Review Reports - Crucial Factors Influencing the Efficiency of Androgenesis in Oat (Avena sativa L.) Through Anther and Microspore Cultures

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The authors have done a good job of studying the issue of oat androgenesis.

However, there are some remarks on the design of the article and the style of narration:

1) Donor plants growing conditions

This section needs to be rewritten to make it consistent. Source 21, 24 gives information about donor plants growing without light intensity and details about fertilization. In the description of source 25 the intensity is given, on fertilizers it is indicated that seeds were sown in soil mixture with 1 g of fertilizer (for what volume of soil?). In general, it is necessary to systematize the data.

2). Panicle pretreatment

It is not customary to say in an article: I will. The article is a collective work, who of your authors is this “I”? usually write: Over the years, the approach to pretreatment has changed, then some of the most striking examples or want to pay attention to the following examples...without “I” or “we”.

2) Conclusions and future directions.

In the conclusion the authors again provide descriptions of the techniques, these descriptions should be in the relevant sections of the review, but not in the discussion. The discussion should include e.g. contradictions in different methodologies or general conclusions, e.g. which of the published methodologies is the most effective, which points of the methodologies are present in all authors, what the researcher should pay attention to.

In general, the review is not written very well, there are no transitions, no “smooth logical narrative”. It often looks like quotations from other articles without proper transition or commentary.

Author Response

Comments 1:

Donor plants growing conditions

Response 1:

Thank you for poniting this out. I agree with this comment. It is justified to provide more details regarding donor plants according to references: [21, 24, 25]. You can find the correction in the revised: page nr 2 and 3, chapter nr 2, line nr: 94-105.

Comment 2:

2). Panicle pretreatment

Response 2:

I totally agree with you. While checking the article it was my oversight of this error, in scientific papers the passive form is used.

You can find the correction in the revised: pag nr 3, chapter nr 3, line nr: 107-108.

Comment 3:

2) Conclusions and future directions.

Response 3:

In the text, I have included in the chapter “Conclusions” of the manuscript that comprise the following components:

various factors influence the effectiveness of androgenesis, including biochemical and physiological factors of the plant, genotype, nutrients, growth factors, and physicochemical conditions.
the developmental stage of the microspore.
the type and composition of the medium, growth factors – a comparison of media compositions used by different researchers.

The citation from the manuscript is below:

“The first oat regenerants through androgenesis were produced by Rines [17], who used MS medium devoid of hormones but supplemented with 10% sucrose. This method demonstrated the highest frequencies of anther callus initiation among all the media tested. However, successful seedling production was achieved only when anthers were initially placed on a modified potato extract medium containing 2.0 mg L⁻¹ 2,4-D and 0.5 mg L⁻¹ kinetin. Kiviharju et al. [27] demonstrated that adding high concentrations of maltose (10% and 14%) doubled the induction of embryo-like structures (ELS) in A. sativa, but did not result in the regeneration of plants. Subsequent studies highlighted two key media for inducing ELS and plant regeneration: C17 medium [34] and W14 [31], each with varying concentrations of the auxin 2,4-D and the cytokinin- kinetin. Kiviharju et al. [19] discovered that lower concentrations of 2,4-D (2-3 mg L⁻¹) and kinetin (0.2-0.5 mg L⁻¹) in MS and W14 media were adequate for inducing embryo formation. Warchol et al. [24] observed that the greatest number of ELS was formed on W14 medium supplemented with 2.0 mg L⁻¹ 2,4-D and 0.5 mg L⁻¹ kinetin.”

comparison of different pretreatments

The citation is below from the manuscript:

“The efficiency of reprogramming through androgenesis is significantly enhanced by cold pretreatment at 4°C in darkness for 2 or 3 weeks combined with a thermal shock at 32°C for 24 hours [24], compared with another research, cold pretreatment 0, 7, 14, 21 or 28 days at 4°C and next incubation at 32°C for 0, 1, 3, 5 or 7 days [54]. Kiviharju et al. [27] indicated that prolonged cold pretreatment did not enhance the efficacy of oat anther culture. Furthermore, an extended period of cold treatment (6-8 weeks) may result in a high incidence of albino regenerates [27]. Heat pretreatment (32°C) for 5 days, as studied by Kiviharju et al. [54], influenced the type of anther response, with cultured anthers showing embryogenesis, whereas those without heat treatment only produced callus.”

A thorough analysis was conducted based on the available but limited literature due to the of Avena sativa L. to androgenesis. Researchers conducted experiments on various plant lines/genotypes, which exhibited differing levels of recalcitrance in Avena sativa L.

Moreover, the methodology provides specific guidelines and recommendations regarding the most favorable conditions for pretreatment, the optimal choice of culture media, and the application of the most advantageous growth factors.

The scientist can choose the best method according to this manuscript to his/her possibilities in the experiment's reality.

Author Response File: Author Response.docx

Reviewer 2 Report

Comments and Suggestions for Authors

Comments and remarks:

The present manuscript presents a review on methods utilized in the production of oat (Avena sativa L.) double haploids using anther and microspore cultures technics. The attempt carried out to inducing androgenesis in this valuable cereal crop are revised and factors influencing the efficacy of the method are presented and discussed. An alternative in the production of homozygous lines to conventional time-consuming and expensive breeding methods is the production of doubled haploid plants derived from (haploid) male gametophytes or their microspores (androgenesis). However, in the case of oat, this method of plant regeneration remains not very effective compared to the most common cereals, probably due to the known reluctance of this cereal to in vitro culture. Thus, the analysis of the studies carried out so far on the in vitro regeneration of oats is important to improve this method in its further application in the breeding program of this valuable crop.

The manuscript is compiled according to the requirements of "Agronomy" for review articles.

The main question addressed in the review is the efficiency of androgenesis in oat (Avena sativa L.) induced by anther and microspore cultures. It examines the factors influencing the effectiveness of this technique as a method of producing double haploids in oats.

The main use of haploids is in the production of doubled haploids applied in breeding programs to reduce the inbreeding generations required to stabilize a hybrid genotype from 7-8 to just one, thereby reducing costs and improving the efficiency of the selection. The application of this method in oat up to now is not very effective compared to the most common cereals, and the analysis of the studies carried out so far on the in vitro regeneration of oat, accentuating on the factors influencing the efficacy of the method, is important to improve this method in its further application in the breeding program of oat. The key determinants of androgenetic response in in vitro cultures of oat include genotype of donor plants, growth conditions; the nuclear stage of male gametes; anther pretreatment; the composition of the in vitro culture medium and the various physical factors during tissue culture as well as their interactions were analyzed in the manuscript.

The conclusions made are consistent with the facts exposed and the arguments presented.

The references on which the review is based are relevant to the topic of the manuscript.

The research reviewed so far on the discussed topic is adequately summarized and clearly presented, focusing on the point of interest for further research on the problem addressed. In addition, cultivation conditions, which are the main factors affecting in vitro oat cultures, are listed in a table, contributing to the presentation and clarity of the existing data.

The following remarks and recommendations should be made:

Abstract:

- The last sentence of this part: “This review briefly presents the two methods utilized in the production of oat DHs, the factors influencing the acquisition of DH plants, and the efficacy of these methods.” should be redacted because the two methods cited are not specified, and in the main text only one method (androgenesis) is set forth and its cultivation factors are revised. My suggestion is the following: “This review presents the methods through anther and microspore cultures utilized in the production of oat DHs revealing the crucial factors influencing the efficiency of this method in oat (Avena sativa L.).”

Introduction

- The author name (Avena sativa L.) should be puted in line 67: “Oat (Avena sativa L.) belong to …”, and omitted from line 74:” Oat is regarded as …”

- The parts between lines 63-66 should be omitted because repeat the information in line 74

- At the end of this part it should be added a sentence to sieve as introduction to following part and outlaning the aim of review similarly the described in Abstract, as: “A continuation the factors influencing the acquisition of DH plants in oat through anther and microspore cultures are listed, and the efficacy of the method is discussed.”

Conclusions

- The sentence of lines 227-229 redundant because concerns other species /not the target one/.

- - The part between lines 237-253 should be placed after the part ending in line 236, as its continuation, uniting the sentences included in the quoted lines, omitting the new lines

Edit the first paragraph of the quoted part /lines 237-242/ as follows: "The highest frequencies of anther callus initiation were achieved on MS medium supplemented with 10% sucrose and deprived of hormones, with anthers previously introduced on modified medium with potato extract containing 2.0 mg L⁻¹ 2,4-D and 0.5 241 mg L⁻¹ kinetin [17]."

Author Response

Comments 1:

Abstract:

The last sentence of this part: “This review briefly presents the two methods utilized in the production of oat DHs, the factors influencing the acquisition of DH plants, and the efficacy of these methods.” should be redacted because the two methods cited are not specified, and in the main text only one method (androgenesis) is set forth and its cultivation factors are revised. My suggestion is the following: “This review presents the methods through anther and microspore cultures utilized in the production of oat DHs revealing the crucial factors influencing the efficiency of this method in oat (Avena sativa L.).”

Response 1.

Thank you for bringing this to my attention. I have changed the sentence according your modification.

You can find the correction on the page nr 1, chapter: Abstract, lines: 22-24.

Comment 2:

Introduction

- The author name (Avena sativa L.) should be puted in line 67: “Oat (Avena sativa L.) belong to …”, and omitted from line 74:” Oat is regarded as …”

- The parts between lines 63-66 should be omitted because repeat the information in line 74

At the end of this part it should be added a sentence to sieve as introduction to following part and outlaning the aim of review similarly the described in Abstract, as: “A continuation the factors influencing the acquisition of DH plants in oat through anther and microspore cultures are listed, and the efficacy of the method is discussed.”

Response2:

Thank you for your correction. I've changed everything what you mentioned above, in revised manusript I crossed out/deleted the text below:

On the one hand, the production of such valuable traits in one generation would be a great achievement for breeders, on the other hand, among cereal plants, oats are considered oats (Avena sativa L.) are regarded as one of the most recalcitrant cereal crops in the context of doubled haploid production

Oat (Avena sativa L.) belong to the family Poaceae, subfamily Pooideae, and tribe Aveneae[8,13]; they are extensively cultivated in the temperate zones of Eurasia and North America. The genus encompasses both cultivated and wild species, exhibiting variations in ploidy levels and genomic compositions [13].

Haploids of hexaploid oat thus have a 21 chromosome complements consisting of three genomic sets of seven chromosomes [14]. The three genomic chromosome sets in hexaploid oat, designated like a the A, C, and D genomes [15].

Oat is regarded as one of the most recalcitrant cereal crops in the context of DH production.

You can find the correction on the page nr 2, chapter: Introduction, lines: 63-69, and line 74 in the revised manuscript.

Comments 3:

Response 3:

I agree with You, it should be link between "Introduction" and further paragraphs.

I've added your sentence to the end of "Introduction".

The text is below:

Key determinants of the androgenetic response in in vitro culture include the genotype of donor plants, growth conditions; the nuclear stage of male gametes; anther pretreatment; the composition of the in vitro culture medium, and various physical factors during tissue culture, as well as their interactions [23-25].

A continuation the factors influencing the acquisition of DH plants in oat through anther and microspore cultures are listed, and the efficacy of the method is discussed.

You can find the correction on the page nr 2, chapter: Introduction, lines: 91-92 in the revised manuscript.

Comment 4:

Conclusions

- The sentence of lines 227-229 redundant because concerns other species /not the target one/.

Response 4:

Thank you again for your sugestion.

I've checked these lines and references:

[24] includes species: A. sativa - The efficiency of embryogenesis of anther culture was compared using four cultivars of oat (Avena sativa L.): ‘Akt’, ‘Bingo’, ‘Bajka’, and ‘Chwat’.
[27] includes species: A. sativa and A.sterilis
[54] includes species: Anther culture ability was tested for 44 oat (Avena sativa L.), six naked oat (A. sativa L., naked type) and 15 wild oat (Avena sterilis L.) genotypes, in addition to progeny of five intraspecific crosses of A. sativa and two interspecific crosses of A. sativa x A. sterilis.

I think that, these species fit the whole picture perfectly, they give a general overview of oat androgenesis on selected species and cultivars.

Comment 5:

- The part between lines 237-253 should be placed after the part ending in line 236, as its continuation, uniting the sentences included in the quoted lines, omitting the new lines.

Response 5:

Thank You for your suggestion, but I think that it's better to use a new paragraph.

Between the lines 238-240 is comparison with the pretreatment of species H.vulgare, and from the lines 238-240, it is inclued completely different subject - a crucial role of medium (ingradients), that's why I think is better stayed construction of chapter "Conclusions" with not corrected the paragraphs.

Comment 6:

Response 6:

Thank you for your comment. You have a better idea to rebild the sentences.

The corrected text is below:

The first oat regenerants through androgenesis were produced by Rines [17]. The highest frequencies of anther callus initiation were achieved on MS medium supplemented with 10% sucrose and deprived of hormones, with anthers previously introduced on modified medium with potato extract containing 2.0 mg L⁻¹ 2,4-D and 0.5 mg L⁻¹ kinetin [17]."

You can find the correction on the page nr 7, chapter: Conclusions, lines: 249-252 in the revised manuscript.

Author Response File: Author Response.docx

Reviewer 3 Report

Comments and Suggestions for Authors

The manuscript entitled “Crucial factors influencing the efficiency of androgenesis in oat 2 (Avena sativa L.) through anther and microspore cultures” focuses on Oat's DH production. Several parts are summarized more comprehensively. Following are specific suggestions

1. The use of spaces in Tabel 1 is inappropriate; please pay attention to the modification. For example, ‘MS 26,1 ±3,’and ‘W14 33,6± 9’。

2. The reference format is incorrect; please pay attention to the correction. For example, Line 312，Line 340， Line341，Line363 Line 373,Line 381.

3. About the preamble. The authors emphasize the rapid breeding approach, mentioning DH and microspore culture. However, there is no mention of parthenogenesis, etc. Why? Is it because the advantages of parthenogenesis are more pronounced, or are the disadvantages of other methods more significant? It would be more logical to add the answer to this question in the preamble.

4. There is a lack of discussion on the vitality and health of DH offspring. Write a separate part of the statement of the life force of the progeny produced in different ways.

5. Some of the contents of 'Conclusions and Future Directions' are highly repetitive and slightly redundant with the previous chapters. It would be better to be a little more streamlined. The authors say improving the method further to achieve reproducibility is good. However, the authors do not present their opinions on androgenesis; they simply collect and organize the data.

6. It's better to add/cite more recent literature in this area.

Author Response

Comment 1:

The use of spaces in Tabel 1 is inappropriate; please pay attention to the modification. For example, ‘MS 26,1 ±3,’and ‘W14 33,6± 9’。

Response 1:

Thank you for pointing this out, I have already corrected it and marked it in the text.

You can find the correction in the revised: in the table 1, page nr 5.

Comment 2:

The reference format is incorrect; please pay attention to the correction. For example, Line 312，Line 340， Line341，Line363 Line 373,Line 381.

Response 2:

Thank you for pointing this out, I have already corrected all references.

Comment 3:

About the preamble. The authors emphasize the rapid breeding approach, mentioning DH and microspore culture. However, there is no mention of parthenogenesis, etc. Why? Is it because the advantages of parthenogenesis are more pronounced, or are the disadvantages of other methods more significant? It would be more logical to add the answer to this question in the preamble.

Response 3:

In the introduction, I deliberately focused on oat (Avena sativa L.) androgenesis, specifically through the application of anther and microspore cultures, while intentionally omitting other methods of obtaining doubled haploids (DH), such as gynogenesis and wide crossing. Gynogenesis in oats is a highly inefficient method, whereas wide crossing demonstrates greater efficiency. Numerous publications are dedicated to wide crossing in oats, providing the scientist with ample information on this subject.

Mentioning about these methods would require expanding the scope of the discussion, even if only briefly, which I think could detract from the overall focus on androgenesis in oats— the central subject of the manuscript.

Comment 4:

There is a lack of discussion on the vitality and health of DH offspring. Write a separate part of the statement of the life force of the progeny produced in different ways.

Response 4:

Thank you for your suggestion.

The topic is discussed in several places in the manuscript, after your suggestions, I also included additional information in the text.

It was included in the following chapters, indicated on the green and albino regenerants (the vitality and health):

“Introduction” line 84-85.
“Media composition and growth factors” line 165-166 – I added extra information about DH offspring according to your sugestion.
“Efficiency of androgenesis” line 207-209 - I added extra information about DH offspring according to your sugestion.

I added extra vers in the table nr 1, it highlighted.

Comment 5:

Some of the contents of 'Conclusions and Future Directions' are highly repetitive and slightly redundant with the previous chapters. It would be better to be a little more streamlined. The authors say improving the method further to achieve reproducibility is good. However, the authors do not present their opinions on androgenesis; they simply collect and organize the data.

Response 5:

We have included in the chapter “Conclusions and future directions” of the manuscript that comprise the following components:

various factors influence the effectiveness of androgenesis, including biochemical and physiological factors of the plant, genotype, nutrients, growth factors, and physico-chemical conditions.
the developmental stage of the microspore.
comparison of different pretreatments

The citation is below from the manuscript:

“The efficiency of reprogramming through androgenesis is significantly enhanced by cold pretreatment at 4°C in darkness for 2 or 3 weeks combined with a thermal shock at 32°C for 24 hours [24], compared with another research, cold pretreatment 0, 7, 14, 21 or 28 days at 4°C and next incubation at 32°C for 0, 1, 3, 5 otr 7 days [54]. Kiviharju et al. [27] indicated that prolonged cold pretreatment did not enhance the efficacy of oat anther culture. Furthermore, an extended period of cold treatment (6-8 weeks) may result in a high incidence of albino regenerates [27]. Heat pretreatment (32°C) for 5 days, as studied by Kiviharju et al. [54], influenced the type of anther response, with cultured anthers showing embryogenesis, whereas those without heat treatment only produced callus."

the type and composition of the medium, growth factors – a comparison of media compositions used by different researchers.

The citation from the manuscript is below:

“The first oat regenerants through androgenesis were produced by Rines [17], who used MS medium devoid of hormones but supplemented with 10% sucrose. This method demonstrated the highest frequencies of anther callus initiation among all the media tested. However, successful seedling production was achieved only when anthers were initially placed on a modified potato extract medium containing 2.0 mg L⁻¹ 2,4-D and 0.5 mg L⁻¹ kinetin. Kiviharju et al. [27] demonstrated that adding high concentrations of maltose (10% and 14%) doubled the induction of embryo-like structures (ELS) in A.sativa, but did not result in the regeneration of plants. Subsequent studies highlighted two key media for inducing ELS and plant regeneration: C17 medium [34] and W14 [31], each with varying concentrations of the auxin 2,4-D and the cytokinin- kinetin. Kiviharju et al. [19] discovered that lower concentrations of 2,4-D (2-3 mg L⁻¹) and kinetin (0.2-0.5 mg L⁻¹) in MS and W14 media were adequate for inducing embryo formation. Warchol et al. [24] observed that the greatest number of ELS was formed on W14 medium supplemented with 2.0 mg L⁻¹ 2,4-D and 0.5 mg L⁻¹ kinetin.”

comparison of different pretreatments

The citation is below from the manuscript:

“The efficiency of reprogramming through androgenesis is significantly enhanced by cold pretreatment at 4°C in darkness for 2 or 3 weeks combined with a thermal shock at 32°C for 24 hours [24], compared with another research, cold pretreatment 0, 7, 14, 21 or 28 days at 4°C and next incubation at 32°C for 0, 1, 3, 5 otr 7 days [54]. Kiviharju et al. [27] indicated that prolonged cold pretreatment did not enhance the efficacy of oat anther culture. Furthermore, an extended period of cold treatment (6-8 weeks) may result in a high incidence of albino regenerates [27]. Heat pretreatment (32°C) for 5 days, as studied by Kiviharju et al. [54], influenced the type of anther response, with cultured anthers showing embryogenesis, whereas those without heat treatment only produced callus.”

Considering these aspects and the diversity of the experiments, it seems reasonable to leave the choice of optimal conditions for specific experiments to the researcher.

Comment 6:

It's better to add/cite more recent literature in this area.

Response 6:

Currently, the literature from androgenesis of Avena sativa is limited, with more studies available on androgenesis using anther cultures than isolated microspore cultures (only two).

The latest article is:

Pathi K.M; Thorben S. From Patri Dish to Field: Plant Tissue Culture and genetic engineering of oats for improved agricultural outcomes. Plants. 2023, 12 (10), 3872.
Warchol M.; Skrzypek E.; Juzoń-Sikora K.; Dragana Jakovlevic. Oat (Avena sativa L.) in vitro cultures: prospects and challenges for breeding. Agronomy 2023, 13(10), 2604.

The knowledge from these articles is included in this manuscript as well.

Author Response File: Author Response.docx

Reviewer 4 Report

Comments and Suggestions for Authors

Dear Authors,

Thank you for submitting your manuscript titled “Crucial factors influencing the efficiency of androgenesis in oat (Avena sativa L.) through anther and microspore cultures.” Overall, this review provides a comprehensive and in-depth discussion of androgenesis techniques for oat, with a clear structure and extensive literature citations, making it suitable for researchers in the field. However, I recommend further improvements and detailed analyses in certain key areas to enhance the quality and practical applicability of the study.

Major Revision Suggestions:

1. Supplement Data Support: Include more tables and figures to visually represent the effects under different experimental conditions, enhancing the credibility of the results.

2. Enhance Methodological Details: Add specific experimental steps in each section, particularly when describing the composition and treatment conditions of various media, providing necessary information on time, temperature, and concentrations.

3. Strengthen the Conclusion: Revise the conclusion to clearly indicate the main challenges facing current technology and possible solutions, emphasizing future research prospects and suggesting key areas for researchers to focus on.

Wishing you success in your research endeavors.

Best regards,

Reviewer

Author Response

Thank you very much for your good opinion about the manuscript.

In response to your suggestions:

Comment 1:

Supplement Data Support: Include more tables and figures to visually represent the effects under different experimental conditions, enhancing the credibility of the results.

Response1:

In the manuscript I presented only one summary table, it contains only a few examples of the effectiveness of androgenesis. I have deliberately included few examples, which reflect the nature of the process and allow for the clarity and transparency of androgenesis in A. sativa L.

I agree with you that article should contain more tables and figures, this would make the article seem more attractive and interesting, but at the moment the construction of the manuscript would be shaken.

Would you agree to leave only one collective table in the article?

Comment 2:

Enhance Methodological Details: Add specific experimental steps in each section, particularly when describing the composition and treatment conditions of various media, providing necessary information on time, temperature, and concentrations.

Response2:

I have tried to include all relevant information regarding time, temperature, and concentrations when necessary.

Please provide specific examples from the text, where I should write more details.

Comment 3:

Strengthen the Conclusion: Revise the conclusion to clearly indicate the main challenges facing current technology and possible solutions, emphasizing future research prospects and suggesting key areas for researchers to focus on.

Response3:

Thank you for your suggestion.

I have included in the chapter “Conclusions and future directions” of the manuscript that comprise the following components:

various factors influence the effectiveness of androgenesis, including biochemical and physiological factors of the plant, genotype, nutrients, growth factors, and physico-chemical conditions.
the developmental stage of the microspore.
comparison of different pretreatments
the type and composition of the medium, growth factors – a comparison of media compositions used by different researchers.

The conclusions compare the methodologies and indicate which method is the most effective, both in terms of pretreatment and the use of media and growth factors.

To the last paragraph in the “Conclusions” I extra added the text below:

To improve the efficiency of androgenesis, further research is needed to standardize the methodology for a specific A. sativa L. variety. The constant change of the donor plant genotype does not allow for the optimization of the experiment. The work indicates what factors influence the success of androgenesis and the researcher has to choose the best cultivation conditions.

Author Response File: Author Response.docx

Reviewer 5 Report

Comments and Suggestions for Authors

The idea of the manuscript is good and data is well presented. I have following minor suggestions

'Conclusions and Future Directions' section need to improve. Authors should make a conclusion based on the information presented in this article, and then recommend future directions/suggestions. There are too many paragraphs (few sentences in each paragraph). Its better to mention as 2-3 paragraphs with detail information.

References./citations need to re-check

Author Response

Comments 1:

Response 1:

Thank you very much for your high rating and suggestions.

In the chapter "Conclusions and Future Directions" I reduced the number of paragraphs to improve the coherence of the chapter.

Comments 2:

References./citations need to re-check

Response 2 :

Thank you very much for your suggestions.

According to your recommendation, I have checked all the references and made corrections.

Author Response File: Author Response.docx

Round 2

Reviewer 3 Report

Comments and Suggestions for Authors

Authors have done the suggested changes, so ms could be accepted for publication

Author Response

Comment:

Authors have done the suggested changes, so ms could be accepted for publication.

Response:

Thank you very much for your acceptance.

In the attachment is a the latest revised version of the manuscript.

Reviewer 4 Report

Comments and Suggestions for Authors

Dear Authors,

Major Revision Suggestions:

1. The manuscript presents comprehensive information but could benefit from clearer organization. Each section should begin with a concise overview of the key points discussed to guide the reader.

2. While the background is informative, I suggest expanding the literature review on recent advancements in androgenesis techniques, especially in relation to oats, to provide a more robust context. In addition, Please carefully standardize the format of each reference, for example, reference 17 (line 321), 19(line 326), 25(line 339) and 50(line 392)are not formatted correctly.

3. It would be beneficial to discuss the limitations of current methods in more depth, particularly the challenges faced in achieving high efficiency in oat DH production.

4. The conclusion section should highlight specific future research directions or innovative techniques that could be explored to overcome current limitations.

Wishing you success in your research endeavors.

Best regards,

Reviewer

Author Response

Comment 1

The manuscript presents comprehensive information but could benefit from clearer organization. Each section should begin with a concise overview of the key points discussed to guide the reader.

Response 1

Thank you very much for bringing this to my attention.

I’ve added some sentences to the beginning of each chapter, with a concise overview of the key points discussed to guide the reader (it's marked in violet color).

In the chapter: “Media composition and growth factors” is to emphasize the importance of used medium to the in-vitro culture. “The composition of the culture medium is a key factor influencing the formation of embryo-like structures (ELS). Numerous studies have evaluated various components of media, such as carbon sources, macronutrients, micronutrients, and the type and concentration of growth regulators, which can decisively influence pollen embryogenesis [25,31-33]”.

Comment 2

While the background is informative, I suggest expanding the literature review on recent advancements in androgenesis techniques, especially in relation to oats, to provide a more robust context. In addition, Please carefully standardize the format of each reference, for example, reference 17 (line 321), 19(line 326), 25(line 339) and 50(line 392)are not formatted correctly.

Response 2

Currently, the literature from androgenesis of Avena sativa is limited, with more studies available (for example available on the website: www.webofscience) on androgenesis using anther cultures than isolated microspore cultures (only two).

The latest article is:

Pathi K.M; Thorben S. From Patri Dish to Field: Plant Tissue Culture and genetic engineering of oats for improved agricultural outcomes. Plants. 2023, 12 (10), 3872.
Warchol M.; Skrzypek E.; Juzoń-Sikora K.; Dragana Jakovlevic. Oat (Avena sativa L.) in vitro cultures: prospects and challenges for breeding. Agronomy 2023, 13(10), 2604.

The knowledge from these articles is included in this manuscript as well.

The references have checked again and corrected.

Comment 3

It would be beneficial to discuss the limitations of current methods in more depth, particularly the challenges faced in achieving high efficiency in oat DH production.

Response 3

Thank You very much for your suggestion. I added extra information in the chapter “Conclusions and future perspectives”, it’s marked in the violet color.

Comment 4

The conclusion section should highlight specific future research directions or innovative techniques that could be explored to overcome current limitations.

Response 4

Thank You very much for your suggestion. I added extra information in the chapter “Conclusions and future perspectives”, it’s marked in the pink color.