The Role of Somaclonal Variation in Plant Genetic Improvement: A Systematic Review

Round 1

Reviewer 1 Report

The manuscript entitled “The role of somaclonal variation in plant genetic improvement: a systematic review” provides a wide review of literature concerning the application of the phenomenon of somaclonal variation in plant breeding. The Authors developed a detailed protocol of a search string with clear and specific assumptions, and finału chose 249 articles published in the last 16 years which deal with the above mentioned topic.

I really appreciate the idea of preparing such a review with the use of the open access software and by following all the stages of planning, execution and summarization. My main objection to this work is the inclusion to the pool of accepted articles, those papers, which concern not only somaclonal variation, but also induced mutations. 

According to Ghag et al. (2014), somaclonal variations result from the changes that occur in the genome during dedifferentiation and redifferentiation pathways (including alteration of chromosome numbers and structures and other chromosomal irregularities). Also, during regeneration cycles the extensive reprogramming can occur in the cell, which may result in loss of genes or their function, turning on-of the silent genes or change in the expression pattern of some recessive genes. Apart from that, quantitative and qualitative changes in the plant genome can result from the enhanced transposon activity occurring during in vitro culture conditions. Therefore, in my opinion, papers reporting plant induced mutations by physical agents (ion beam irradiation, gamma and UV rays) and chemical ones (e.g. EMS, NMU, SA, SN), as well as taking into account induction of polyploidy (using colchicine or trifluralin) or demethylation (using 5-Azacitidyne) should not be taken into consideration in the review concerning somaclonal variation. At least 28 articles should not be included in the pool of accepted articles. Thus, additional excluding parameters should be applied in the system of papers selection and evaluation or such reports should be manually rejected. I would like to encourage the Authors to make such adjustments, to correct the results and to do a resubmission.

Other remarks:

Line 27: I would recommend to use the full terms "plant growth regulator" and "plant growth regulators" and their abbreviations, PGR and PGRs, respectively, instead of “the plant regulator” or “the plant regulators”

Lines 64-56: I would never say that the somaclonal variation induction technique is one of the methods used for tissue culture. Somaclonal variation is a phenomenon, which occurs in tissue culture but it is rather unpredictable, although we know the factors that promote it. 

Line 162. The abbreviation SR should be introduced at the first use of a phrase “systematic review” and used consistently later in the text, without repeating the abbreviated phrase in full. 

Line 179: It should be "In Vitro Cellular and Developmental Biology - Plant", instead of "Developmental Biology". These are two completely different journals.

Line 206: what kind of plants?

Figure 3: South Korea should be indicated on the map, instead of North Korea.

Line 208 (Figure 3 description): To be precise - it is not the frequency of cultures created, but the number of articles concerning somaclonal variation published in the last 16 years and the main species being the objects of the study.

Lines 209 and 212; not frequency, but numbers

Line 215: It should be “A lower number of somaclones was generated for other crops”, instead of „Other crops generated a lower number of somaclones”

Line 217: „Only articles that made clear…” - This information should be moved to Material and Methods

Line 220: “studies included”, not “studiesincluded”

Line 229: “The largest number of studies was aimed at generating somaclones for the 229 molecular study of genetic diversity among clones” – I rather doubt in such an aim, in my view the reverse is true, i.e. the somaclones were generated (deliberately or not) and the molecular studies were then undertaken to evaluate the variation / genetic diversity generated.

Lines 242-145: The articles do not use plant growth regulators, they just report the use, please, correct this.

Lines 250-252: ”KIN and IBA were used for the evaluation of molecular and agronomic traits, TDZ was used for molecular evaluations of genetic diversity “ - this is a mental shortcut, please rewrite this, because plant growth regulators are not used for the evaluations. They are used to induce somaclonal variation, which can be then evaluated.

Line 264: “between the regulators”, not “between the regulator”

Line 266: “BAP regulator” – Please, do not repeat the word “regulator” after the abbreviation of a particular auxin or cytokinin, it’s a redundancy.

Lines 267-268: There are no verbs in these sentences. Please, introduce them or join these sentences with the previous one, concerning BAP.

Line 275: It should be “1-naphthaleneacetic acid” or “α-naphthaleneacetic acid” instead of “A-naphthalene acetic acid”

Lines 300-303: The Latin names of genus or species should be written in italics.

Lines 300-303: “The crops with the most reports...” - This sentence is unclear, please rewrite it. The crops can not include crops.

Line 303: “Leaves, seeds and rhizomes were used as explant sources” - It is also unclear whether the leaves, seeds and rhizomes were used as explants or they were the source of explants? Please, specify this. 

Lines 347-348: The sentence "In the last year, only analyses applying single nucleotide polymorphism (SNPs) markers have been reported" sounds much better.

Lines 349-352: I do not understand these two sentences. Generally speaking, all molecular markers are used for detection of somaclonal variation. Depending on the mechanisms behind somaclonal variation (e.g. DNA methylation or DNA sequence changes) we use different markers to identify them.

Line 361: the phrase "genes related to somaclonal variation" is a mental shortcut. 

Line 362: It should be "expression of genes related to" instead of "gene expression related to".

Line 365: It should be "the size of each gene name indicates the number of articles that described expression of this gene" instead of "the size of each word indicates the frequency of articles that obtained gene expression"

Line 369: "this gene evaluated dwarphism" -> "this gene was related to dwarfism"

Line 370: The genes do not have the function of coding genes! Please correct this.

Line 372: The genes are not expressive, they just can be expressed.

Line 373: The genes do not have any sensor domains.

Lines 418-419: "The changes described in the articles initially involved genetic diversity analysis…" - this sentence is unclear.

Lines 478-479: As I have already mentioned, irradiation is not a method of somaclonal variation induction. It is used for mutagenesis.

Lines 495-497: The number of subcultures is not a method, but a factor, which has an important effect on somaclonal variation.

Line 506: Please, provide the name of the species for these cultivars.

Line 507-508: "In this study we identified that the stem apices were…" -> "According to the literature studied, the stem apices were the most popular explants for induction of somaclonal variation in banana".

Line 512: " The successful use of seeds as explants…" - I do not understand this sentence and the articles cited do not support this strange thesis.

Lines 517-520: "The studies that used the irradiation method to generate mutants were not included in this review; only the studies in which irradiation generated somaclonal mutants were included…" - Can you explain the difference between irradiation that generates mutants and irradiation that generates somaclonal mutants? 

Line 522: The reference with the number 35 describes only Zoysia matrella mutagenesis.

Line 536: "...these variations occur in all cultures subjected to in vitro condition" - this is not true, somaclonal variation can occur, but does not have to

Line 557: "Many studies showed that…" - if many studies showed something, a few examples (i.e. appropriate citations) should be provided.

Line 569: The morphological changes can not be focused on plants. They can be observed in plants. The studies on ornamentals can be focused on induction and identification of such morphological changes.

Lines 612-614. I can not agree. Classical AFLP analysis does not detect DNA methylation. Only modifications like MSAP or metAFLP, in which a pair of isoschizomers is used, and one of these enzymes is methylation-sensitive, can do that. Also, contrary to RAPD or ISSR, larger amounts of DNA are needed for AFLP analysis, because a restriction digestion has to be conducted in its first step, not PCR. 

Lines 615-617: This sentence is incomprehensible.

Line 643: "The study of Lee et al. [83] also used gene expression analysis…." -> "In the study of Lee et al. [83], gene expression analysis was used…"

 

Some comments to supplementary tables:

Table S1

It should be “Prunus spp.” instead of “Prunnus spp.” 

Table S2

It should be “genetic diversity” instead of “genetical diversity” 

Please, do not format the whole text under the table S2 as superscript because it makes it unreadable.

Also, please, unify the notation for more than 1 molecular marker, because it is unclear why some of them were written like “RAPD e ISSR”, some of them like “RAPD, ISSR” and the other like “RAPD/ISSR”

Table S3

Please, change the title of the table, because genes cannot be associated with the strategies. You may use the phrase like "Genes associated with somaclonal variation in different crops".

The column called “Function” contains confusing substance. Please, decide whether the function of a particular gene will be provided here or maybe something more like the reason of analyzing such genes. ”Uncovering possible dwarfing mechanisms” or “To evaluate 'Guijiao 9' resistance genes” are not the gene functions. 

 

 

 

Author Response

We would like to thank all the reviewers for their corrections/suggestions to improve the manuscript. 

The manuscript entitled “The role of somaclonal variation in plant genetic improvement: a systematic review” provides a wide review of literature concerning the application of the phenomenon of somaclonal variation in plant breeding. The Authors developed a detailed protocol of a search string with clear and specific assumptions, and finału chose 249 articles published in the last 16 years which deal with the above mentioned topic.

I really appreciate the idea of preparing such a review with the use of the open access software and by following all the stages of planning, execution and summarization. My main objection to this work is the inclusion to the pool of accepted articles, those papers, which concern not only somaclonal variation, but also induced mutations. 

According to Ghag et al. (2014), somaclonal variations result from the changes that occur in the genome during dedifferentiation and redifferentiation pathways (including alteration of chromosome numbers and structures and other chromosomal irregularities). Also, during regeneration cycles the extensive reprogramming can occur in the cell, which may result in loss of genes or their function, turning on-of the silent genes or change in the expression pattern of some recessive genes. Apart from that, quantitative and qualitative changes in the plant genome can result from the enhanced transposon activity occurring during in vitro culture conditions. Therefore, in my opinion, papers reporting plant induced mutations by physical agents (ion beam irradiation, gamma and UV rays) and chemical ones (e.g. EMS, NMU, SA, SN), as well as taking into account induction of polyploidy (using colchicine or trifluralin) or demethylation (using 5-Azacitidyne) should not be taken into consideration in the review concerning somaclonal variation. At least 28 articles should not be included in the pool of accepted articles. Thus, additional excluding parameters should be applied in the system of papers selection and evaluation or such reports should be manually rejected. I would like to encourage the Authors to make such adjustments, to correct the results and to do a resubmission.

We understand your concern regarding the insertion of these articles and we agree to remove all articles that used the induction of mutations (mutagenesis) by means of physical agents to obtain genetic variability and made the necessary changes after the elimination of a total of 17 articles. Regarding chemical agents, we kept the articles in our study, since they correspond to all the others that make up our review. Although, the concepts mentioned in your comment are correct, our objective in conducting this review was precisely the view of somaclonal variation as a tool to promote genetic diversity useful to plant breeding programs, meaning that the different protocols adopted, mostly with the supplementation of chemical agents, should be shown,  even for providing data for queries from other researchers who wish to use somaclonal variation to obtain improved crops.  Manuscripts that covered somaclonal variation in the field, or reports of useful variations from in vitro culture without the use of chemical agents, were not found nor inserted in our systematic review.

Other remarks:

Line 27: I would recommend to use the full terms "plant growth regulator" and "plant growth regulators" and their abbreviations, PGR and PGRs, respectively, instead of “the plant regulator” or “the plant regulators”

Changes were made throughout the text accordingly.

Lines 64-56: I would never say that the somaclonal variation induction technique is one of the methods used for tissue culture. Somaclonal variation is a phenomenon, which occurs in tissue culture but it is rather unpredictable, although we know the factors that promote it. 

New clearer sentences were inserted in the text.

Line 162. The abbreviation SR should be introduced at the first use of a phrase “systematic review” and used consistently later in the text, without repeating the abbreviated phrase in full.

Changes were made throughout the text but we opted to use the term “systematic review” in full in figure legends and table titles since they should be self-explanatory.

Line 179: It should be "In Vitro Cellular and Developmental Biology - Plant", instead of "Developmental Biology". These are two completely different journals.

Changes were made accordingly.

Line 206: what kind of plants?

This was a translation error which we corrected.

Figure 3: South Korea should be indicated on the map, instead of North Korea.

This information was added to the map.  

Line 208 (Figure 3 description): To be precise - it is not the frequency of cultures created, but the number of articles concerning somaclonal variation published in the last 16 years and the main species being the objects of the study.

Change made accordingly in the text.

Lines 209 and 212; not frequency, but numbers

Change made accordingly in the text.

Line 215: It should be “A lower number of somaclones was generated for other crops”, instead of „Other crops generated a lower number of somaclones”

Change made accordingly in the text.

Line 217: „Only articles that made clear…” - This information should be moved to Material and Methods

Change made accordingly in the text.

Line 220: “studies included”, not “studiesincluded”

Change made accordingly in the text.

Line 229: “The largest number of studies was aimed at generating somaclones for the 229 molecular study of genetic diversity among clones” – I rather doubt in such an aim, in my view the reverse is true, i.e. the somaclones were generated (deliberately or not) and the molecular studies were then undertaken to evaluate the variation / genetic diversity generated.

Change made accordingly in the text.

Lines 242-145: The articles do not use plant growth regulators, they just report the use, please, correct this.

Change made accordingly in the text.

Lines 250-252: ”KIN and IBA were used for the evaluation of molecular and agronomic traits, TDZ was used for molecular evaluations of genetic diversity “ - this is a mental shortcut, please rewrite this, because plant growth regulators are not used for the evaluations. They are used to induce somaclonal variation, which can be then evaluated.

Changes made accordingly in the text.

Line 264: “between the regulators”, not “between the regulator”

Change made accordingly in the text.

Line 266: “BAP regulator” – Please, do not repeat the word “regulator” after the abbreviation of a particular auxin or cytokinin, it’s a redundancy.

Change made accordingly in the text.

Lines 267-268: There are no verbs in these sentences. Please, introduce them or join these sentences with the previous one, concerning BAP.

Change made accordingly in the text.

Line 275: It should be “1-naphthaleneacetic acid” or “α-naphthaleneacetic acid” instead of “A-naphthalene acetic acid”

Change made accordingly in the text.

Lines 300-303: The Latin names of genus or species should be written in italics.

Change made accordingly throughout the text.

Lines 300-303: “The crops with the most reports...” - This sentence is unclear, please rewrite it. The crops can not include crops.

Changes made accordingly in the text for better understanding.

Line 303: “Leaves, seeds and rhizomes were used as explant sources” - It is also unclear whether the leaves, seeds and rhizomes were used as explants or they were the source of explants? Please, specify this. 

We made clear in the text that leaves, seeds and rhizomes were used as sources of explants.

Lines 347-348: The sentence "In the last year, only analyses applying single nucleotide polymorphism (SNPs) markers have been reported" sounds much better.

Change made accordingly in the text.

Lines 349-352: I do not understand these two sentences. Generally speaking, all molecular markers are used for detection of somaclonal variation. Depending on the mechanisms behind somaclonal variation (e.g. DNA methylation or DNA sequence changes) we use different markers to identify them.

Paragraph was re-written for better understanding. 

Line 361: the phrase "genes related to somaclonal variation" is a mental shortcut. 

Change made accordingly in the text.

Line 362: It should be "expression of genes related to" instead of "gene expression related to".

Change made accordingly in the text.

Line 365: It should be "the size of each gene name indicates the number of articles that described expression of this gene" instead of "the size of each word indicates the frequency of articles that obtained gene expression"

Change made accordingly in the text.

Line 369: "this gene evaluated dwarphism" -> "this gene was related to dwarfism"

Change made accordingly in the text.

Line 370: The genes do not have the function of coding genes! Please correct this.

Change made accordingly in the text.

Line 372: The genes are not expressive, they just can be expressed.

In this sentence we were referring to the size of the gene (word) in the word cloud but we understand that the word “expressive” was not used correctly and this was change in the text.

Line 373: The genes do not have any sensor domains.

Change made accordingly in the text.

Lines 418-419: "The changes described in the articles initially involved genetic diversity analysis…" - this sentence is unclear.

Change made accordingly in the text.

Lines 478-479: As I have already mentioned, irradiation is not a method of somaclonal variation induction. It is used for mutagenesis.

We agree with your observation and all data regarding manuscripts that report the use of mutagenesis were removed from our systematic review.

Lines 495-497: The number of subcultures is not a method, but a factor, which has an important effect on somaclonal variation.

Change made accordingly in the text.

Line 506: Please, provide the name of the species for these cultivars.

Change made accordingly in the text.

Line 507-508: "In this study we identified that the stem apices were…" -> "According to the literature studied, the stem apices were the most popular explants for induction of somaclonal variation in banana".

Change made accordingly in the text.

Line 512: " The successful use of seeds as explants…" - I do not understand this sentence and the articles cited do not support this strange thesis.

Change made accordingly in the text and references added.

Lines 517-520: "The studies that used the irradiation method to generate mutants were not included in this review; only the studies in which irradiation generated somaclonal mutants were included…" - Can you explain the difference between irradiation that generates mutants and irradiation that generates somaclonal mutants? 

We understand that irradiation generates mutations. In the case of somaclonal variation the most frequent modifications are related to DNA methylation, however, although rarer, mutations related to the sequence of base pairs can also occur during the phenomenon of somaclonal variation. However, as suggested, we removed from our analysis the data related to the articles that report the use of mutagenesis and, therefore, this will not be discussed in depth in our systematic review.

Line 522: The reference with the number 35 describes only Zoysia matrella mutagenesis.

This was an error – changes were made accordingly.

Line 536: "...these variations occur in all cultures subjected to in vitro condition" - this is not true, somaclonal variation can occur, but does not have to

Correction made accordingly.

Line 557: "Many studies showed that…" - if many studies showed something, a few examples (i.e. appropriate citations) should be provided.

References provided in the text.

Line 569: The morphological changes can not be focused on plants. They can be observed in plants. The studies on ornamentals can be focused on induction and identification of such morphological changes.

Change made accordingly in the text.

Lines 612-614. I can not agree. Classical AFLP analysis does not detect DNA methylation. Only modifications like MSAP or metAFLP, in which a pair of isoschizomers is used, and one of these enzymes is methylation-sensitive, can do that. Also, contrary to RAPD or ISSR, larger amounts of DNA are needed for AFLP analysis, because a restriction digestion has to be conducted in its first step, not PCR. 

Changes made accordingly in the text.

Lines 615-617: This sentence is incomprehensible.

Change made accordingly in the text.

Line 643: "The study of Lee et al. [83] also used gene expression analysis…." -> "In the study of Lee et al. [83], gene expression analysis was used…"

 Change made accordingly in the text.

 

Some comments to supplementary tables:

Table S1

It should be “Prunus spp.” instead of “Prunnus spp.” 

Change made accordingly to table SI.

Table S2

It should be “genetic diversity” instead of “genetical diversity” 

Change made accordingly to table S2.

Please, do not format the whole text under the table S2 as superscript because it makes it unreadable.

Also, please, unify the notation for more than 1 molecular marker, because it is unclear why some of them were written like “RAPD e ISSR”, some of them like “RAPD, ISSR” and the other like “RAPD/ISSR”

Change made accordingly to table S2.

Table S3

Please, change the title of the table, because genes cannot be associated with the strategies. You may use the phrase like "Genes associated with somaclonal variation in different crops".

Change made accordingly to table S3.

The column called “Function” contains confusing substance. Please, decide whether the function of a particular gene will be provided here or maybe something more like the reason of analyzing such genes. ”Uncovering possible dwarfing mechanisms” or “To evaluate 'Guijiao 9' resistance genes” are not the gene functions.

Change made accordingly to table S3.

Reviewer 2 Report

Dear Authors,

The presented review encompasses comprehensive information concerning somaclonal variation, understood as variation stably inherited during the generative cycle. The data arrangement gives good insight into where who, and what was studied concerning the issue.
Please consider including the "tissue culture-induced variation" term in your search criteria for a better arrangement of the issue. For sure, this would extend your database significantly. Furthermore, more important directions will be discussed by you (including albinism, the role of metal ions in variation induction, biochemical aspects of the phenomenon, as well as attempts to discover phenomena forming the background for SV and TCIV.

Sincerely

Author Response

Dear Authors,

The presented review encompasses comprehensive information concerning somaclonal variation, understood as variation stably inherited during the generative cycle. The data arrangement gives good insight into where who, and what was studied concerning the issue.
Please consider including the "tissue culture-induced variation" term in your search criteria for a better arrangement of the issue. For sure, this would extend your database significantly. Furthermore, more important directions will be discussed by you (including albinism, the role of metal ions in variation induction, biochemical aspects of the phenomenon, as well as attempts to discover phenomena forming the background for SV and TCIV.

Sincerely

We kindly inform you that our search criteria was made as recommended in the systematic review methodology after many test cycles in which various forms and combinations of “terms” were tested in different databases and with the participation of researchers with the necessary knowledge of the theme and its updates. Regardless, we performed a test by inserting the suggested term into our strategy and noticed that in some databases the number of items found reduced by half compared to the number found in our original search. Thus, we consider the term "tissue culture-induced variation" to be extensive and very restrictive to compute a search string with the proposed theme.

Reviewer 3 Report

-          The introduction is too long and very confusing. Some terms are inappropriate explained (i.e cisgenesis).

-          The purpose of the article is not very clear: is it a review regarding the role of somaclonal variability for the genetic improvement of plants or is it a presentation of a methodology for creating a review-type article?

-          There are too many graphs and tables showing the "statistical" results of querying different databases. The use of the gene cloud image seems inappropriate.

-          The chapter "Discussions" repeats most of the information presented in the previous chapters.

Author Response

The introduction is too long and very confusing. Some terms are inappropriate explained (i.e cisgenesis).

As noted, we made  corrections in many paragraphs of the introduction, with substantial changes to make it shorter and clearer, removing some terms and inserting appropriate references.

        The purpose of the article is not very clear: is it a review regarding the role of somaclonal variability for the genetic improvement of plants or is it a presentation of a methodology for creating a review-type article?

We seek to make our objective clear when conducting this systematic review. We believe that our method was very well described. A systematic review aims to answer a main research question and it is clearly described initially in the abstract, in lines 18-20, and later in the material and methods, which already gives clear the objective of the work. The methodology of systematic review for several areas of knowledge is already well disseminated and known in the field of scientific research. We only mentioned the use of a software, which was reported in other studies of this research group (https://doi.org/10.3390/jof7040249 https://doi.org/10.3389/fpls.2021.657916 https://doi.org/10.3390/plants12020305. Perhaps you can give us an idea of which locations in the manuscript this could be clearer.

       There are too many graphs and tables showing the "statistical" results of querying different databases. The use of the gene cloud image seems inappropriate.

The number of charts and tables were made to best synthesize the appropriate way to portray the information of a set that comprised 232 articles on the subject. As well as a cloud of words, which is very common in systematic reviews, in order to didactically illustrate the results of the work and the research trend, enabling several audiences to understand the result. Furthermore, our review can be considered a data source for future research, where researchers who wish to use the phenomenon of somaclonal variation as a technique of genetic improvement, will be able to consult for many crops, the protocols that are being adopted and this will facilitate discussions and improvement of the technique, as well as future research.

       The chapter "Discussions" repeats most of the information presented in the previous chapters.

      We have resumed some results to facilitate understanding of the discussion, but after an extensive review of the manuscript to meet the demands of reviewers in general, we believe that the text has improved considerably.

Reviewer 4 Report

The role of somaclonal variation in plant genetic improvement: a

systematic review

 

Thank you,

As ra reviewer I have following suggestions:

I appreciate your efforts, you collected a lot of material, pool up many protocols.

Introduction:

Give some history of somaclonal variation.

Additionally, learn sources of somaclonal variations in plant tissue culture, where they are applied, and techniques to identify them in your cultures.

 

In materials and method and subsequent chapters:

 

-       To mitigate this problem in Regeneration system:

-       The regeneration systems (explants used to regenerate plants) in terms of genetic stability can be arranged in descending order as: micropropagation by preformed structures (such as shoot tips or nodal explants) > adventitiously derived shoots > somatic embryogenesis > organogenesis from callus, cell, and protoplast cultures.

-       you may add these points if possible.

 

 

Good luck

Author Response

The role of somaclonal variation in plant genetic improvement: a

systematic review

 

Thank you,

As ra reviewer I have following suggestions:

I appreciate your efforts, you collected a lot of material, pool up many protocols.

Introduction:

Give some history of somaclonal variation.

Additionally, learn sources of somaclonal variations in plant tissue culture, where they are applied, and techniques to identify them in your cultures.

 

In materials and method and subsequent chapters:

 

-       To mitigate this problem in Regeneration system:

-       The regeneration systems (explants used to regenerate plants) in terms of genetic stability can be arranged in descending order as: micropropagation by preformed structures (such as shoot tips or nodal explants) > adventitiously derived shoots > somatic embryogenesis > organogenesis from callus, cell, and protoplast cultures.

-       you may add these points if possible.

 

 

Good luck

 

We inserted, as suggested, a brief history of somaclonal variation in the introduction, as well as the additional information requested in this session. Regarding the placements related to the mitigation of problems in the regeneration system in terms of genetic stability, this is not the focus of our study.  On the contrary, we tried to positively expose what happens when there is genetic variability in the in vitro cultivation process, and that it can be applied as a tool for plant breeding. We do not understand whether, in fact, this was the question, and therefore we ask that if necessary, that this comment be better explained, in order to meet and improve our study.

Round 2

Reviewer 1 Report

Unfortunately, I cannot recommend this manuscript for publication. The corrected review still includes a pool of articles which describes induced mutations and polyploidy (44, 64, 71, 81, 97, 104, 131, 162, 176, 191, 212, 253, 255) in the bibliography. As I have already mentioned in the first review, the chemical agents which have proven action as mutagens or inhibitors of mitosis, and do not have any other impact in plant tissue culture (e.g. they are not used as plant growth regulators, carbon sources, sources of macro- and micronutrients, vitamins, osmotic regulators or gelling agents), should not be considered as a source of somaclonal variation. These include substances such as ethyl methanesulfonate (EMS), methyl methanesulfonate (MMS), N-nitroso-N-methyl urea (NMU), sodium azide (SA), and sodium nitrite (SN), as well as hypomethylation and mutagenic agent 5-Azacytidine, and polyploidy inducers like colchicine and trifuralin. If the Authors want to review different protocols that promote the enhancement of genetic diversity useful in plant breeding programs with the use of such chemicals, they should not entitle such review "The role of somaclonal variation in plant genetic improvement", because such title misleads the readers.

Also, although Authors insist that they have removed all articles that describe the induction of mutations by means of physical agents and thus they have reduced the number of accepted articles from 249 to 232, they have left two papers which report the use of gamma rays and ion beam irradiation. And the rest of the above-mentioned papers have been removed only from the list of references and from the tables, whereas all the column and pie charts remained exactly the same as before and some of their description still refers to 249 accepted articles (lines 251, 267, 286, 339). Also, the digital library contains the same set of articles as previously described (line 153) and the number 249 is cited in the chapters “Results” (line 159), “Discussion” (line 385), and “Conclusions” (line 637) as well as in title of the Table S4 (line 687). The irradiation as a method of induction of somaclonal variation is mentioned in the section 3.4, whereas the chemical mutagens are not mentioned at all, despite the fact that the papers reporting their use have not been rejected (lines 215-217).

The Authors also insist that they have made all appropriate changes concerning the use of the terms “plant growth regulator” and “plant growth regulators as well as their abbreviations, PGR and PGRs, but such changes have been implemented only in lines 27 and 234. The terms “plant regulator” and “plant regulators” still remain in many other places (lines: 77, 216, 217, 248, 264, 456, 461, 474, 478, 507, 526, 655, 658, and Table 1). The abbreviation PGR has been introduced in the Abstract but it has never been used again.

The journal title “In Vitro Cellular and Developmental Biology – Plant” has been corrected only in the text (Line 168), but the Figure 2 still contains the name developmentalbiology plant.

Despite assurances, the remarks that “genes do not have the function of coding gene” and “they do not have any sensor domains” have not been corrected (lines 349 and 353).

Line 258: The sentence “For 2,4-D, 2 mg/l (26), 1 mg/l (19) and 3 mg/l (13) (Figure 8)” still does not have the verb.

There is a paragraph before the Figure 8, which is strongly centered.

Lines 337-340: This sentence has not been written in English.

Lines 354-355: Instead of “…NPR1 genes that express signaling of the salicylic acid hormone (SA) and play an essential role in plant immunity”, it should be: “…NPR1 genes which function as a master regulator of the plant hormone salicylic acid (SA) signaling and play an essential role in plant immunity”

 

Author Response

Comments and suggestions to authors

Unfortunately, I cannot recommend this manuscript for publication. The corrected review still includes a pool of articles which describes induced mutations and polyploidy (44, 64, 71, 81, 97, 104, 131, 162, 176, 191, 212, 253, 255) in the bibliography. As I have already mentioned in the first review, the chemical agents which have proven action as mutagens or inhibitors of mitosis, and do not have any other impact in plant tissue culture (e.g. they are not used as plant growth regulators, carbon sources, sources of macro- and micronutrients, vitamins, osmotic regulators or gelling agents), should not be considered as a source of somaclonal variation. These include substances such as ethyl methanesulfonate (EMS), methyl methanesulfonate (MMS), N-nitroso-N-methyl urea (NMU), sodium azide (SA), and sodium nitrite (SN), as well as hypomethylation and mutagenic agent 5-Azacytidine, and polyploidy inducers like colchicine and trifuralin. If the Authors want to review different protocols that promote the enhancement of genetic diversity useful in plant breeding programs with the use of such chemicals, they should not entitle such review "The role of somaclonal variation in plant genetic improvement", because such title misleads the readers.

Dear reviewer, once again we would like to thank you for your comments and corrections for the improvement of our manuscript. We understand their placement in relation to the performance of these reagents as methods of promoting genetic variability and not specifically as sources of somaclonal variation and we chose to accept the exclusion of the articles. Initially we kept these articles to contemplate all data related to the promotion of genetic variability in plant genetic improvement, but we decided to keep the focus of the review only on the genetic variability resulting from somaclonal variation, and no further modifications of the manuscript regarding title and other discussions were necessary, as commented. So, we removed these articles from our analysis and did what was necessary. We would like to note that this does not always imply the change of some frequencies in the charts, because the amount of articles removed is not enough to change the general data as main countries, cultures…etc.

Also, although Authors insist that they have removed all articles that describe the induction of mutations by means of physical agents and thus they have reduced the number of accepted articles from 249 to 232, they have left two papers which report the use of gamma rays and ion beam irradiation. And the rest of the above-mentioned papers have been removed only from the list of references and from the tables, whereas all the column and pie charts remained exactly the same as before and some of their description still refers to 249 accepted articles (lines 251, 267, 286, 339). Also, the digital library contains the same set of articles as previously described (line 153) and the number 249 is cited in the chapters “Results” (line 159), “Discussion” (line 385), and “Conclusions” (line 637) as well as in title of the Table S4 (line 687). The irradiation as a method of induction of somaclonal variation is mentioned in the section 3.4, whereas the chemical mutagens are not mentioned at all, despite the fact that the papers reporting their use have not been rejected (lines 215-217).

All specifications were taken into consideration in the text as well as tables and figures.

The Authors also insist that they have made all appropriate changes concerning the use of the terms “plant growth regulator” and “plant growth regulators as well as their abbreviations, PGR and PGRs, but such changes have been implemented only in lines 27 and 234. The terms “plant regulator” and “plant regulators” still remain in many other places (lines: 77, 216, 217, 248, 264, 456, 461, 474, 478, 507, 526, 655, 658, and Table 1). The abbreviation PGR has been introduced in the Abstract but it has never been used again.

Dear reviewer, there has been a failure to understand your corrections. We had thought that these modifications were satisfactory only in the indicated places, but we carried out a thorough review of the manuscript and included the abbreviations throughout the text. We hope to meet this request and improvement of our SR.

The journal title “In Vitro Cellular and Developmental Biology – Plant” has been corrected only in the text (Line 168), but the Figure 2 still contains the name developmental biology plant.

Changes made accordingly after new analysis of the figure.

Despite assurances, the remarks that “genes do not have the function of coding gene” and “they do not have any sensor domains” have not been corrected (lines 349 and 353).

 

Inserted in the manuscript

Line 258: The sentence “For 2,4-D, 2 mg/l (26), 1 mg/l (19) and 3 mg/l (13) (Figure 8)” still does not have the verb.

There was an error in the translation and this correction was inserted in the text.

There is a paragraph before the Figure 8, which is strongly centered.

Formatting carried out accordingly.

Lines 337-340: This sentence has not been written in English.

There was a translation error and has now been correct in the text.

Lines 354-355: Instead of “…NPR1 genes that express signaling of the salicylic acid hormone (SA) and play an essential role in plant immunity”, it should be: “…NPR1 genes which function as a master regulator of the plant hormone salicylic acid (SA) signaling and play an essential role in plant immunity”

Changes made in the text in this section of the manuscript.

Reviewer 3 Report

After the improvements made, I believe that the article can be published in this form.

Author Response

Dear reviewer! Many thanks for the suggestions and corrections. Your contribution was very important to improve the manuscript.