chart in figures 1 and 3 lacks statistical significance test marks; The illustration in figure 2 is not strong and lacks a scale; In the reference list, the format of the journal name is not uniform. Therefore, this manuscript fails to meet the academic standards of this journal. Furthermore, the manuscript has the following problems

Response. Our investigation is devoted to the development of an effective protocol for indirect somatic shoot organogenesis using the model of three Russian white cabbage genotypes (Brassica oleracea convar. capitata (L.) Alef.), but not three Brassica rapa genotypes. We have corrected the manuscript title for better underlying. We have clarified that this protocol was developed based on Russian white cabbage genotypes.

Responses to subsequent comments are provided below.

Remark 1: The study appears rather conventional and fails to clearly articulate its innovative aspects and outstanding contributions compared to existing literature.

Response 1: We revised the resubmitted manuscript and tried to justify the novelty of our study. As the reviewer rightly noted, our study is traditional for similar studies. However, the literature does not contain many experimental data on induced morphogenesis of white cabbage that are universal for different genotypes and explant types. At the same time, Russian genotypes of white cabbage are not represented at all. Thus, we have corrected the title of the article (L. 1-4) and the text to better justify the novelty.

Remark 2: Depth of Data: The experimental data presented seem insufficient to robustly support the conclusions. It is recommended to add experimental data.

Response 2: Unfortunately, the reviewer did not indicate which experimental data were not presented. Our manuscript presents the complete cycle from obtaining aseptic seeds and conditioned donor explants to adaptation to soil conditions of in vitro rooted plantlets regenerated from the hypocotyl- and cotyledon-derived callus tissues. The efficiency and duration of each stage, as well as the culture conditions are summarized in Fig. 4. The frequencies of callus formation and somatic organogenesis of shoots or roots, as well as mean number regenerants per explant are systematized in tables, and fig. 2. These characteristics are traditional for such studies.

Remark 3: Abstract: The core issues, methods, key findings, and main conclusions of the study are not clearly summarized (For example, lines 16-19 are not clear and can be ambiguous).

Response 3: Thank you for your valuable comment. We agree with the reviewer's remark. We have improved the Abstract sections, which are marked in the resubmitted file (L. 10-33).

Remark 4: Technical flow chart: The current flow chart fails to clearly and intuitively outline the core steps and technical key points of the experiment, and is not readable. It is suggested to redesign it.

Response 4: The fig. 4 systematizes the developed protocol of induced shoot organogenesis, which presents efficiency and duration of each stage, as well as the culture conditions. In our opinion, the final scheme is useful for readers and is often used in similar studies, for example, [Gerszberg, A. Tissue Culture and Genetic Transformation of Cabbage (Brassica oleracea var. capitata): an Overview. Planta 2018, 248, 1037–1048.] and [Demidenko, D.V.; Varlamova, N.V.; Soboleva, T.M.; Shitikova, A.V.; Khaliluev, M.R. An Efficient and Rapid Protocol for Somatic Shoot Organogenesis from Juvenile Hypocotyl-Derived Callus of Castor Bean cv. Zanzibar Green. BioTech 2024, 13, 25].

Remark 5: Figure 1 and Figure 3: The bar chart lacks a statistical significance test annotation; please add the necessary statistical analysis annotation.

Response 5: Thank you for your valuable comment. We agree with the reviewer's remark. We have supplemented Figure 1 (L. 177) and Figure 4 (L. 276) with statistical analysis. The captions to Figs. have also been supplemented information.

 

Remark 6: Figure 2: The picture is not illustrative, and the key picture lacks a scale, so readers cannot judge the actual size. Please add the scale.

Response 6: Thank you for your valuable comment. We agree with the reviewer's remark. We have added scale bars to Figure 2 (L. 207) and Figure S1 (Supplement file).

Remark 7: Discussion: This section is out of focus and fails to closely focus on the core findings (For example, lines 334-395 do not highlight the argument, do not conduct in-depth analysis, and do not have new insights). Recommend a new discussion.

Response 7: Thank you for your valuable comment. We agree with the reviewer's remark. We have rewritten and significantly improved the Discussion section for better understanding (L. 365-415).

Remark 8: References: The format of journal names in the reference list is not uniform ( For example, lines 436-437 are inconsistent with the format of lines 438-439 ). Please unify the format according to the requirements of journals.

Response 8: Thank you for your valuable comment. Reference list has been unified according to the journal rules.

Once again, we are so grateful for your review and valuable comments. In addition, we also send a resubmitted Word document with your and other reviewers' comments. We hope that the resubmitted manuscript version has become more understandable for readers.

Best regards,

Marat Khaliluev.

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

The authors have conducted an extensive study, but many questions and concerns arise.
1. Only three locally genotypes from Russia were tested, and the applicability of this protocol to cabbage varieties from other geographical origins or with significantly different genetic backgrounds has not been verified. Its universality still requires further validation.
2. It seems that the content presented in Table 2 and Table 3 is identical, please verify this. In Table 2, there are significant differences in regeneration capacity among different genotypes, 78.0% for the Parus and 36.3% for the Podarok. How can this be used to illustrate the universality of the protocol?
3. In the seed sterilization method, the specific concentration of sodium hypochlorite is 12.5%, does it refers to the available chlorine content?
4. The authors consider this protocol to be a highly effective and universal protocol for in vitro indirect shoot organogenesis. However, no direct comparison has been made with existing mainstream protocols, making it difficult to quantitatively evaluate the efficiency of this method.
5.  The author repeatedly mentioned this protocol can be applied for genetic transformation. How to prove this? There is no direct evidence showing that this system can be used for transgenesis< !--StartFragment -->, and its practical application value requires further verification.

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Author Response

Dear Reviewer,

On behalf of ourselves and the co-authors, we thank you for your appreciation of our manuscript and valuable comments. We thank the reviewer for his high assessment of our manuscript. We are confident that your comments and corrections will make our manuscript better.

Remark 1: Only three locally genotypes from Russia were tested, and the applicability of this protocol to cabbage varieties from other geographical origins or with significantly different genetic backgrounds has not been verified. Its universality still requires further validation.

Response 1: Thank you for your valuable comment. We agree with the reviewer's remark. Indeed, 3 genotypes of Russian origin were applied in our study. We have revised the discussion section, which provides a comparison with foreign genotypes (Chinise [38], Polish [21] and other origin [22]) the morphogenesis of which was also induced from hypocotyl and cotyledon explants on a culture medium of similar PGRs composition (L. 376-415). Additionally, in the title of the article we have clarified that this protocol was developed based on Russian white cabbage genotypes (L. 1-4).

Remark 2: It seems that the content presented in Table 2 and Table 3 is identical, please verify this. In Table 2, there are significant differences in regeneration capacity among different genotypes, 78.0% for the Parus and 36.3% for the Podarok. How can this be used to illustrate the universality of the protocol?

Response 2: We thank the reviewer for valuable comment. We made a mistake in the title of table 3. Table 3 summarizes the values of somatic organogenesis of roots on the induction media (L. 309-310). Table 2, in the bottom row (L. 258), presents the mean values of the shoot organogenesis frequency for all explant types and variants of the induction medium (MS1-MS4), including those that were not optimal. These average values, which for the DH line and cvs. Parus and Podarok are 61.5, 78 and 36.3%, respectively, allow us to compare only the differences between genotypes. The high efficiency of our protocol is demonstrated by an objective assessment of the average values presented for the MS3 induction medium for each genotype and explant type (55.2-89.1%), as well as the average values (73%). We have also included this in the Discussion section (L. 391-394) for better understanding.

Remark 3: In the seed sterilization method, the specific concentration of sodium hypochlorite is 12.5%, does it refers to the available chlorine content?

Response 3: We thank the reviewer for valuable comment. This is our mistake. We have made corrections to 2.2 section of Materials and Methods (L. 102-107) and to the caption to Figure 1 (L178-180).

Remark 4: The authors consider this protocol to be a highly effective and universal protocol for in vitro indirect shoot organogenesis. However, no direct comparison has been made with existing mainstream protocols, making it difficult to quantitatively evaluate the efficiency of this method.

Response 4: We thank the reviewer for valuable comment. We have rewritten part of the Discussion section in which we compare our results with those obtained previously (L. 377-415). Additionally, in the resubmitted article we focus on the efficiency and rapid of the presented protocol.

Remark 5: The author repeatedly mentioned this protocol can be applied for genetic transformation. How to prove this? There is no direct evidence showing that this system can be used for transgenesis< !--StartFragment -->, and its practical application value requires further verification.

Response 5: We thank the reviewer for valuable comment. We have made corrections to the Discussion section (L. 399-406). It is believed that achieving regeneration efficiency of more than 50% is considered effective in experiments on genetic transformation of cabbage. Additionally, we applied regeneration protocol for induction of shoot regeneration during Agrobacterium-mediated transformation using pCambia binary vector, containing selective nptII gene and reporter gus gene. We obtained independent kanamycin-resistant lines of the studied cabbage genotypes with expression of the reporter gene.

Once again, we are so grateful for your review and valuable comments. In addition, we also send a resubmitted Word document with your and other reviewers' comments. We hope that the resubmitted manuscript version has become more understandable for readers.

Best regards,

Marat Khaliluev.

Author Response File: Author Response.pdf

Round 2

Reviewer 2 Report

Comments and Suggestions for Authors

1. Authors are required to supplement the agronomic traits or breeding background of the 3 selected genotypes in Section 2.1, so as to verify their representativeness for "a wide range of genotypes".
2. The experiment did not set up a control medium "without AgNO₃", making it impossible to verify the promoting effect of AgNO₃ on shoot organogenesis. Have the authors considered supplementing this control experiment? If not, can they theoretically explain why the addition of AgNO₃ is necessary? In addition, what is the basis for selecting the concentration of 5 mg/L?
3. The concentration of NaOCl used for sterilization is stated as "<5%", which is too broad a range—could the actual concentration range be 1%-5%? Furthermore, supplementary information is needed regarding the purity of 70% ethanol and the purpose of adding Tween 20.
4. The authors propose that this protocol can be used for "transgenesis or CRISPR/Cas editing", yet no preliminary transformation data are provided. Can the results of transformation verification experiments for this protocol be supplemented? If not, the reference to "CRISPR/Cas editing" should be deleted wherever it is mentioned.
5. In the abstract, the expression "1,47-4,93 shoots per explant" uses commas—are these intended to be points (i.e., "1.47-4.93")?

Author Response

Dear Reviewer,

On behalf of ourselves and the co-authors, we thank you for your appreciation of our manuscript and valuable comments. We thank the reviewer for his high assessment of our manuscript. We are confident that your comments and corrections will make our manuscript more understanding.

Remark 1: Authors are required to supplement the agronomic traits or breeding background of the 3 selected genotypes in Section 2.1, so as to verify their representativeness for "a wide range of genotypes".

Response 1: We thank you for your valuable comment. We agree with the reviewer's remark. We supplemented agronomic traits and breeding background of the 3 selected genotypes in Section 2.1 (L. 98-106). Additionally, we delete a “a wide range” from Abstract section (L. 30). We have also added an Acknowledgements section in which we thank the breeders for providing the plant material.

Remark 2: The experiment did not set up a control medium "without AgNO₃", making it impossible to verify the promoting effect of AgNO₃ on shoot organogenesis. Have the authors considered supplementing this control experiment? If not, can they theoretically explain why the addition of AgNO₃ is necessary? In addition, what is the basis for selecting the concentration of 5 mg/L?

Response 2: We thank you for your valuable comment. Initially, we developed a regeneration protocol for subsequent Agrobacterium-mediated transformation. Previously published high effective protocols for Agrobacterium-mediated transformation of Brassica successfully used silver nitrate at a concentration of 5 mg/L to modulate organogenesis ([36] Bhalla, P.L.; Singh, M.B. Agrobacterium-mediated transformation of Brassica napus and Brassica oleracea. Nat. Protoc. 2008, 3, 181–189; [42] Neves, M.; Correia, S.; Cavaleiro, C.; Canhoto, J. Modulation of Organogenesis and Somatic Embryogenesis by Ethylene: An Overview. Plants 2021, 10, 1208; [43] Paladi, R.K.; Rai, A.N.; Penna, S. Silver Nitrate Modulates Organogenesis in Brassica juncea (L.) through Differential Antioxidant Defense and Hormonal Gene Expression. Sci. Hort. 2017, 226, 261–267.) (L. 385). Therefore, we did not test culture media without silver nitrate. Additionally, theoretically explanation for applying AgNO₃ at concentration of 5 mg/L we discussed in L. 381-386.

Remark 3: The concentration of NaOCl used for sterilization is stated as "<5%", which is too broad a range—could the actual concentration range be 1%-5%? Furthermore, supplementary information is needed regarding the purity of 70% ethanol and the purpose of adding Tween 20.

Response 3: Thank you for your valuable comment. We agree with the reviewer's remark. We asked the manufacturer of commercial bleach Belizna for the NaOCl concentration, which should not exceed 5%. Depending on the batch, the NaOCl concentration is no more than 5% but no less than 3% for 12 months from production. We used only fresh commercial bleach Belizna for preparation of sterilizing solution. We replaced “<5%” with “3-5%” (L. 110 and 113). Additionally, we prepared a 70% ethanol solution based on 96% ethanol. 0.1% solution of TWEEN20 was used as a non-ionic surfactant to improve the surface wettability of seeds, which has been successfully applied as an additional component of sterilizing solutions (for example, [35]. Sparrow, P.A.C.; Irwin, J.A.; Goldsack, C.M.; Østergaard, L. Brassica Transformation: Commercial Application and Powerful Research Tool. Transgenic Plant J. 2007, 1(2), 330-339). We have added the corresponding clarification in L. 115.

Remark 4: The authors propose that this protocol can be used for "transgenesis or CRISPR/Cas editing", yet no preliminary transformation data are provided. Can the results of transformation verification experiments for this protocol be supplemented? If not, the reference to "CRISPR/Cas editing" should be deleted wherever it is mentioned.

Response 4: We applied regeneration protocol for induction of shoot regeneration during Agrobacterium-mediated transformation using pCambia binary vector, containing selective nptII gene and reporter gus gene. We obtained independent kanamycin-resistant lines of the studied cabbage genotypes with expression of the reporter gene. We believe that it is not advisable to supplement this regeneration protocol with data on genetic transformation, as we will present it later in a future article. Therefore, we agree with the reviewer's remark and have made corrections (L. 31-32 and 489-490).

Remark 5: In the abstract, the expression "1,47-4,93 shoots per explant" uses commas—are these intended to be points (i.e., "1.47-4.93")?

Response 5: Thank you for your valuable comment. We agree with the reviewer's remark. We replaced “1,47-4,93” with “1.47-4.93” (Lines 19 and 408-409).

 

Once again, we are so grateful for your review and valuable comments. In addition, we also send a resubmitted Word document.

Best regards,

Marat Khaliluev.

Author Response File: Author Response.pdf

Round 3

Reviewer 2 Report

Comments and Suggestions for Authors

I have no further comments on your paper. The revision was correctly preformed and the final version of this paper is ready for publication. 

Author Response

Dear Reviewer,

On behalf of ourselves and the co-authors, we thank you for your appreciation of our manuscript and valuable comments.

Best regards,

Marat Khaliluev.