Contemporary Advances and Future Perspectives in Rosaceae Plant Regeneration

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The manuscript titled “Contemporary Advances and Future Perspectives in Rosaceae Plant Regeneration”, with Qi Zang et al., provides a comprehensive review of conventional and emerging plant regeneration strategies in economically and ornamentally valuable Rosaceae species. The objective is to evaluate biological and technical barriers in regenerative capacity and to explore how molecular biotechnology, genome editing, and epigenetic mechanisms can enhance regeneration efficiency. The scope spans across various plant types in the Rosaceae family—including apples, pears, strawberries, and roses—emphasizing genotype-specific responses, explant types, environmental factors, and hormonal regulation. The most compelling finding is the proposal of integrating gene-editing technologies (e.g., CRISPR/Cas9) with tissue culture-independent transformation systems as a forward-looking strategy to overcome recalcitrant regeneration challenges in woody Rosaceae species.

This manuscript is likely to have a meaningful impact in the fields of horticultural biotechnology and plant tissue culture. By compiling dispersed findings into a single, detailed resource, it will serve as a reference point for future empirical research and practical applications in Rosaceae regeneration. The discussion on integrating gene regulatory networks, epigenetics, and genome editing broadens its relevance beyond Rosaceae, potentially influencing research in other recalcitrant plant families. The manuscript discusses genotypic variability, plant growth regulators, explant types, environmental conditions, and molecular aspects.

However, the manuscript does exhibit some weaknesses. Despite its comprehensive coverage, the review lacks a strong critical lens when discussing limitations in applying advanced technologies such as CRISPR or non-tissue culture methods to Rosaceae species; the discussion is largely speculative without acknowledging current feasibility barriers. Additionally, minor grammatical inconsistencies and some overly long sentences may hinder clarity for non-native readers. Furthermore, certain figures (e.g., Figure 1 on page 3) lack detailed legends necessary for standalone comprehension.

One critical omission in this otherwise comprehensive review is the lack of discussion on in vitro maintenance of explants under low-sucrose conditions and enhanced gas exchange, which has become a highly relevant topic in modern micropropagation research. Recent studies have emphasized that reducing exogenous carbohydrate supplementation, particularly sucrose, in combination with improved ventilation or temporary immersion systems, can stimulate autotrophic growth, improve stomatal function, and enhance photosynthetic capacity of cultured plantlets. These approaches not only reduce physiological disorders such as hyperhydricity but also improve the overall acclimatization success of regenerated plants. Given the increasing attention this area has received in recent years, particularly for optimizing large-scale and cost-effective micropropagation protocols, it is a significant oversight that this aspect is not addressed in the current review. Inclusion of this topic would strengthen the review's relevance and completeness, particularly for researchers aiming to bridge in vitro propagation with ex vitro performance.

To guide the authors in improving this manuscript I send some queries to guide in your revision:

Q1: Can the authors improve the resolution and clarity of Figure 1 and expand the caption to ensure it is interpretable without the main text?
Q2: Have all cited regenerative genes (e.g., WUS, BBM, WOX) been validated in Rosaceae species specifically, or are some extrapolated from model plants?
Q3: Can the discussion on CRISPR and non-tissue culture-based methods include limitations or current implementation barriers in Rosaceae?
Q4: Is it possible to include a schematic or table summarizing which regeneration methods are most suitable for each Rosaceae genus?
Q5: Have the authors considered including quantitative data (e.g., regeneration efficiency percentages) across studies in Table 1?
Q6: Could the authors clarify the inclusion criteria for selecting references and studies summarized in the review?
Q7: Is the role of epigenetics adequately distinguished between model plant findings and Rosaceae-specific results?
Q8: Can the manuscript be edited for grammatical consistency and sentence length in some sections to improve readability?
Q9: Are there any recent (post-2023) advances or pilot studies in CRISPR for Rosaceae that could be briefly discussed?
Q10: Would it be beneficial to offer a concise future research roadmap or priority actions in the conclusion?

Q11: Could the authors discuss the role of in vitro gas exchange in improving plantlet quality, particularly in relation to stomatal development and photosynthetic activation during micropropagation?

Q12: Have recent advancements in ventilation systems or temporary immersion bioreactors been considered as strategies to enhance gas exchange and reduce physiological disorders such as hyperhydricity in Rosaceae tissue cultures?

Comments on the Quality of English Language

The manuscript is generally well-written in scientific English, with formal tone and appropriate academic structure. However, it would benefit from stylistic refinement in certain sections. Sentence constructions are sometimes overly complex, and a few verb tense inconsistencies appear in the results and discussion sections. The use of passive voice is appropriate for the genre but is occasionally overused. Some paragraphs would benefit from clearer topic sentences and tighter cohesion.

Author Response

The manuscript titled “Contemporary Advances and Future Perspectives in Rosaceae Plant Regeneration”, with Qi Zang et al., provides a comprehensive review of conventional and emerging plant regeneration strategies in economically and ornamentally valuable Rosaceae species. The objective is to evaluate biological and technical barriers in regenerative capacity and to explore how molecular biotechnology, genome editing, and epigenetic mechanisms can enhance regeneration efficiency. The scope spans across various plant types in the Rosaceae family—including apples, pears, strawberries, and roses—emphasizing genotype-specific responses, explant types, environmental factors, and hormonal regulation. The most compelling finding is the proposal of integrating gene-editing technologies (e.g., CRISPR/Cas9) with tissue culture-independent transformation systems as a forward-looking strategy to overcome recalcitrant regeneration challenges in woody Rosaceae species.

This manuscript is likely to have a meaningful impact in the fields of horticultural biotechnology and plant tissue culture. By compiling dispersed findings into a single, detailed resource, it will serve as a reference point for future empirical research and practical applications in Rosaceae regeneration. The discussion on integrating gene regulatory networks, epigenetics, and genome editing broadens its relevance beyond Rosaceae, potentially influencing research in other recalcitrant plant families. The manuscript discusses genotypic variability, plant growth regulators, explant types, environmental conditions, and molecular aspects.

However, the manuscript does exhibit some weaknesses. Despite its comprehensive coverage, the review lacks a strong critical lens when discussing limitations in applying advanced technologies such as CRISPR or non-tissue culture methods to Rosaceae species; the discussion is largely speculative without acknowledging current feasibility barriers. Additionally, minor grammatical inconsistencies and some overly long sentences may hinder clarity for non-native readers. Furthermore, certain figures (e.g., Figure 1 on page 3) lack detailed legends necessary for standalone comprehension.

One critical omission in this otherwise comprehensive review is the lack of discussion on in vitro maintenance of explants under low-sucrose conditions and enhanced gas exchange, which has become a highly relevant topic in modern micropropagation research. Recent studies have emphasized that reducing exogenous carbohydrate supplementation, particularly sucrose, in combination with improved ventilation or temporary immersion systems, can stimulate autotrophic growth, improve stomatal function, and enhance photosynthetic capacity of cultured plantlets. These approaches not only reduce physiological disorders such as hyperhydricity but also improve the overall acclimatization success of regenerated plants. Given the increasing attention this area has received in recent years, particularly for optimizing large-scale and cost-effective micropropagation protocols, it is a significant oversight that this aspect is not addressed in the current review. Inclusion of this topic would strengthen the review's relevance and completeness, particularly for researchers aiming to bridge in vitro propagation with ex vitro performance.

To guide the authors in improving this manuscript I send some queries to guide in your revision:

Q1: Can the authors improve the resolution and clarity of Figure 1 and expand the caption to ensure it is interpretable without the main text?

Response: Thanks！We have replaced Fig. 1 with clearer picture, and all figures have been included complete labels, abbreviations, and defined pathways in the revised manuscript.

 

Q2: Have all cited regenerative genes (e.g., WUS, BBM, WOX) been validated in Rosaceae species specifically, or are some extrapolated from model plants?

Response: Thanks！Those regenerative genes (e.g., WUS/WOX/BBM/LEC/AGL/ESR/FUS/STM) been validated in Rosaceae species specifically, and we have clarified them in the section of Regeneration-Related Genes and Their Regulatory Networks. In addition, relevant references have also been added in the revised manuscript, and those genes were marked with red borders in Figure 3.

 

Q3: Can the discussion on CRISPR and non-tissue culture-based methods include limitations or current implementation barriers in Rosaceae?

Response: Thanks！We have discussed the limitations or current implementation barriers on CRISPR and non-tissue culture-based methods in Rosaceae and added relevant literatures in the revise manuscript.

 

Q4: Is it possible to include a schematic or table summarizing which regeneration methods are most suitable for each Rosaceae genus?
Response: Thanks！In the revised paper, we have added a schematic which summarizes the most suitable regeneration methods for different Rosaceae genus (apple/pear/strawberry/rose, etc.), including the typical explants, regeneration pathways, and the most effective plant growth regulators strategy.

 

Q5: Have the authors considered including quantitative data (e.g., regeneration efficiency percentages) across studies in Table 1?

Response: Thanks！We have added quantitative data (e.g., regeneration efficiency percentages, transformation rates, rooting rates, survival rates) in Table 1, and provided the Table note including complete labels, and abbreviations in the revised manuscript.

 

Q6: Could the authors clarify the inclusion criteria for selecting references and studies summarized in the review?
Response: Thanks！To ensure the review is comprehensive and representative, we applied the following criteria during our literature search and selection process:

1. Literature Search:Our search was conducted systematically using major academic databases such as Web of Science, PubMed, and Scopus, with a combination of keywords related to our core themes, including: Rosaceae, in vitro regeneration, callus, somatic embryogenesis, organogenesis, totipotency, epigenetics, DNA methylation, Fragaria, Prunus, Malus, etc.
2. Time Frame:We primarily focused on studies published from (g., 2015) to the present (2025), with an emphasis on recent advances, while also including seminal older publications that established foundational concepts.
3. Study Types:We focused on peer-reviewed original research articles (e.g., experimental studies on epigenetic changes during in vitro culture) published in reputable scientific journals. Review articles were consulted for background and context but were not the primary source for summarizing experimental findings. Seminal studies that established key concepts were included alongside recent high-impact research (approximately 2000-2025) to present a current and evolving perspective.
4. Relevance Screening:Titles and abstracts were screened for direct relevance to the central focus of our review (g., epigenetic regulation, particularly DNA methylation, in the tissue culture and regeneration of fruit crops, with a special emphasis on Rosaceous species). Studies not directly addressing these interconnected topics were excluded.
5. Quality and Impact:Within the relevant research literature, we selected studies that presented robust experimental evidence, had clear methodology, and were published in reputable journals. We also aimed to highlight studies that reported novel findings or presented contrasting results (such as the differential effects of 5-AzaC in strawberry vs. peach) to illustrate the diversity of regenerative responses and regulatory strategies within the Rosaceae family. 

We acknowledge that our selection, while systematic, may not be exhaustive of all existing literature. The goal was to construct a coherent narrative that accurately reflects the current state of knowledge and key debates in this specific field. We are happy to consider including any additional key studies the reviewers and editors may recommend to further strengthen our review.

 

Q7: Is the role of epigenetics adequately distinguished between model plant findings and Rosaceae-specific results?

Response: Thanks！We have clearly indicated the epigenetics parts (e.g., DNA methylation and H3K27me3 histone modification) involved in the regulation of Rosaceae regeneration in the section of Epigenetic Modification Promotes Plant Regeneration, and relevant references have also been added in the revised manuscript. Furthermore, in Figure 3, we have marked them with red borders.

 

Q8: Can the manuscript be edited for grammatical consistency and sentence length in some sections to improve readability?

Response: Thanks！Prior to the initial submission, we have had the manuscript professionally polished by a native English-speaking editor. In order to improve article quality, we have now engaged the editing service again for a thorough second round of language polishing focused on improving readability and fluency. We hope it now could meet the journal's standards for publication.

 

Q9: Are there any recent (post-2023) advances or pilot studies in CRISPR for Rosaceae that could be briefly discussed?

Response: Thanks！We have discussed the recent (post-2023) advances or pilot studies in CRISPR for Rosaceae in the CRISPR/Cas9 System section of revised manuscript.

 

Q10: Would it be beneficial to offer a concise future research roadmap or priority actions in the conclusion?

Response: Thanks！We have added the content of “Future priorities / roadmap” in the Conclusion section, as well as a roadmap in the revised manuscript.

 

Q11: Could the authors discuss the role of in vitro gas exchange in improving plantlet quality, particularly in relation to stomatal development and photosynthetic activation during micropropagation?

Response: Thanks！We have discussed the role of gas exchange in the section of Effects of Environmental Factors on the Regeneration of Rosaceae of the revised paper.

 

Q12: Have recent advancements in ventilation systems or temporary immersion bioreactors been considered as strategies to enhance gas exchange and reduce physiological disorders such as hyperhydricity in Rosaceae tissue cultures?

Response: Thanks！Yes, temporary immersion bioreactors are highly beneficial and compelling. They are currently recognized as key strategies for effectively improving the quality of Rosaceae tissue cultures by optimizing gas exchange, such as plum (Prunus domestica L.) (Gago, et al. 2022). We have added relevant conent and literature in the section of Environmental Factors on the Regeneration of Rosaceae of revised paper.

(1) Gago, D.; Sánchez, C.; Aldrey, A.; Christie, C.B.; Bernal, M.Á.; Vidal, N. Micropropagation of plum (Prunus domestica L.) in bioreactors using photomixotrophic and photoautotrophic Conditions. Horticulturae, 2022, 8, 286. https://doi.org/10.3390/horticulturae8040286.

Reviewer 2 Report

Comments and Suggestions for Authors

Dear authors,

This review is quite comprehensive and covers several topics on plant regeneration in the Rosaceae family. I suggested some improvements to Figure 1 and the inclusion of the topic Somaclonal Variation (adding more references).

Comments for author File: Comments.pdf

Author Response

This review is quite comprehensive and covers several topics on plant regeneration in the Rosaceae family. I suggested some improvements to Figure 1 and the inclusion of the topic Somaclonal Variation (adding more references).

Response: Thanks！We have replaced Fig. 1 with clearer picture, and all figures have been included complete labels, abbreviations, and defined pathways in the revised manuscript. In addition, we have included the content on somaclonal variation, including examples of somatic cell variations in Rosaceae plants, methods for evaluating and screening variations, etc., and have added relevant literature in the revised version.

 

Comments on the Quality of English Language

The manuscript is generally well-written in scientific English, with formal tone and appropriate academic structure. However, it would benefit from stylistic refinement in certain sections. Sentence constructions are sometimes overly complex, and a few verb tense inconsistencies appear in the results and discussion sections. The use of passive voice is appropriate for the genre but is occasionally overused. Some paragraphs would benefit from clearer topic sentences and tighter cohesion.

Response: Thanks！Prior to the initial submission, we have had the manuscript professionally polished by a native English-speaking editor. In order to improve article quality, we have now engaged the editing service again for a thorough second round of language polishing focused on improving readability and fluency. We hope it now could meet the journal's standards for publication.

Reviewer 3 Report

Comments and Suggestions for Authors

The submitted review article provides a comprehensive and timely synthesis of current knowledge on plant regeneration in the Rosaceae family. The manuscript demonstrates substantial breadth, covering classical in vitro regeneration pathways, key physiological and molecular determinants, and emerging biotechnological approaches. The structure is well framed, the scope is appropriate for a review paper, and the authors succeed in integrating a large body of literature into a coherent narrative, which can be intreresting  for researchers and practitioners working in plant biotechnology and horticultural sciences

 

The introduction loosely outlines the biological importance of plant regeneration and its relevance for breeding, genetic transformation, and germplasm conservation in Rosaceae.

1/ However, this Introduction section currently remains rather general and largely descriptive. It lacks concrete factual and quantitative economic context that would better justify the significance of the topic from an applied perspective. I suggest that in particular, the manuscript would really benefit from the inclusion of basic economic indicators illustrating the importance of Rosaceae in horticulture and agriculture, such as global or regional production values, market volumes, or turnover figures. Even a concise overview of key crops would strengthen the motivation of readers to take into account facts stated in the review. 1a/ For example, apples could be highlighted in relation to their dominant position in the fruit production and marketing sector, 1b/ while roses could be briefly discussed with respect to their economic relevance in the ornamental plant industry. Such targeted examples would anchor the biological discussion more firmly in real-world agricultural and horticultural production systems.

The core sections dealing with regeneration pathways, genotypic effects, explant selection, environmental factors, and plant growth regulators are generally well written and informative. The discussion of obstacles to regeneration is also appropriate and relevant.

2/ However, in the subsection addressing browning (page 12), the authors thoroughly describe the biochemical basis of phenolic oxidation and common mitigation strategies. Nevertheless, an important and widely used practical approach is missing: the frequent transfer of explants onto fresh medium, it works for example easily and efficiently in apple explants the second day after culture establishement/beginning. This simple intervention is often one of the fastest and most effective ways to limit browning and associated toxicity during early culture stages, and its omission represents a minor but notable gap in an otherwise solid discussion.

3/ More broadly, the manuscript would benefit from a deeper conceptual treatment of callus cultures beyond their role as intermediate regeneration stages. Callus systems can be viewed as plant explant-based bioreactors with significant potential for experimental manipulation, scale-up, and standardization. Related to this, the text only marginally addresses the possibilities of process automation, temporary immersion systems, and robotic handling in in vitro culture. Given current trends in plant biotechnology, a more explicit discussion of automated and semi-automated regeneration platforms, as well as regeneration phenomena associated with these advanced experimental approaches, would considerably enhance the forward-looking perspective of the review.

 

Author Response

The submitted review article provides a comprehensive and timely synthesis of current knowledge on plant regeneration in the Rosaceae family. The manuscript demonstrates substantial breadth, covering classical in vitro regeneration pathways, key physiological and molecular determinants, and emerging biotechnological approaches. The structure is well framed, the scope is appropriate for a review paper, and the authors succeed in integrating a large body of literature into a coherent narrative, which can be intreresting for researchers and practitioners working in plant biotechnology and horticultural sciences

The introduction loosely outlines the biological importance of plant regeneration and its relevance for breeding, genetic transformation, and germplasm conservation in Rosaceae.

 

1/ However, this Introduction section currently remains rather general and largely descriptive. It lacks concrete factual and quantitative economic context that would better justify the significance of the topic from an applied perspective. I suggest that in particular, the manuscript would really benefit from the inclusion of basic economic indicators illustrating the importance of Rosaceae in horticulture and agriculture, such as global or regional production values, market volumes, or turnover figures. Even a concise overview of key crops would strengthen the motivation of readers to take into account facts stated in the review. 1a/ For example, apples could be highlighted in relation to their dominant position in the fruit production and marketing sector, 1b/ while roses could be briefly discussed with respect to their economic relevance in the ornamental plant industry. Such targeted examples would anchor the biological discussion more firmly in real-world agricultural and horticultural production systems.

The core sections dealing with regeneration pathways, genotypic effects, explant selection, environmental factors, and plant growth regulators are generally well written and informative. The discussion of obstacles to regeneration is also appropriate and relevant.

Response: Thanks！We have added a concise overview of global or regional production values, market volumes, or turnover figures of key Rosaceae crops, and relevant literature in the revised manuscript.

 

2/ However, in the subsection addressing browning (page 12), the authors thoroughly describe the biochemical basis of phenolic oxidation and common mitigation strategies. Nevertheless, an important and widely used practical approach is missing: the frequent transfer of explants onto fresh medium, it works for example easily and efficiently in apple explants the second day after culture establishement/beginning. This simple intervention is often one of the fastest and most effective ways to limit browning and associated toxicity during early culture stages, and its omission represents a minor but notable gap in an otherwise solid discussion.

Response: Thanks！We have added the content of the impact of frequently transferring explants onto fresh culture media on browning in the section of Obstacles to Plant Regeneration in Rosaceae of the revised manuscript.

 

3/ More broadly, the manuscript would benefit from a deeper conceptual treatment of callus cultures beyond their role as intermediate regeneration stages. Callus systems can be viewed as plant explant-based bioreactors with significant potential for experimental manipulation, scale-up, and standardization. Related to this, the text only marginally addresses the possibilities of process automation, temporary immersion systems, and robotic handling in in vitro culture. Given current trends in plant biotechnology, a more explicit discussion of automated and semi-automated regeneration platforms, as well as regeneration phenomena associated with these advanced experimental approaches, would considerably enhance the forward-looking perspective of the review.

Response: Thanks！We have added a more explicit discussion of callus systems, temporary immersion systems and automated and semi-automated regeneration platforms in the section of Effects of Environmental Factors on the Regeneration of Rosaceae of the revised paper.

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

No more coments