Somatic Embryogenesis and Indirect In Vitro Plant Regeneration in Amorphophallus konjac K. Koch by One-Step Seedling Formation
Round 1
Reviewer 1 Report
Due to the medical properties of the Konjac (Amorphophallus konjac K. Koch), it is worth developing an efficient cloning system for this species. Somatic embryogenesis is a favourable phenomenon for this process because embryos contain both root and shoot meristems and easier regenerated in in vitro culture. However, the course of this process through indirect embryogenesis is not positive due to the genetic instability of the callus tissue and somaclonal variability, that may occur.
The work can be valuable, however, the way the methodology and results are presented requires a lot of improvement, and the conclusions must be supported by statistical calculations.
Moreover, the topic of the work " Direct and Indirect Somatic Embryogenesis Induction and In vitro Propagation in Amorphophallus konjac K. Koch" is not adequate to its content - the regeneration technique proposed by the authors occurs only through indirect embryogenesis, and the aim of the experiment is to develop an efficient method of embryogenesis, not to analyse of the process of embryogenesis.
The abstract needs to be corrected because the information in it is illegible, e.g. " Our results showed that the optimal embryogenic callus was achieved on full strength Morashige and Skoog (MS) basal salts medium supplemented with 0.5 mg/L6-benzylaminopurine 21 (6-BA), 0.5mg/L naphthaleneacetic acid (NAA), 2.0 mg/L 2, 4-dichlorophenoxyacetic acid (2,4-D) and 30 g/L sucrose; the induction rate was 90.6%." suggests that the addition of above hormones gives the optimal callus, while callus types I and II do not regenerate. The sentence " From three types of embryogenic callus induced only the type Ⅲ embryogenic callus further regenerated plantlet with induction rate counted for 55.6%." – instead of “55.6%" in tab. 2 is 55.73%. Moreover, phrases like “Morshige”; "small masive" or "full strength MS" indicate linguistic shortcomings. Please describe the methods and results correctly and extend the discussion.
In addition, I propose to include in the work a drawing showing the scheme of the experiment, thanks to which the reader will better understand the next stages of the experiment. Poza tym nazwa one step… nie jest adekwatna – etapów doświadczenia związanych z pasażowaniem jest kilka. Also, the name “one-step seedling regeneration system” is inadequate - there are several stages of the experiment - several passages of explants.
In summary, the work contains numerous descriptive and linguistic errors. Nevertheless, I believe that the research done is worth publishing and can be re-reviewed after in-depth revisions
Comments for author File: 
Comments.pdf
Author Response
To: Horticulturae
Date: November 4, 2021 30, 2020
RE: Response to reviewer #1 comments
Dear Editor of Horticulturae
Thank you for the recommendations from the reviewers to our manuscript entitled:
"Somatic Embryogenesis and Indirect Plant Regeneration in Amorphophallus konjac K. Koch by One-step Seedling Formation" (Manuscript ID: horticulturae-1438724).
We appreciate all the comments from the three reviewers. We found the comments very helpful for improving the manuscript, and we have carefully revised the manuscript according to the suggestions. Please find our responses to the reviewers’ comments below, and all the revised parts marked in red in the revised manuscript.
Please inform us if there are any further requirements or comments.
Sincerely,
Yuan Qin
Professor,
Dean College of Life Science
Center for Genomic and Biotechnology
Haixia Institute of Science and Technology, Fujian Agriculture and Forestry University,
Fujian, Fuzhou, China 350005
Email: [email protected];
Phone: 86-591-83595382
Due to the medical properties of the Konjac (Amorphophallus konjac K. Koch), it is worth developing an efficient cloning system for this species. Somatic embryogenesis is a favourable phenomenon for this process because embryos contain both root and shoot meristems and easier regenerated in in vitro culture. However, the course of this process through indirect embryogenesis is not positive due to the genetic instability of the callus tissue and somaclonal variability, that may occur. The work can be valuable, however, the way the methodology and results are presented requires a lot of improvement, and the conclusions must be supported by statistical calculations.
Response: We have improved and checked the manuscript thoroughly the methodology and results have been received.
Moreover, the topic of the work " Direct and Indirect Somatic Embryogenesis Induction and In vitro Propagation in Amorphophallus konjac K. Koch" is not adequate to its content - the regeneration technique proposed by the authors occurs only through indirect embryogenesis, and the aim of the experiment is to develop an efficient method of embryogenesis, not to analyse of the process of embryogenesis.
Responnnse; however we had initially regenerate the seedlings directly in order to get aseptic leave explants but as you pointout the primary purpose of our research is to regenrate plant from callus thus we have modified the title according to the content of the manuscript.
The abstract needs to be corrected because the information in it is illegible, e.g. " Our results showed that the optimal embryogenic callus was achieved on full strength Morashige and Skoog (MS) basal salts medium supplemented with 0.5 mg/L6-benzylaminopurine 21 (6-BA), 0.5mg/L naphthaleneacetic acid (NAA), 2.0 mg/L 2, 4-dichlorophenoxyacetic acid (2,4-D) and 30 g/L sucrose; the induction rate was 90.6%." suggests that the addition of above hormones gives the optimal callus, while callus types I and II do not regenerate. The sentence " From three types of embryogenic callus induced only the type Ⅲ embryogenic callus further regenerated plantlet with induction rate counted for 55.6%." – instead of “55.6%" in tab. 2 is 55.73%. Moreover, phrases like “Morshige”; "small masive" or "full strength MS" indicate linguistic shortcomings. Please describe the methods and results correctly and extend the discussion.
Response: Thanks for your suggestion abstract and entire manuscript improved revised according to your comment.
In addition, I propose to include in the work a drawing showing the scheme of the experiment, thanks to which the reader will better understand the next stages of the experiment.
Response: We have proof of an experimental model for our work; please see figure 4.
Poza tymnazwa one step… nie jest adekwatna – etapówdoświadczeniazwiązanych z pasażowaniem jest kilka. Also, the name “one-step seedling regeneration system” is inadequate - there are several stages of the experiment - several passages of explants.
Response: As we mentioned in the conclusion section, the tissue culture of plants mainly adopts multiple procedures such as explants to callus formation, differentiation of callus to seedlings, rooting induction from seedlings. But by one step seedling formation, we regenerate micro-corm from callus, and the corm regenerates root and shoot in the same medium. Which shorten the plant regeneration.
In summary, the work contains numerous descriptive and linguistic errors. Nevertheless, I believe that the research done is worth publishing and can be re-reviewed after in-depth revisions.
Response: Thank you very much for your positive comment. We have already invited experttts in the field to help us to improve this manuscript. We have revised the whole manuscript revised are marked in red.
Author Response File: 
Author Response.docx
Reviewer 2 Report
The manuscript by Li et. al. titled ‘Direct and Indirect Somatic Embryogenesis Induction and In vitro Propagation in Amorphophallus konjac K. Koch has focused on one of the important plants under family Araceae.
Authors have explained tissue culture strategies to get seedlings in less time using the somatic embryogenesis (SE) approach for this important plant which might help in future regeneration efforts and transgenics of this plant using SE.
There are following few points authors need to address to further improve this manuscript.
- In the title of the paper please use italic font for ‘In-vitro’ as well as for the botanical name of the plant. Moreover, also check throughout the manuscript for the same.
- Please check the legends of Figure 1, it has errors e.g. What does mean Table 14 days? Details for 'b' and 'c' are missing. similarly, also check for 'e', 'f', 'g' and 'h' and rectify it. Line No. 110-112.
- Line No 174, How much? specify.
- Check the following lines for minor errors, Line No. 177, 192; Line No. 169, should it be 23 days?
Author Response
To: Horticulturae
Date: November 4, 2021 30, 2020
RE: Response to reviewer #2 comments
Dear Editor of Horticulturae
Thank you for the recommendations from the reviewers to our manuscript entitled:
"Somatic Embryogenesis and Indirect Plant Regeneration in Amorphophallus konjac K. Koch by One-step Seedling Formation" (Manuscript ID: Horticulturae-1438724).
We appreciate all the comments from the three reviewers. We found the comments very helpful for improving the manuscript, and we have carefully revised the manuscript according to the suggestions. Please find our responses to the reviewers’ comments below, and all the revised parts marked in red in the revised manuscript.
Please inform us if there are any further requirements or comments.
Sincerely,
Yuan Qin
Professor,
Dean College of Life Science
Center for Genomic and Biotechnology
Haixia Institute of Science and Technology, Fujian Agriculture and Forestry University,
Fujian, Fuzhou, China 350005
Email: [email protected];
Phone: 86-591-83595382
The manuscript by Li et. al. titled ‘Direct and Indirect Somatic Embryogenesis Induction and In vitro Propagation in Amorphophallus konjac K. Koch has focused on one of the important plants under family Araceae.
Authors have explained tissue culture strategies to get seedlings in less time using the somatic embryogenesis (SE) approach for this important plant which might help in future regeneration efforts and transgenics of this plant using SE.
There are following few points authors need to address to further improve this manuscript.
- In the title of the paper please use italic font for ‘In-vitro’ as well as for the botanical name of the plant. Moreover, also check throughout the manuscript for the same.
Response: We have revised all the concerns in the manuscript accordingly.
- Please check the legends of Figure 1, it has errors e.g. What does mean Table 14 days? Details for 'b' and 'c' are missing. similarly, also check for 'e', 'f', 'g' and 'h' and rectify it. Line No. 110-112.
Response: We are so sorry for the mixed description. We have correct in new version.
- Line No 174, How much? specify.
- Response: It has been recify in the new version.
- Check the following lines for minor errors, Line No. 177, 192; Line No. 169, should it be 23 days?
- Response: It is revised
Author Response File: Author Response.docx
Reviewer 3 Report
The manuscript “Direct and Indirect Somatic Embryogenesis Induction and In vitro Propagation in Amorphophallus konjac K. Koch” by Li, et al., talks about direct and indirect somatic embryogenesis induction and in vitro propagation in Konjac (Amorphophallus konjac K. Koch) plants.
Overall, this manuscript is well written and thoroughly described. However, the language used in the manuscript is not up to the standard for publication, and several major and minor mistakes are shown, and many of the phrases are not clear. It should go through a complete proof-reading by a native speaker. I have mentioned below some examples from abstract section
Line 16: Konjac (Amorphophallus konjac K. Koch) is a well-known tuberous herbaceous vegetable that belongs to an important medicinal family ‘Araceae’.
Line 17: ‘small and massive’ cannot be written together. Revise the sentence
Line 18: ‘an underground tuber’
Line 21: ‘Murashige & Skoog (MS)’ check the spelling
Line 25: A proliferation rate of 40% was achieved on MS basal medium ….
Line 28: The seeding induction rate achieved was 100% ….
Line 31: the last sentence does not make any sense. It should be re written
Line 20: say something about different treatments before talking about the results
Line 73-74: needs references
The introduction of the manuscript is well written and thoroughly described. However, authors should mention the agronomic importance of the konjac plants. Moreover, there are some irrelevant statements in this section like lines 51-54, and some references are outdated as well.
At the end of the introduction section, say something about the future prospects of this study and how the researchers and farmer’s community can benefit from your research.
The M&M section has some confusions. The authors have mentioned three different sterilization methods but did not say anything about which method was more successful? Have you used all three methods to different tubers or to the same tubers? It is confusing. Moreover, it should be NaOCl instead of NaClO in table 5.
Lines 219-221: the sentence should be written in a past tense
Line 223: sterilization with detergents?
Line 237: did you cultured the explants on MS basal medium with or without hormones?
Line 240: which different concentrations of PGRs?
Line 243: combination of which PGRs?
Line 249: the different concentrations of hormones should be mentioned in a tabular format here as well, in order to easily understand the treatments.
The authors have mentioned the term ‘embryogenic callus type III’ but did not explain that on which grounds have they categorize it? Also did not say anything about Type-I and Type-II?
Line 263: how did you measure 1.0 g micro-corms?
The M&M section needs some serious attention because there are a lot of confusions and grammatical errors as well.
The other major drawback of this manuscript is the discussion section. Results and Discussion section needs some serious revisions the authors should provide more precise and strong justifications to support the arguments/results obtained during the experiments. Moreover, there are many sentences and grammatical errors throughout this section as well.
I have following major concerns in this section as well.
All the figures need scale bars to be added, in order to easily understand the size of corms and callus and other material.
All the results mentioned in tables should be revised to add the letters of statistical analysis. Moreover, the statistical significance statement should also be mentioned in the captions.
Please transform the percentage data in angular values (arc sin transformation) and then subject them to ANOVA.
To sum up, this experiment can find an interest for the specialists in this field but I think without properly explaining the methodology and results and discussions, it is not sufficient to be accepted for publication in its current position.
Moreover, the references are not formatted as per the author’s guidelines provided by the journal.
Please follow the format.
Author Response
To: Horticulturae
Date: November 4, 2021 30, 2020
RE: Response to reviewer #3 comments
Dear Editor of Horticulturae
Thank you for the recommendations from the reviewers to our manuscript entitled:
"Somatic Embryogenesis and Indirect Plant Regeneration in Amorphophallus konjac K. Koch by One-step Seedling Formation" (Manuscript ID: Horticulturae-1438724).
We appreciate all the comments from the three reviewers. We found the comments very helpful for improving the manuscript, and we have carefully revised the manuscript according to the suggestions. Please find our responses to the reviewers’ comments below, and all the revised parts marked in red in the revised manuscript.
Please inform us if there are any further requirements or comments.
Sincerely,
Yuan Qin
Professor,
Dean College of Life Science
Center for Genomic and Biotechnology
Haixia Institute of Science and Technology, Fujian Agriculture and Forestry University,
Fujian, Fuzhou, China 350005
Email: [email protected];
Phone: 86-591-83595382
The manuscript “Direct and Indirect Somatic Embryogenesis Induction and In vitro Propagation in Amorphophallus konjac K. Koch” by Li, et al., talks about direct and indirect somatic embryogenesis induction and in vitro propagation in Konjac (Amorphophallus konjac K. Koch) plants.
Overall, this manuscript is well written and thoroughly described. However, the language used in the manuscript is not up to the standard for publication, and several major and minor mistakes are shown, and many of the phrases are not clear. It should go through a complete proof-reading by a native speaker. I have mentioned below some examples from abstract section.
Response: Thank you very much for your suggestion; we have already invited English native speakers to help us improve this manuscript. All the revised parts are marked in red.
Line 16: Konjac (Amorphophallus konjac K. Koch) is a well-known tuberous herbaceous vegetable that belongs to an important medicinal family ‘Araceae’.
Response: Revised please see new format.
Line 17: ‘small and massive’ cannot be written together. Revise the sentence
Response: We have revised the point.
Line 18: ‘an underground tuber’
Response: We have rephrased the sentences.
Line 21: ‘Murashige& Skoog (MS)’ check the spelling
Response: We have revised the point.
Line 25: A proliferation rate of 40% was achieved on MS basal medium ….
Response: We have revised the point.
Line 28: The seeding induction rate achieved was 100% ….
Response: We have double check the point.
Line 31: the last sentence does not make any sense. It should be re written
Response: We have drope the sentences.
Line 20: say something about different treatments before talking about the results
Response: we revised accordingly.
Line 73-74: needs references
Response: It has been corrected in the new version.
The introduction of the manuscript is well written and thoroughly described. However, authors should mention the agronomic importance of the konjac plants. Moreover, there are some irrelevant statements in this section like lines 51-54, and some references are outdated as well.
Response: It has corrected in new version.
At the end of the introduction section, say something about the future prospects of this study and how the researchers and farmer’s community can benefit from your research.
Response: We have revised the Introduction, Results and Discussion section; the revised are marked in red.
The M&M section has some confusions. The authors have mentioned three different sterilization methods but did not say anything about which method was more successful? Have you used all three methods to different tubers or to the same tubers? It is confusing. Moreover, it should be NaOCl instead of NaClO in table 5.
Response: We have done it and checked the paper thoroughly according to your suggestion. As well as a model proof to clarify whole methodology.
Lines 219-221: the sentence should be written in a past tense
Response: We have revised the point.
Line 223: sterilization with detergents?
Response: We have repherase the term.
Line 237: did you cultured the explants on MS basal medium with or without hormones?
Response: without PGRs, We have revised the point.
Line 240: which different concentrations of PGRs?
Response: without PGRs.
Line 243: combination of which PGRs?
Response: revised in the new format.
Line 249: the different concentrations of hormones should be mentioned in a tabular format here as well, in order to easily understand the treatments.
Response: since all the PGRs ratios in results tables in order to prevent repetitive we did not add separate table for each treatment in methods section.
The authors have mentioned the term ‘embryogenic callus type III’ but did not explain that on which grounds have they categorize it? Also did not say anything about Type-I and Type-II?
Response: we have added those two parts in new version.
Line 263: how did you measure 1.0 g micro-corms?
Response: about 1.0 g of corm were measured by and electronic balance.
The M&M section needs some serious attention because there are a lot of confusions and grammatical errors as well.
Response: Thank you very much for your suggestion. In the revised version, we have tried to solve grammatical and English language mistakes. The entire manuscript was enriched with detailed information.
The other major drawback of this manuscript is the discussion section. Results and Discussion section needs some serious revisions the authors should provide more precise and strong justifications to support the arguments/results obtained during the experiments. Moreover, there are many sentences and grammatical errors throughout this section as well.
I have following major concerns in this section as well.
All the figures need scale bars to be added, in order to easily understand the size of corms and callus and other material.
Response; we have added the scale bars in new format.
All the results mentioned in tables should be revised to add the letters of statistical analysis. Moreover, the statistical significance statement should also be mentioned in the captions.
Response: In the revised version, we have tried to solve such mistakes, the tables changed in new format.
Please transform the percentage data in angular values (arc sin transformation) and then subject them to ANOVA.
Response; we added the statistical analysis in the new format.
To sum up, this experiment can find an interest for the specialists in this field, but I think without properly explaining the methodology and results and discussions, it is not sufficient to be accepted for publication in its current position.
Moreover, the references are not formatted as per the author’s guidelines provided by the journal.
Please follow the format.
Response: The reference has cited machinery. We used EndNote X9 for citation and MDPI citation style selected. By the way, double checked through the manuscript. Your can revised section is marked in red.
Author Response File: Author Response.docx
Round 2
Reviewer 1 Report
Thank you for considering my comments. The inserted fragments still need to be corrected in spelling, e.g. spacing in the abstract; Fig. 1 - (a) should be bolded instead A, Fig. 4 - should be "Proposed" instead of "proposed"; figure 4 requires graphic correction - the text enters the pictures; etc. Authors should review the entire text for this purpose. Under tables with statistical analysis authors should put description, e.g. "groups
followed by the same letter are not statistically significant by Duncan’s multiple range test (p < 0.01)". All abbreviations used in tables and figures should be described below them. I am still not sure if the name "one-step seedling regeneration" is adequate - the procedure is carried out in a few steps - several passages and on several media. It is not explained how much presented procedure can shorten the time needed for cloning compared to the traditional method.
Author Response
Thank you for considering my comments. The inserted fragments still need to be corrected in spelling, e.g. spacing in the abstract; Fig. 1 - (a) should be bolded instead A, Fig. 4 - should be "Proposed" instead of "proposed"; figure 4 requires graphic correction - the text enters the pictures; etc. Authors should review the entire text for this purpose.
Response; In the new format, we solve such errors please see the new format
Under tables with statistical analysis authors should put description, e.g. "groups
followed by the same letter are not statistically significant by Duncan’s multiple range test (p < 0.01)". All abbreviations used in tables and figures should be described below them.
Response; Thanks for your suggestion we add the description.
I am still not sure if the name "one-step seedling regeneration" is adequate - the procedure is carried out in a few steps - several passages and on several media. It is not explained how much-presented procedure can shorten the time needed for cloning compared to the traditional method.
Response; This term is used in plenty of Chinese articles, we have pointed out in several sections of the manuscript the difference and advantages of one-step seedling formation. Line; 93-98, 336-339. Anyway if you have any term to replace please suggest for us it will be very helpful.
Reviewer 3 Report
Dear Authors,
I am very pleased to see that all suggestions and corrections have been made.
I find the manuscript and the protocol described in it very interesting from a scientific point of view.
Best regards
Author Response
Thank you very much for your positive comment on our manuscript.
Best
