Recent Progress in Genetic Transformation and Gene Editing Technology in Cucurbit Crops

Round 1

Reviewer 1 Report

The manuscript described recent progress in genetic transformation and gene editing technology in Cucurbit crops which is comprehensive and provides an important reference for the study of cucurbit crops. However, I would like to ask for the following changes:

1.      The species order in Table 1 and Table 2 should be consistent with each other.

2.      The width of the first column in Table 2 needs to be adjusted to ensure that the world “watermelon” is in the same row.

3.      Some of the successful application of CRISPR/Cas9 in watermelon listed in line297-306 were missing in Table 2, such as ClWIP, ClCOMT1, ClLEC1. In addition, some important progress has also been missed. The gene list is as follows: ClVST1, LAGA2, ClSWEET3, ClTST2, ClATM1.

Author Response

The manuscript described recent progress in genetic transformation and gene editing technology in Cucurbit crops which is comprehensive and provides an important reference for the study of cucurbit crops. However, I would like to ask for the following changes:

1. The species order in Table 1 and Table 2 should be consistent with each other.

Re: We sincerely thank you for careful reading. As suggested by you, we have corrected the species order in Table 1 and Table 2 to keep them consistent.

2. The width of the first column in Table 2 needs to be adjusted to ensure that the world “watermelon” is in the same row.

Re: Thanks for your careful checks. We are sorry for our carelessness. Based on your suggestion, we have adjusted the width of the first column of Table 2 to ensure that the word “watermelon” is in the same row.

3. Some of the successful application of CRISPR/Cas9 in watermelon listed in line297-306 were missing in Table 2, such as ClWIP, ClCOMT1, ClLEC1. In addition, some important progress has also been missed. The gene list is as follows: ClVST1, LAGA2, ClSWEET3, ClTST2, ClATM1.

Re: We sincerely appreciate your valuable comments. We have checked the literature carefully and found that we had missed many important progresses. As you suggested, we have added more references to the successful application of CRISPR/Cas9 in watermelon in line 309-313 of 3.1 and Table 2.

Author Response File: Author Response.docx

Reviewer 2 Report

Cucurbit crops have many major horticultural crops with high nutritional and economic values. Gene transformation and gene editing has made great progress in these crops. As a result, the manuscript is of great importance. Overall, the manuscript is well written and fluent. However, some issues need to be solved before publication. 1. In Table 2, functions of some genes should be changed. Regulation of male and female fertility and ovule development--Csa3M850670; Fruit elongation--Csa2G337260; Fruit neck length--Csa2G285890 2. In discussion 4.1, the authors focus on screening germplasms with high transformation rates. In my opinion, construction of transformation systems free of genotypes in Cucurbit crops is also a promising direction, since this attempt has been achieved in cotton and other crops. 3. In addition to the presentation in the discussion part, the manuscript could give more prospects that may be applied in transformation or gene editing in Cucurbit crops, such as transgene without foreign gene and so on. 4. The application fields of transformation and gene editing in Cucurbit crops in the future may also be highlighted in the discussion part. 5. The reference styles should be uniform throughout the manuscript. For example, in some cases, the authors give the full name of the journal, whereas in other places the abbreviated names were displayed.

Author Response

Cucurbit crops have many major horticultural crops with high nutritional and economic values. Gene transformation and gene editing has made great progress in these crops. As a result, the manuscript is of great importance. Overall, the manuscript is well written and fluent. However, some issues need to be solved before publication.

1. In Table 2, functions of some genes should be changed. Regulation of male and female fertility and ovule development--Csa3M850670; Fruit elongation--Csa2G337260; Fruit neck length--Csa2G285890

Re: We appreciate your valuable opinions. We note that some gene function or phenotype descriptions given in Table 2 are not appropriate or scientific. Therefore, we have revised and unified the description. We’ve changed “Reduced male and female fertility with malformed pollen and suppressed ovule development--Csa3M850670” to “Reduced male and female fertility and ovule development--Csa3M850670”, “Suppressed the elongation of the fruit--Csa2G337260” to “Fruit elongation--Csa2G337260”, “Regulated fruit neck length--Csa2G285890” to “Fruit wart--Csa2G285890” and “The length of fruit neck--Csa4G038760, Csa2G379350” to “Fruit neck length--Csa4G038760, Csa2G379350”.

2. In discussion 4.1, the authors focus on screening germplasms with high transformation rates. In my opinion, construction of transformation systems free of genotypes in Cucurbit crops is also a promising direction, since this attempt has been achieved in cotton and other crops.

Re: We believe this is an excellent suggestion. It is undoubtedly a promising direction to construct a genotype-free transformation system in cucurbit crops. However, in view of the fact that the regeneration mode of cucurbit crops is different from that of cotton and the current research results show that various species can be transformed, there is no widely adaptable germplasm, and the transformation efficiency is relatively backward. For such species with low genetic transformation efficiency, screening excellent receptor germplasm and optimizing the most suitable transformation system are the most effective and direct ways to improve the plant transformation efficiency. We have made some changes in 4.1. Of course, for future molecular breeding, it is the goal of all researchers to establish a transformation system free from the limitation by genotype.

3. In addition to the presentation in the discussion part, the manuscript could give more prospects that may be applied in transformation or gene editing in Cucurbit crops, such as transgene without foreign gene and so on.

Re: Thanks for your suggestion. In addition to the transgenes without foreign genes, we have also added possible application prospects. See line 363-373 for detailed modifications.

4. The application fields of transformation and gene editing in Cucurbit crops in the future may also be highlighted in the discussion part.

Re: We have re-written part 4 based on your suggestion. We've added the content concerning further development of gene editing technologies in cucurbit crops in 4.2, such as the use of CRISPR/Cas9 technology for spatial or temporal genomic changes, and rapid identification of gene functions. See line 397-406 for detailed modifications.

5. The reference styles should be uniform throughout the manuscript. For example, in some cases, the authors give the full name of the journal, whereas in other places the abbreviated names were displayed.

Re: We appreciate your advice and agree with your assessment. Accordingly, throughout the manuscript, we have given the full name of the journal and revised the reference styles uniformly.

 

Author Response File: Author Response.docx

Reviewer 3 Report

Dear Authors,

I have one major and one minor comment.

Major Comment:

I think that the discussion is repeating the statements presented earlier in the review. When I started reading it, it was unclear why re-iterate on the same topic once again. I suggest to write a more concise discussion, omitting the paragraphs that re-iterate your main text. Focus on challenges that still need to be addressed and the future perspectives and possibly the agricultural impact.

Minor Comment:

Cefotaxime (line 251) is not an antioxidant, research it and correct.

Author Response

1. Major Comment:

I think that the discussion is repeating the statements presented earlier in the review. When I started reading it, it was unclear why re-iterate on the same topic once again. I suggest to write a more concise discussion, omitting the paragraphs that re-iterate your main text. Focus on challenges that still need to be addressed and the future perspectives and possibly the agricultural impact.

Re: Thank you for your suggestion. We have simplified and omitted some contents of the discussion section by putting the focus on the establishment of a genotype-free transformation system and the further development of gene editing technology in cucurbit crops. We believe that these two aspects are more important: One is an urgent problem to be solved, and the other has a good development prospect, which deserves further study. At the same time, both of them have a great significance for the application of cucurbit genetic transformation. These changes can be seen in 4.1 and 4.2.

2. Minor Comment:

Cefotaxime (line 251) is not an antioxidant, research it and correct.

Re: Thank you for pointing out our mistake. In order to correct the error, we searched relevant information and found that cefotaxime is the third generation cephalosporin antibiotic having broad-spectrum activity against Gram-negative bacteria and positive bacteria. We have classified it as an Agrobacterium contamination inhibitor and inserted it in line 266. In addition, we added sodium thiosulfate to the antioxidants, as detailed in line 257.

Author Response File: Author Response.docx

Reviewer 4 Report

Comments and Suggestions for Authors:

Manuscript ID: agronomy-2254000

Type of manuscript: Perspective

Title: Recent Progress in Genetic Transformation and Gene Editing Technology in Cucurbit Crops

 

In this manuscript, authors had been an extensive review of the state of the art in terms of the recent advances in improving the genetic transformation efficiency of cucurbit crops. As well as about the application of gene editing technology to cucurbit crops, including CRISPR. This work would provide a reference for improving genetic transformation efficiency and gene editing technology for cucurbit crops.

The review is well carried out, it includes all the advances on the subject so far, but in my opinion, the authors need to attend to some suggestions so that it can be published:

-          In Section 2. Methods to improve the genetic transformation efficiency of cucurbit crops, in lines 100 -103, authors wrote “Researchers have recently optimized the transformation process based on previous work, including genotype screening, Agrobacterium infection methods, the application of morphogenic genes, and the diversification of screening markers, thus improving the transformation efficiency (Figure 1)”.  Which researchers are referring to, the group writing this review? or the figure comes from another group and/or article. If this were the case, they should give the reference, if on the contrary it is the product of the same authors they should explain it in the text, for example "as a result of our investigations we have developed efficient methods for the genetic transformation of cucurbit crops (Figure 1)”

-          Starting from line 136, Table 1 is presented. In the last row, the information highlighted in blue appears in the germplasm column. Which is the reason?

-          Throughout the manuscript, for example in line 66, in lines 173 to 180, 184, 290, 292, the names of the genes appear in capital letters and italics, I think that if what is intended is to highlight them, it would suffice to write them in italics

For all those reason my overall recommendation is: Accept after minor revision

Comments for author File: Comments.docx

Author Response

In this manuscript, authors had been an extensive review of the state of the art in terms of the recent advances in improving the genetic transformation efficiency of cucurbit crops. As well as about the application of gene editing technology to cucurbit crops, including CRISPR. This work would provide a reference for improving genetic transformation efficiency and gene editing technology for cucurbit crops. The review is well carried out, it includes all the advances on the subject so far, but in my opinion, the authors need to attend to some suggestions so that it can be published:

1. In Section 2. Methods to improve the genetic transformation efficiency of cucurbit crops, in lines 100 -103, authors wrote “Researchers have recently optimized the transformation process based on previous work, including genotype screening, Agrobacterium infection methods, the application of morphogenic genes, and the diversification of screening markers, thus improving the transformation efficiency (Figure 1)”. Which researchers are referring to, the group writing this review? or the figure comes from another group and/or article. If this were the case, they should give the reference, if on the contrary it is the product of the same authors they should explain it in the text, for example "as a result of our investigations we have developed efficient methods for the genetic transformation of cucurbit crops (Figure 1)”

Re: First of all, we thank you for your critical suggestions. The content in line 103-106 is a summary we concluded from the work of recent years on improving the genetic transformation methods for cucurbitaceae crops, including but not limited to genotype screening, Agrobacterium infection methods, application of morphogenetic genes, and diversification of screening markers. From the above aspects, we summarized and formed Figure 1 for reference. At the same time, we added the literature content of [17-31] to facilitate the search for relevant evidence, and gave examples of relevant “researchers” to ensure the followability of the conclusion.

2.Starting from line 136, Table 1 is presented. In the last row, the information highlighted in blue appears in the germplasm column. Which is the reason?

Re: The highlighted blue text has no special meaning, which is a result our negligence during review, failing to remove the mark timely. The mark in the last row of Table 1 has been modified.

3.Throughout the manuscript, for example in line 66, in lines 173 to 180, 184, 290, 292, the names of the genes appear in capital letters and italics, I think that if what is intended is to highlight them, it would suffice to write them in italics

Re: Thank you for your careful review, we have changed the names of the genes to show them  only in italics. However, for the format reason, the revised version does not show the blue mark, see line 69, 179 to 184, 185, 296, 298 for detailed modifications.

 

 

Author Response File: Author Response.docx