Embryogenic callus induction from immature zygotic embryos and genetic transformation of Larix kaempferi 3x Larix gmelinii 9

Sufang Zhang; Shanshan Yan; Peiqi An; Qing Cao; Chen Wang; Junhui Wang; Hanguo Zhang; Lei Zhang

doi:10.1371/journal.pone.0258654

Peer Review History

Original SubmissionMay 21, 2021
4 Jun 2021 Decision Letter - Vijay Kumar, Editor PONE-D-21-16841 Embryogenic callus induction from immature zygotic embryos and genetic transformation of Larix kaempferi 3x Larix gmelinii 9 PLOS ONE Dear Dr. Zhang, Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process. Please submit your revised manuscript by Jul 19 2021 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file. 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See the following link for instructions on providing the original image data: https://journals.plos.org/plosone/s/figures#loc-original-images-for-blots-and-gels. In your cover letter, please note whether your blot/gel image data are in Supporting Information or posted at a public data repository, provide the repository URL if relevant, and provide specific details as to which raw blot/gel images, if any, are not available. Email us at plosone@plos.org if you have any questions. 3. Your ethics statement should only appear in the Methods section of your manuscript. If your ethics statement is written in any section besides the Methods, please move it to the Methods section and delete it from any other section. Please ensure that your ethics statement is included in your manuscript, as the ethics statement entered into the online submission form will not be published alongside your manuscript. [Note: HTML markup is below. Please do not edit.] Reviewers' comments: Reviewer's Responses to Questions Comments to the Author 1. Is the manuscript technically sound, and do the data support the conclusions? The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented. Reviewer #1: Partly ******** 2. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: No ****** 3. Have the authors made all data underlying the findings in their manuscript fully available? The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified. Reviewer #1: Yes ****** 4. Is the manuscript presented in an intelligible fashion and written in standard English? PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here. Reviewer #1: No ******** 5. Review Comments to the Author Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters) Reviewer #1: In this study, immature zygotic embryos of the hybrid larch Larix kaempferi 3 x Larix gmelinii 9 were used as explants, embryogenic callus induction and genetic transformation was claimed. The manuscript is not presented in an intelligible fashion and written in standard English. In general the manuscript is wordy and truncated. English language/usage has to be carefully revised. It is truncated and very difficult to the reader to follow it The Introduction does not clearly state the aim of the research and the present knowledge. It should be deeply revised. Authors should revise the way of citing authors from the very Introduction. Line 81: Smith discovered ( ???? ); Line 88 : Sederoff applied (Sederoff et al.) ; Line 90: Huang obtained (* Huang et al. ) ; Line 91: Shin infected the hypocotyls (Shin et al.)…; Line 93: Levee used (Leéve at al.); just to mention a few. By the way, what does ´Shin infected' really mean? Line 26: Please define BM at first mention Line 30: ABA should be defined at first mention Line: PEG should´ve been defined at first Lines 32 and 41 81,82, 84, 85, 86,87, 88, 92, 94 : Agrobacterium should appear in italics! Line: What does “ transformation rate of resistance callus” mean? Resistant??? Line 52/53: It is suggested: Since embryos of L. x eurolepis and L.x leptoeuropaea at precotyledonary developmental stage were used as an.... Data on “ Optimal sterilisation conditions for explants” are so elementary and irrelevant to the scope of the manuscript. It should be briefly mentioned in Material and Methods. Note that they have not even been discussed. References should be carefully standardized. On top of that some key references should be considered, moreover the recent publication by Song et al. 2020 should be really looked into: Ismail G., Schnabl H., Zoglauer K., Boehm R. (2004). Agrobacterium-mediated transformation of Larix decidua: an assessment of factors influencing the efficiency of gus gene transfer. J. Appl. Bot. Food Qual. 78 83–90 Song Y., Zhang H., Li S., Li S. (2016). Relationship between the induction of embryogenic callus of larch and the morphology of immature embryos. J. North-East Forest. Univ. 44 25–30. Song Y, Bai X, Dong S, Yang Y, Dong H, Wang N, Zhang H, Li S. Stable and Efficient Agrobacterium-Mediated Genetic Transformation of Larch Using Embryogenic Callus. Front Plant Sci. 2020 Nov 25;11:584492. doi: 10.3389/fpls.2020.584492. Lines 76-78: Please delete it since it is way obvious the statement since it does not add any information: “ As is well known, the premise of plant somatic embryogenesis is that embryogenic callus is induced and can proliferate stably; thus, it is necessary to induce stably proliferating embryogenic callus in hybrid larch. Line 330: After the co-culture, numerous Agrobacterium were symbiotic on the surface of embryogenic callus. Symbiotic, REALLY? Line 858-859: What does ‘ E: cotyledons germinate’ mean? Figures 1, 2, 4, 8 and 9 do lack size reference bars. The Discussion sounds more like a Literature Review.... (see lines 357 to 372). It is poorly elaborated as presented. Lines 358 and 362: Stasolla summarized ... Stasolla believe... Authors shouls sitck to a scientific writing style. Even though it should appear Satasolla ang Yeung [ ] Line 372: … stage was the progeny stage [32]. What does progeny stage mean? Lines 372-389: In what sense authors want to stress the collection dates/times? Does it really matter? In my opinion the stage is what really matters. Above all, authors do not provide a convincing explanation to justify this difference. The discussion is way superficial. Line 386: This finding is also consistent with the results reported in previous studies. Please, specify them! And this also applies to the dose-response data (lines 392-423) during induction step. The Discussion again sounds as a literature review. The finding are not really compared and contrasted with existing and referenced literature. If one have a look from line 441-454 this very same style appears denoting a literature review on the topic (Agrobacterium-mediated transformation). For transformation purposes why authors did not consider adding acetosyringone or coniferyl alcohol as vir gene inducers? How about the Agro strain GV 3101 used as compared to others - C58 /EHA 105? It would be interesting to consider for the Discussion. Overall, the ms lacks concision and clearness, and it is just borderline. That said it is advocated that the manuscript should be carefully revised, and as presented does not merit to be published. ******** 6. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files. If you choose “no”, your identity will remain anonymous but your review may still be made public. Do you want your identity to be public for this peer review?** For information about this choice, including consent withdrawal, please see our Privacy Policy. Reviewer #1: No [NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.] While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org. Please note that Supporting Information files do not need this step. https://doi.org/10.1371/journal.pone.0258654.r001
Revision 1
13 Aug 2021 Author Response Dear editor: Thanks to the editors and reviewers for valuable comments on the manuscript "Embryogenic callus induction from immature zygotic embryos and genetic transformation of Larix kaempferi 3 x Larix gmelinii 9". The author has made detailed revisions in accordance with the comments. If there are any inadequacies, please correct or point out them. The answers are as follows: Author's responses to the editor 1. Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. The PLOS ONE style templates can be found at https://journals.plos.org/plosone/s/file?id=wjVg/PLOSOne_formatting_sample_main_body.pdf and Answer: My manuscript meets PLOS ONE's style requirements, including those for file naming. 2.PLOS ONE now requires that authors provide the original uncropped and unadjusted images underlying all blot or gel results reported in a submission’s figures or Supporting Information files. This policy and the journal’s other requirements for blot/gel reporting and figure preparation are described in detail at https://journals.plos.org/plosone/s/figures#loc-blot-and-gel-reporting-requirements and https://journals.plos.org/plosone/s/figures#loc-preparing-figures-from-image-files. When you submit your revised manuscript, please ensure that your figures adhere fully to these guidelines and provide the original underlying images for all blot or gel data reported in your submission. See the following link for instructions on providing the original image data: https://journals.plos.org/plosone/s/figures#loc-original-images-for-blots-and-gels. In your cover letter, please note whether your blot/gel image data are in Supporting Information or posted at a public data repository, provide the repository URL if relevant, and provide specific details as to which raw blot/gel images, if any, are not available. Email us at plosone@plos.org if you have any questions. Answer: I have resubmit the figures accroding to the Plos one requirement， and my blot/gel image data has been named ‘S1_raw_image’ and uploaded as a Supporting Information file. In my cover letter, “my blot/gel image data are in Supporting Information“ has been noted. 3. Your ethics statement should only appear in the Methods section of your manuscript. If your ethics statement is written in any section besides the Methods, please move it to the Methods section and delete it from any other section. Please ensure that your ethics statement is included in your manuscript, as the ethics statement entered into the online submission form will not be published alongside your manuscript. Answer: My ethics statement have been moved to the Methods section of my manuscript. Author's responses to the reviewers Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters) Reviewer #1: In this study, immature zygotic embryos of the hybrid larch Larix kaempferi 3 x Larix gmelinii 9 were used as explants, embryogenic callus induction and genetic transformation was claimed. (1) The manuscript is not presented in an intelligible fashion and written in standard English. In general the manuscript is wordy and truncated. English language/usage has to be carefully revised. It is truncated and very difficult to the reader to follow it The Introduction does not clearly state the aim of the research and the present knowledge. It should be deeply revised. Answer: English language has been revised in my manuscript. The ”Introduction” about the aim of the research and the present knowledge has been revised. (2) Authors should revise the way of citing authors from the very Introduction. Line 81: Smith discovered ( ???? ); Line 88 : Sederoff applied (Sederoff et al.) ; Line 90: Huang obtained (* Huang et al. ) ; Line 91: Shin infected the hypocotyls (Shin et al.)…; Line 93: Levee used (Leéve at al.); just to mention a few. Answer: The way of citing authors have been revised from the very Introduction and other sections in this manuscript. (3) By the way, what does ´Shin infected' really mean? Answer: ´Shin infected' has been revised as shown in this manuscript. (4) Line 26: Please define BM at first mention Answer: ”Supplement table 1” has been submited separately to define BM. (5) Line 30: ABA should be defined at first mention Answer: ”ABA” has been defined in this manuscript. (6) Line: PEG should´ve been defined at first Answer: ”PEG” has been defined in this manuscript. (7) Lines 32 and 41 81,82, 84, 85, 86,87, 88, 92, 94 : Agrobacterium should appear in italics! Answer: The“Agrobacterium” appeared in ”Lines 32 and 41 81,82, 84, 85, 86,87, 88, 92, 94 and other sections” have been revised in italics. (8) Line: What does “ transformation rate of resistance callus” mean? Resistant??? Answer: ”transformation rate of resistance callus” has been replaced with ”transformation rate of callus”. (9): Line 52/53: It is suggested: Since embryos of L. x eurolepis and L.x leptoeuropaea at precotyledonary developmental stage were used as an.... Answer: “ Since Klimaszewska used embryos of L.x eurolepis and L.x leptoeuropaea at precotyledonary developmental stage as an ....“ has been replaced with “Since embryos of L. x eurolepis and L.x leptoeuropaea at precotyledonary developmental stage were used as an....” (10) Data on “ Optimal sterilisation conditions for explants” are so elementary and irrelevant to the scope of the manuscript. It should be briefly mentioned in Material and Methods. Note that they have not even been discussed. Answer: “Optimal sterilisation conditions for explants” was correponding to the “Disinfection and inoculation of explants” in “material and methods” section, so this section was retained. (11) References should be carefully standardized. On top of that some key references should be considered, moreover the recent publication by Song et al. 2020 should be really looked into: Ismail G., Schnabl H., Zoglauer K., Boehm R. (2004). Agrobacterium-mediated transformation of Larix decidua: an assessment of factors influencing the efficiency of gus gene transfer. J. Appl. Bot. Food Qual. 78 83–90 Song Y., Zhang H., Li S., Li S. (2016). Relationship between the induction of embryogenic callus of larch and the morphology of immature embryos. J. North-East Forest. Univ. 44 25–30. Song Y, Bai X, Dong S, Yang Y, Dong H, Wang N, Zhang H, Li S. Stable and Efficient Agrobacterium-Mediated Genetic Transformation of Larch Using Embryogenic Callus. Front Plant Sci. 2020 Nov 25;11:584492. doi: 10.3389/fpls.2020.584492. Answer: References have been standardized, and the recent publication by Song et al. 2020 have been looked into and was some references have been added to this manscript. (12) Lines 76-78: Please delete it since it is way obvious the statement since it does not add any information: “ As is well known, the premise of plant somatic embryogenesis is that embryogenic callus is induced and can proliferate stably; thus, it is necessary to induce stably proliferating embryogenic callus in hybrid larch. Answer: “ As is well known, the premise of plant somatic embryogenesis is that embryogenic callus is induced and can proliferate stably; thus, it is necessary to induce stably proliferating embryogenic callus in hybrid larch“ has been deleted. (13) Line 330: After the co-culture, numerous Agrobacterium were symbiotic on the surface of embryogenic callus. Symbiotic, REALLY? Answer: ”symbiotic” has been replaced with “appeared”. (14) Line 858-859: What does ‘ E: cotyledons germinate’ mean? Answer: ”cotyledons germinate” has been replaced with “somatic embryo germination”. (15) Figures 1, 2, 4, 8 and 9 do lack size reference bars. Answer: ”Figures 1, 2, 4, 8 and 9” has been added the size reference bars. (16) The Discussion sounds more like a Literature Review.... (see lines 357 to 372). It is poorly elaborated as presented. Answer: The ”Discussion” section has been revised in this manscript. (17) Lines 358 and 362: Stasolla summarized ... Stasolla believe... Authors shouls sitck to a scientific writing style. Even though it should appear Satasolla ang Yeung [ ] Answer: They have been revised in this manscript. (18) Line 372: … stage was the progeny stage [32]. What does progeny stage mean? Answer: ”the progeny stage” was incorrect, it has been deleted and revised in this manscript. (19) Lines 372-389: In what sense authors want to stress the collection dates/times? Does it really matter? In my opinion the stage is what really matters. Above all, authors do not provide a convincing explanation to justify this difference. The discussion is way superficial. Answer: The collection date actually corresponds to the development stage, and this difference actually was presented in Table 5. It also has been revised in this manscript. (20) Line 386: This finding is also consistent with the results reported in previous studies. Please, specify them! Answer: They have been specified in this manscript. (21) And this also applies to the dose-response data (lines 392-423) during induction step. The Discussion again sounds as a literature review. The finding are not really compared and contrasted with existing and referenced literature. Answer: ” the dose-response data” and ”Discussion” have been revised in this manscript. (22) If one have a look from line 441-454 this very same style appears denoting a literature review on the topic (Agrobacterium-mediated transformation). Answer: They have been revised in this manscript. (23) For transformation purposes why authors did not consider adding acetosyringone or coniferyl alcohol as vir gene inducers? How about the Agro strain GV 3101 used as compared to others - C58 /EHA 105? It would be interesting to consider for the Discussion. Answer: We did not consider adding acetosyringone or coniferyl alcohol as vir gene inducers because of refering to the study on Larix olgensis and the reference [54] has been added in this manscript. In addition, GV3101 and other stains has been added in discussion section. Yours Sincerely Sufang Zhang Attachments Attachment Submitted filename: Response to Reviewers.docx https://doi.org/10.1371/journal.pone.0258654.r002
4 Oct 2021 Decision Letter - Vijay Kumar, Editor Embryogenic callus induction from immature zygotic embryos and genetic transformation of Larix kaempferi 3x Larix gmelinii 9 PONE-D-21-16841R1 Dear Dr. Zhang, We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements. Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication. An invoice for payment will follow shortly after the formal acceptance. To ensure an efficient process, please log into Editorial Manager at http://www.editorialmanager.com/pone/, click the 'Update My Information' link at the top of the page, and double check that your user information is up-to-date. If you have any billing related questions, please contact our Author Billing department directly at authorbilling@plos.org. If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they’ll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org. Kind regards, Vijay Kumar Academic Editor PLOS ONE Additional Editor Comments (optional): authors has revised the manuscript significantly and now I recommend it for acceptance in the current state. Reviewers' comments: https://doi.org/10.1371/journal.pone.0258654.r003
Formally Accepted
7 Oct 2021 Acceptance Letter - Vijay Kumar, Editor PONE-D-21-16841R1 Embryogenic callus induction from immature zygotic embryos and genetic transformation of Larix kaempferi 3x Larix gmelinii 9 Dear Dr. Zhang: I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department. If your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact onepress@plos.org. If we can help with anything else, please email us at plosone@plos.org. Thank you for submitting your work to PLOS ONE and supporting open access. Kind regards, PLOS ONE Editorial Office Staff on behalf of Dr. Vijay Kumar Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0258654.r004

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