Establishment of an Efficient Regeneration System of Rosa ‘Pompon Veranda’
Round 1
Reviewer 1 Report
Comments and Suggestions for AuthorsThis manuscript offers a valuable contribution to the field of rose in vitro propagation. The work is well-structured and easy to follow. To enhance its quality for publication, several points require clarification and revision. The specific recommendations are detailed below:
Abstract:
- In plant tissue culture, the standard term is “contamination rate,” not “pollution rate.” Please correct this terminology throughout the manuscript.
Materials and Methods:
- Please specify the composition of the “bud initiation medium” mentioned on Line 81.
- Please specify the type and concentration of the “aqueous detergent solution” used (Lines 86-87).
- Please state the age of the plantlets selected for the rooting experiment.
- The reference to “Section 1.2.1” on Line 138 appears to be incorrect. Please check and correct the cross-reference.
Results:
- In Section 3.1, please identify the types of contaminants observed (e.g., bacteria, fungi) and include a discussion of these findings.
- The reference to “Section 1.2.2” in Section 3.2 appears to be incorrect. Please correct the cross-reference.
- The results in Section 3.2 would be more clearly presented in a table or figure.
- For Section 3.4, consider including representative images of the callus generated from each treatment to visually support the results.
- In Table 6, please include data on the number of shoots generated per explant for each treatment.
- To strengthen Table 7, please consider adding other relevant growth parameters, such as the number of leaves per shoot and average shoot length.
Discussion:
- The discussion in Section 4.1 should be more focused. Instead of general information, please provide a detailed comparison of the treatments, elaborating on how different concentrations and exposure times specifically influenced the results.
- Similarly, the discussion on shoot proliferation (Section 4.2) needs more depth. Please discuss how different types and concentrations of Plant Growth Regulators (PGRs) specifically affected shoot formation, comparing the outcomes of each treatment.
- In Section 4.3, please elaborate on the potential physiological or developmental reasons why leaf explants were more responsive to the culture medium and PGRs compared to other explant types.
- The discussion in Section 4.4 would be strengthened by elaborating on the effects of 2,4-D. Please discuss the observed phenomena and speculate, based on existing literature, on what might happen at concentrations higher than 5 mg/L and why.
Author Response
Comments 1：In plant tissue culture, the standard term is “contamination rate,” not “pollution rate.” Please correct this terminology throughout the manuscript.
Response 1：Thank you for pointing out this issue. Based on your suggestion, we have made revisions to multiple identical misuse of words in the manuscript.
Comments 2：Please specify the composition of the “bud initiation medium” mentioned on Line 81.
Response 2：We have supplemented the composition of the shoot initiation medium in the original text, namely MS + 1.0 mg∙L-1 6-BA + 0.01 mg∙L-1 NAA + 0.1 mg∙L-1 GA3. Thank you for your comment.
Comments 3：Please specify the type and concentration of the “aqueous detergent solution” used (Lines 86-87).
Response 3：We have supplemented the type and concentration of the water-soluble reagent in the revised manuscript, specifically 0.1% (w/v) carbendazim solution. Thank you for your suggestion.
Comments 4：Please state the age of the plantlets selected for the rooting experiment.
Response 4：We have supplemented the relevant information in the manuscript. The materials used for the rooting experiment were tissue culture seedlings that had been transferred from the shoot initiation medium to the proliferation medium and grown for 25 days. Thank you for your valuable suggestion.
Comments 5：The reference to “Section 1.2.1” on Line 138 appears to be incorrect. Please check and correct the cross-reference.
Response 5：Thank you for your careful guidance. We have revised it to "Section 2.2.1."
Comments 6：In Section 3.1, please identify the types of contaminants observed (e.g., bacteria, fungi) and include a discussion of these findings.
Response 6：Thank you for your valuable suggestions. We have added explanations in the manuscript, clarifying that the contamination type is fungal contamination. Corresponding content has also been added to the discussion section: Taking all factors into consideration, the optimal disinfection protocol for R.‘Pompon Veranda’ was found to be treatment with 75% alcohol for 1 minute followed by 15% NaClO for 15 minutes. This method effectively reduced fungal contamination and mortality. Although some fungal contamination still occurred with this treatment, the contamination rate remained within a controllable range compared to other protocols and did not affect subsequent experiments. In the future, we can further optimize the sterilization of explants by studying factors such as the sampling season (e.g., spring or autumn), sampling conditions (e.g., sunny weather, avoiding rainy days), and the state of the sampled branches (e.g., tender green shoots in spring).
Comments 7：The reference to “Section 1.2.2” in Section 3.2 appears to be incorrect. Please correct the cross-reference.
Response 7：Thank you for your careful guidance. We have revised it to "Section 2.2.2."
Comments 8：The results in Section 3.2 would be more clearly presented in a table or figure.
Response 8：Thank you for your suggestion. This experiment was performed based on the previously established rapid propagation system of R. 'Samantha' by our research group (Gao et al., 2005), with minor adjustments (increasing the NAA concentration from 0.004 mg∙L-1 to 0.01 mg∙L-1). It was found that the proliferation coefficient of R. 'Pompon Veranda' could reach 4.04, with the plants growing very vigorously, which could well meet the requirements of subsequent experiments. Therefore, no in-depth experiment was conducted at this section.
The specific information of the relevant reference is as follows:
Gao LP, Bao MZ. Callus Induction and Plant Regeneration of Rosa hybrida ‘Samantha’[J]. Acta Horticulture Sinica, 2005, 32(3): 534-536.
Comments 9：For Section 3.4, consider including representative images of the callus generated from each treatment to visually support the results.
Response 9：Thank you for your suggestion. We have supplemented representative images of callus induction by different concentrations of 2,4-D in the revised manuscript (Figure 3).
Comments 10：In Table 6, please include data on the number of shoots generated per explant for each treatment.
Response 10：Thank you for your valuable comment. We have supplemented the data on the number of shoots generated per explant for each treatment in the revised manuscript.
Comments 11：To strengthen Table 7, please consider adding other relevant growth parameters, such as the number of leaves per shoot and average shoot length.
Response 11：Thank you for your valuable comment. We have supplemented the data on the number of leaves generated per shoot and the average shoot length in the revised manuscript..
Comments 12：The discussion in Section 4.1 should be more focused. Instead of general information, please provide a detailed comparison of the treatments, elaborating on how different concentrations and exposure times specifically influenced the results.
Response 12：Thank you for your valuable comment. We have supplemented the corresponding content in Section 4.1 in the manuscript regarding the impact of different treatments on explant disinfection, as detailed below:
The duration of disinfection and the concentration of the disinfectant both affect the sterilization efficacy and survival rate of explants. Short disinfection time and low disinfectant concentration may result in incomplete sterilization, while prolonged disinfection time and high disinfectant concentration can damage or even poison the explants. In this study, it was found that with protocols 1, 2 and 3 (the disinfectant concentration was 5% NaClO), the contamination rate and mortality rate gradually decreased as the disinfection time increased. With protocols 4, 5 and 6 (the disinfectant concentration was 10% NaClO), the contamination rate and mortality rate initially decreased but then increased with longer disinfection time. With protocols 7, 8 and 9 (the disinfectant concentration was 15% NaClO), both the contamination rate and mortality rate increased with longer disinfection time. However, when the disinfection time is the same, as the disinfection concentration increases, the contamination rate gradually decreases, while the mortality rate first rises and then declines. This indicates that the disinfection effect is not proportional to the disinfection time and concentration, similar to the findings of Sheikh-Assadi's study.
Comments 13：Similarly, the discussion on shoot proliferation (Section 4.2) needs more depth. Please discuss how different types and concentrations of Plant Growth Regulators (PGRs) specifically affected shoot formation, comparing the outcomes of each treatment.
Response 13：Thank you for your suggestion. The tissue culture seedling proliferation experiment in this study was performed based on the rapid propagation system of R. 'Samantha' previously established by our research group (Gao et al., 2005), with minor adjustments (increasing the NAA concentration from 0.004 mg∙L-1 to 0.01 mg∙L-1). It was found that the proliferation coefficient of R. 'Pompon Veranda' could reach 4.04, with the plants growing vigorously, which could well meet the requirements of subsequent experiments. Therefore, no in-depth experiment was conducted at this stage. However, based on your suggestion, we have supplemented the content regarding the effects of different plant growth regulators (PGRs) on shoots proliferation in Section 4.2 in the revised manuscript: when the ratio of auxin to cytokinin is low, it induces the formation of shoots in tissue culture seedlings. The commonly used auxins for inducing shoots formation in tissue culture seedlings are NAA and IBA, and the cytokinin is 6-BA. A concentration range of 6-BA between 0.5-2.0 mg∙L-1 promotes the elongation of shoots, while a concentration greater than 3.0 mg∙L-1 can cause vitrification in tissue culture seedlings. NAA at 0.05-0.2 mg∙L-1 promotes the production of shoots; IBA at 0.1-0.5 mg∙L-1 enhances the polar growth of shoots. Cytokinins combined with low concentrations of auxins promote the formation and proliferation of shoots.
Comments 14：In Section 4.3, please elaborate on the potential physiological or developmental reasons why leaf explants were more responsive to the culture medium and PGRs compared to other explant types.
Response 14：Thank you for your valuable suggestion. In fact, in addition to using small leaves of tissue-cultured seedlings as explants to induce callus, we also attempted to use anthers as explants for callus induction. The results showed that the highest callus induction rate using anthers as explants was only 45.45%. Moreover, during the cultivation process, the callus gradually became hard and turned brown. Additionally, no differentiation of these callus tissues was observed in the later stages of cultivation. On the other hand, obtaining leaves from tissue-cultured seedlings is relatively simple and unaffected by seasons or environmental factors. Furthermore, the callus induction rate using leaves from tissue-cultured seedlings as explants can reach 100%, and the growth status of the callus is much better compared to that of callus induced from anthers. Therefore, small leaves from tissue-cultured seedlings were ultimately used as explants for callus induction.
To more robustly support this conclusion, we have supplemented relevant content in Section 4.3 and included the data related to anther culture as supplementary tables and figures in the revised manuscript.
Comments 15：The discussion in Section 4.4 would be strengthened by elaborating on the effects of 2,4-D. Please discuss the observed phenomena and speculate, based on existing literature, on what might happen at concentrations higher than 5 mg/L and why.
Response 15：Thank you for your valuable suggestion. We have supplemented relevant content in Section 4.4 in the manuscript as follows:
A total of eight treatments were set up in this section. When the concentration of 2,4-D exceeded 5.0 mg∙L-1 (6.0 mg∙L-1 and 7.0 mg∙L-1), the callus exhibited slight waterlogging, which was unfavorable for its differentiation. Previous research suggested that high concentrations of 2,4-D disrupt Ga2+ signaling, hindering cell wall synthesis, while also promoting ethylene synthesis and increasing plasma membrane permeability. This leads to vitrification of the callus, making its surface transparent and waterlogged. Interestingly, however, when the 2,4-D concentration was 8.0 mg∙L-1, its induction effect on callus was nearly identical to that at 5.0 mg∙L-1. Taking into account both economic cost and induction efficiency, it is concluded that 2,4-D (5.0 mg∙L-1 ) is the optimal choice for inducing callus in R. 'Pompon Veranda'.
Author Response File: 
Author Response.pdf
Reviewer 2 Report
Comments and Suggestions for AuthorsThis manuscript, "Establishment of an Efficient Regeneration System of Rosa ‘Pompon Veranda’," presents a valuable and well-written study on the regeneration of Rosa ‘Pompon Veranda’. Addressing the following recommendations will further enhance the clarity, rigour, and impact of the work.
In the abstract, briefly mention the broader implications for rose breeding or biotechnology to highlight the study’s relevance.
Provides a solid background on rose breeding challenges and the need for efficient regeneration systems.
Expand on the limitations of previous regeneration protocols with more quantitative comparisons or specific examples.
Could you please clarify how your approach is novel compared to existing protocols for other Rosa cultivars?
Briefly mention the potential applications of the established system (e.g., gene editing, trait improvement) in the introduction.
Include the exact number of biological and technical replicates for each experiment in the main text, not just in tables.
Specify the source and age of plant material more precisely (e.g., plant age, growth conditions).
Provide catalogue numbers or suppliers for key reagents (e.g., plant growth regulators, media components).
Clarify statistical methods: state the software version, post-hoc tests, and significance thresholds in a dedicated subsection.
Consider adding a flowchart or a schematic summarising the experimental workflow for clarity.
Ensure all tables and figures are self-explanatory with complete legends (e.g., define all abbreviations, units, and statistical notations).
Where possible, provide quantitative data in the text to support qualitative descriptions (e.g., “vigorous growth”).
For each key result, briefly discuss its significance or how it compares to previous studies.
Consider including representative images of callus, shoot, and root development stages with scale bars for visual clarity.
If available, provide data on the efficiency and stability of the regeneration system over multiple subcultures.
Deepen the discussion on the limitations of the current system (e.g., relatively low differentiation rate, genotype dependency).
Suggest specific future directions, such as optimising for other cultivars or integrating with transformation protocols.
Discuss potential challenges in scaling up the protocol for commercial or research applications.
Suggest how this protocol could facilitate genetic transformation or gene editing in Rosa species.
Double-check that all cited studies are referenced in the main text and vice versa.
Standardise terminology (e.g., use either “adventitious buds” or “shoots” consistently).
Ensure all units and concentrations are presented uniformly (e.g., mg∙L⁻¹).
Consider adding a supplementary table summarising key parameters and outcomes from previous rose regeneration studies for comparison.
If the study aims to support future genetic transformation, discuss any preliminary transformation attempts or plans.
Author Response
Comments 1：This manuscript, "Establishment of an Efficient Regeneration System of Rosa ‘Pompon Veranda’," presents a valuable and well-written study on the regeneration of Rosa ‘Pompon Veranda’. Addressing the following recommendations will further enhance the clarity, rigour, and impact of the work.
In the abstract, briefly mention the broader implications for rose breeding or biotechnology to highlight the study’s relevance.
Response 1：Thank you for your comment. According to your suggestion, we have made modifications to the abstract.
Comments 2：Provides a solid background on rose breeding challenges and the need for efficient regeneration systems.
Response 2：Thank you for your comment.
Modern roses (Rosa hybrida) are mostly obtained through hybridization or backcrossing of Rosa plants originating from China, Europe, and the Middle East. Using these traditional breeding methods, breeders have created tens of thousands of new rose cultivars. However, repeated hybridization and backcrossing can lead to a series of problems such as a relatively narrow genetic background of the offspring, so traditional breeding methods have certain limitations.
The advent of plant genetic engineering technology has broken the boundaries of species, and provided a new approach for targeted improvement of rose traits, making it possible to create new and unique rose germplasms in a relatively short period of time. The establishment of efficient rose regeneration is the basic premise for the smooth development of rose genetic engineering research.
Comments 3：Expand on the limitations of previous regeneration protocols with more quantitative comparisons or specific examples.
Response 3：Thank you for your comment. The current limitations of the existing regeneration system include low differentiation rates, technically demanding multi-stage processes, and narrow genotype applicability, which restrict transgenic breeding and functional genomics research. In the future, optimizing differentiation rates through editing key regeneration genes could help overcome these existing limitations. We have supplemented the relative content in different parts of Discussion Section.
Comments 4：Could you please clarify how your approach is novel compared to existing protocols for other Rosa cultivars?
Response 4：Thank you for your question.
The team to which the corresponding author of this study belongs has been dedicated to establishing and improving the regeneration and transformation system of Rosa plants since the 1990s. By comparing the regeneration ability of nearly 100 Rosa plant materials, two ideal genotypes were screened out, which were R. 'Samantha' and rugosa rose 'Baobai'. On this basis, through years of exploration and optimization, stable systems of regeneration and genetic transformation of them have been established. Moreover, these systems have been successfully applied to the functional verification of genes in Rosa plants (Bao et al. 2012; Xing et al. 2014; Shen et al. 2019; Zhang et al., 2021). And R. 'Samantha' has become one of the most important rose transformation receptors internationally. However, it has poor disease resistance, low regeneration and transformation efficiency, and a long transformation cycle. Moreover, it is worth noting that in the past decade, the market share of R. 'Samantha' has been decreasing, so it is urgent to screen new and excellent rose transformation receptor materials to enrich the resource library of rose transformation receptors.
- R.'Pompon Veranda' is a classic miniature rose cultivar distinguished by a pale pink central eye, creamy outer petals, and a delicate tea fragrance. It is prized by consumers for its prolific flowering, extended bloom period, and strong disease resistance. However, the dense distribution of prickles on its stems poses significant challenges for field manegement and postharvest handling. So far, there hasbeen no report on the regeneration system of R. 'Pompon Veranda'. Therefore, the establishment of an in vitro regeneration system for it is of great significance for the molecular mechanism research and quality improvement of important traits such as floral fragrance, and prickle in Rosa
The relative references are as follows:
Bao, Y., Liu, G., Shi, X., Xing, W., Ning, G., Liu, J. et al. (2012) Primary and repetitive secondary somatic embryogenesis in Rosa hybrida ‘Samantha’. Plant Cell, Tissue and Organ Culture (PCTOC), 109 (3), 411-418.
Shen Y, Sun T, Pan Q, Nachaisin A, Ning G (2019). Rrmyb5and rrmyb10‐regulated flavonoid biosynthesis plays a pivotal role in feedback loop responding to wounding and oxidation in rosa rugosa. Plant Biotechnol J 17(11).
Xing, W., Wang, Z., Wang, X., Bao, M. and Ning, G. (2014) Over-expression of an FT homolog from Prunus mume reduces juvenile phase and induces early flowering in rugosa rose. Scientia Horticulturae, 172, 68-72.
Zhang, Y., Wu, Z., Feng, M., Chen, J., Qin, M., Wang, W. et al. (2021) The circadian-controlled PIF8–BBX28 module regulates petal senescence in rose flowers by governing mitochondrial ROS homeostasis at night. The Plant Cell, 33 (8), 2716-2735.
Comments 5：Briefly mention the potential applications of the established system (e.g., gene editing, trait improvement) in the introduction.
Response 5：Thank you for your suggestion. We have supplemented the potential applications of this system in the introduction as follows:
Therefore, the efficient regeneration system of R. ‘Pompon Veranda’ established in this study laid a solid foundation for the establishment of its genetic transformation system. The results can also be used for subsequent studies on quality traits such as floral scent and prickles of Rosa plants.
Comments 6：Include the exact number of biological and technical replicates for each experiment in the main text, not just in tables.
Response 6：Thank you for your suggestion. We have fully supplemented the exact number of biological and technical replicates for each experiment in the manuscript. Please refer to line (116-117、124-126、136-137、146-147、158-159、165-166).
Comments 7：Specify the source and age of plant material more precisely (e.g., plant age, growth conditions).
Response 7：Thank you for your valuable suggestion. We have added supplementary explanations in the manuscript: Branches with shoots were collected from the healthy, current-year apical shoots of R. 'Pompon Veranda' plants grown in natural environment under field conditions for about five years in the Rosa germplasm repository of Huazhong Agriculture University, Wuhan, China.
The materials used for the rooting experiment were tissue culture seedlings that had been transferred from the shoot initiation medium to the proliferation medium and grown for 25 days.
Comments 8：Provide catalogue numbers or suppliers for key reagents (e.g., plant growth regulators, media components).
Response 8：Thank you for your comment. We have supplemented the reagent information in the manuscript: Murashige and Skoog medium (MS, coolaber, Beijing)、N-(Phenylmethyl)-9H-purin-6-amine (6-BA, coolaber, Beijing)、1-Naphthylacetic acid (NAA, coolaber, Beijing)、Gibberellin A3 (GA3, coolaber, Beijing)、MS with half-strength macronutrients (1/2MS, coolaber, Beijing)、2,4-dichlorophenoxyacetic acid (2,4-D, coolaber, Beijing)、cytokinin-like compounds thidiazuron (TDZ, coolaber, Beijing)、3-Indolebutyric acid(IBA, coolaber, Beijing)、Sucrose(coolaber, Beijing).
Comments 9：Clarify statistical methods: state the software version, post-hoc tests, and significance thresholds in a dedicated subsection.
Response 9：Thank you for your comment. We would like to explain that we had already clarified statistical methods in section 2.3 of the first submitted manuscript.
Comments 10：Consider adding a flowchart or a schematic summarising the experimental workflow for clarity.
Response 10：Thank you for your suggestion. We have added the flowchart to Section 1 as Figure 1 in the revised manuscript.
Comments 11：Ensure all tables and figures are self-explanatory with complete legends (e.g., define all abbreviations, units, and statistical notations).
Response 11：Thank you for your comment. We have checked abbreviations, units, and statistical notations, etc., to ensure all tables and figures are self-explanatory with complete legends.
Comments 12：Where possible, provide quantitative data in the text to support qualitative descriptions (e.g., “vigorous growth”).
Response 12：In the previous manuscript, we calculated the multiplication coefficient to reflect the proliferation ability of adventitious shoots. Currently, there are no other available quantitative data to support the description of vigorous growth. Thank you for your suggestion. In the future, we will pay attention to collecting relevant data in the process of further improving the system based on your suggestions.
Comments 13：For each key result, briefly discuss its significance or how it compares to previous studies.
Response 13：In this round of revisions, we have made modifications and improvements in each section of the discussion, including the significance of the research and the comparison with previous studies. We greatly appreciate your suggestion.
Comments 14：Consider including representative images of callus, shoot, and root development stages with scale bars for visual clarity.
Response 14：Thank you for your suggestion. We have added representative callus images in the revised manuscript as Figure 3, and images of roots and shoots can be found in Figure 2 and Figure 5.
Comments 15：If available, provide data on the efficiency and stability of the regeneration system over multiple subcultures.
Response 15：Thank you for your suggestion. The regenerated plants obtained from callus were transplanted into the greenhouse until natural flowering, and no obvious phenotypic variations were observed. Therefore, molecular-level testing has not been conducted on the plants yet. We greatly appreciate your valuable comments and suggestions. In the future, we can further utilize molecular markers to examine the effects of different subculture generations on the genetic stability of tissue-cultured seedlings and their regenerated plants.
Comments 16：Deepen the discussion on the limitations of the current system (e.g., relatively low differentiation rate, genotype dependency).
Response 16：Thank you for your suggestion. We have added the corresponding content in Section 5 (Conclusions and Prospects) in the manuscript.
Comments 17：Suggest specific future directions, such as optimising for other cultivars or integrating with transformation protocols.
Response 17：Thank you for your suggestion. We have added the corresponding content in Section 5 (Conclusions and Prospects) in the manuscript as follows:
At present, there are still many areas in which the system could be optimized. In the future, to further ellevate the differentiation efficiency of callus, we can attempt to adjust the following factors: The basic medium types (WPM, SH, etc.), the ratio of different plant growth regulators, and culture methods (e.g., suspension culture). Furthermore, the regeneration system established in this study lays the foundation for the development of a genetic transformation system. Therefore, we need to conduct sensitivity tests of materials at different stages—callus proliferation, callus differentiation into shoots, and shoots elongation—to antibiotics (Kan, Hyg). We will then investigate the effects of Agrobacterium strain, different OD values of Agrobacterium, infection time, and co-culture duration on the efficiency of genetic transformation. What’s more, we can also try to employ plant developmental regulators (e.g., GROWTH-REGULATING FACTORs, BABYBOOM etc.) to improve the efficiency of regeneration and genetic transformation or even overcome the barrier of genotype dependency.
Comments 18：Discuss potential challenges in scaling up the protocol for commercial or research applications.
Response 18：Thank you for your comment. To scale up the protocol for commercial or research applications, there are several potential challenges, such as somaclonal variation, lower capacity for explant regeneration when compared to model plants, extensive browning, and relatively high cost etc. Its extensive application remains a formidable task. Nonetheless, optimistically, by utilizing appropriate culture media, optimizing the use of plant developmental regulators, and meticulously controlling environmental conditions in vitro, the regeneration system can be facilitated.
Comments 19：Suggest how this protocol could facilitate genetic transformation or gene editing in Rosa species.
Response 19：Thank you for your comment. This protocol could facilitate genetic transformation or gene editing in Rosa species from the following two aspects.
On the one hand, using callus obtained directly from the organogenesis pathway as a transformation receptor, although this approach may easily lead to chimerism. However, by optimizing regeneration conditions such as shortening callus induction time, screening dense callus, optimizing organ induction conditions, and conducting sufficient subculture screening, the probability of obtaining non chimeric transformants can also be increased.
On the other hand, we found that in the process of callus subculture, a small amount of embryogenic callus can also be produced, which can be further proliferated and cultured as a transformation receptor, and it is expected to successfully obtain transformed plants.
In the future, we will continuously optimize this regeneration system based on the protocol, as it is one of the core research directions for improving the efficiency of genetic transformation and gene editing. Without efficient regeneration, even the most advanced gene editing tools are difficult to exert practical effects in Rosa species.
Comments 20：Double-check that all cited studies are referenced in the main text and vice versa.
Response 20：Thank you for your comment. We have checked the manuscript to ensure that all cited references are mentioned in the main text.
Comments 21：Standardise terminology (e.g., use either “adventitious buds” or “shoots” consistently).
Response 21：Thank you for your comment. We have standardized the term as "shoots" throughout the manuscript.
Comments 22：Ensure all units and concentrations are presented uniformly (e.g., mg∙L⁻¹).
Response 22：Thank you for your suggestion. We have checked the manuscript and ensured that all units and concentrations are consistently expressed.
Comments 23：Consider adding a supplementary table summarising key parameters and outcomes from previous rose regeneration studies for comparison.
Response 23：Thank you for your suggestion. We have added a supplementary table containing information relative with rose regeneration studies in the appendix of the manuscript.
Comments 24：If the study aims to support future genetic transformation, discuss any preliminary transformation attempts or plans.
Response 24：Thank you for for your suggestion. We have supplemented the corresponding experimental plans in Section 5:
The regeneration system established in this study lays the foundation for the development of a genetic transformation system. Therefore, in subsequent experiments, we need to conduct sensitivity tests of materials at different stages - callus proliferation, callus differentiation into shoots, and shoots elongation - to antibiotics (Kan, Hyg). We will then investigate the effects of Agrobacterium strain, different OD values of Agrobacterium, infection time, and co-culture duration on the efficiency of genetic transformation.
Author Response File: Author Response.pdf
Reviewer 3 Report
Comments and Suggestions for AuthorsThere are a lot of problems in this manuscript, concerning the language, the description of material and methods (i.e. 30-35 explants per treatment, 3-5 rinses with water etc), the not proper use of the correct scientific terminology (pollution rate) etc. Thus, in my opinion, the manuscript is not suitable for publication in the journal. Bellow are also some specific remarks
line 12 "stem segments with buds" This is the initial material. In addition you used leaflets callus. Please rephrase. Also, the plantlets from callus were the same with the initial plant? Have you checked it?
line15 "pollution rate is only" change is to was
line 21. differentiation rate of callus was 10.87%. This is very low for commercial propagation, maybe by the use of bioreactors
70 "were used as explants" ad initial explants
77 The plants were indoor or outdoor, in ground or pots, age? Please be analytical
81 "on bud initiation medium" more details Also the apical leaves were from the top only, and in which stage specific?
85 more details about explant. tip? length? etc
91 3-5 times. this should be the same for all treatments i.e. 3 times
94 Murashige and Skoog ad reference
94-95 to all the chemicals ad in parenthesis company, city, country. Also the first time all the compound, in parenthesis i.e. BA) and in addition only the BA. The same to all text
101 ad reference
102 more details about medium i.e. PH, sucrose etc the same in 2.2.3
2.2.2 which are the modifications? which are the treatments?
2.2.4 PH
121 30-35 explants this should be the same to all the treatments
table 3 infected
Author Response
Comments 1：line 12 "stem segments with buds" This is the initial material. In addition you used leaflets callus. Please rephrase. Also, the plantlets from callus were the same with the initial plant? Have you checked it?
Response 1：Thank you for your correction. We have added the word "initial" in the corresponding positions in the manuscript (line 16, 78).
In this study, callus was induced by using leaflets of tissue-cultured seedlings as explants. The callus obtained in this way was called leaflets callus.
As the phrase ‘leaflets callus’ was firstly proposed in Section 2.2.4, according to your suggestion, we have added an explanation of it in the manuscript like this: callus was induced by using leaflets of tissue-cultured seedlings as explants. The callus obtained in this way was called leaflets callus. And in the following text, we continue to use this phrase.
What’s more, the regenerated plants obtained from callus were transplanted into the greenhouse until natural flowering, and no obvious phenotypic variations were observed. Therefore, molecular-level testing has not been conducted on the plants yet. We greatly appreciate your valuable comments and suggestions. In the future, we can further utilize molecular markers to examine the effects of different subculture generations on the genetic stability of tissue-cultured seedlings and their regenerated plants.
Comments 2：line15 "pollution rate is only" change is to was
Response 2：Thank you for your correction. We have made the revision in the manuscrip (line 18).
Comments 3：line 21. differentiation rate of callus was 10.87%. This is very low for commercial propagation, maybe by the use of bioreactors
Response 3：Thank you for your valuable comment. There is indeed space for improvement in the callus differentiation rate obtained from this study. We will further optimize the regeneration system based on the existing results, maybe by the use of bioreactors as you suggested.
Comments 4：70 "were used as explants" ad initial explants
Response 4：Thank you for your suggestion. We have added the word "initial" in the corresponding position in the manuscript (line 78).
Comments 5：77 The plants were indoor or outdoor, in ground or pots, age? Please be analytical
Response 5：Thank you for your valuable comment. We have added supplementary explanations in the manuscript: Branches with shoots were collected from the healthy, current-year apical shoots of R. 'Pompon Veranda' plants grown in natural environment under field conditions for about five years in the Rosa germplasm repository of Huazhong Agriculture University, Wuhan, China.
Comments 6：81 "on bud initiation medium" more details Also the apical leaves were from the top only, and in which stage specific?
Response 6：Thank you for your comment. We have added more detailed information to the manuscript as follows:
The shoot initiation medium was slightly adjusted based on previous reports, with the specific formulation being MS + 1.0 mg∙L-1 6-BA + 0.01 mg∙L-1 NAA + 0.1mg∙L-1GA3（line 99-102）。
After stem segment disinfection, tissue culture seedlings were obtained on shoot initiation medium, and then transferred to proliferation medium. After 25-day cultures, the small leaves at the top that were not fully expanded were taken for callus induction (line 94-96).
Comments 7：85 more details about explant. tip? length? Etc
Response 7：Thank you for your suggestion. We have added detailed information about the explants in the manuscript: Cut the current-year apical branches of R. 'Pompon Veranda' into stem segments containing one or two shoot points as the initial experimental explants.
Comments 8：91 3-5 times. this should be the same for all treatments i.e. 3 times
Response 8：Thank you for your valuable comment, which has helped make the wording more precise. Based on your suggestion, we have made the corresponding revisions in the manuscript: After each NaClO treatment, explants were rinsed 3 times with sterile distilled water (one minute per rinse)
Comments 9：94 Murashige and Skoog ad reference
Response 9：Thank you for your valuable suggestion. We have supplemented the relevant reference in the manuscript: Murashige T, and Skoog F. A Revised Medium for Rapid Growth and Bio Assays with Tobacco Tissue Cultures. Plant Physiology, 1962, 15: 473-497.
Comments 10：94-95 to all the chemicals ad in parenthesis company, city, country. Also the first time all the compound, in parenthesis i.e. BA) and in addition only the BA. The same to all text
Response 10：Thank you for your comment. We have supplemented the reagent information in the manuscript: Murashige and Skoog medium(MS, coolaber, Beijing)、N-(Phenylmethyl)-9H-purin-6-amine(6-BA, coolaber, Beijing)、1-Naphthylacetic acid (NAA, coolaber, Beijing)、Gibberellin A3 (GA3, coolaber, Beijing)、MS with half-strength macronutrients (1/2MS, coolaber, Beijing)、2,4-dichlorophenoxyacetic acid (2,4-D, coolaber, Beijing)、cytokinin-like compounds thidiazuron (TDZ, coolaber, Beijing)、3-Indolebutyric acid(IBA, coolaber, Beijing)、Sucrose(coolaber, Beijing)
Comments 11：101 ad reference
Response 11：Thank you for your suggestion. We have supplemented the relevant literature in the manuscrip: Gao LP, Bao MZ. Callus Induction and Plant Regeneration of Rosa hybrida‘Samantha’[J]. Acta Horticulture Sinica, 2005, 32(3): 534-536.
Comments 12：102 more details about medium i.e. PH, sucrose etc the same in 2.2.3
Response 12：Thank you for your suggestion. We have supplemented the relevant medium information in the manuscript: MS with 0.5 mg∙L-1 6 BA, 0.01mg∙L-1 NAA, 30 g∙L-1 Sucrose, pH=5.8; Four treatments were arranged in Section2.2.3, with the pH of each treatment medium at 5.8: 1/2MS with 0.1 mg∙L-1 NAA, 30 g∙L-1 Sucrose; 1/2MS with 0.5 mg∙L-1 NAA, 30 g∙L-1 Sucrose; 1/2MS with 0.1 mg∙L-1 IBA, 30 g∙L-1 Sucrose; 1/2MS with 0.5 mg∙L-1 IBA, 30 g∙L-1 Sucrose.
Comments 13：2.2.2 which are the modifications? which are the treatments?
Response 13：Thank you for your suggestion. This experiment was performed based on the previously established rapid propagation system of R. 'Samantha' by our research group (Gao et al., 2005), with minor adjustments (increasing the NAA concentration from 0.004 mg∙L-1 to 0.01 mg∙L-1). It was found that the proliferation coefficient of R. 'Pompon Veranda' could reach 4.04, with the plants growing very vigorously, which could well meet the requirements of subsequent experiments. Therefore, no in-depth experiment was conducted at this section.
The specific information of the relevant reference is as follows:
Gao LP, Bao MZ. Callus Induction and Plant Regeneration of Rosa hybrida ‘Samantha’[J]. Acta Horticulture Sinica, 2005, 32(3): 534-536.
Comments 14：2.2.4 PH
Response 14：Thank you for your suggestion. We have added the relevant information in the manuscript: pH=5.8.
Comments 15：121 30-35 explants this should be the same to all the treatments
Response 15：Thank you for your valuable suggestion, which has helped make the diction more precise. We have made the revisions to the manuscript.
Comments 16：table 3 infected
Response 16：Thank you for your suggestion. On the basis of reviewing a large number of references, we have consistently used the word "contamination" in the revised manuscript.
Author Response File: Author Response.pdf
Round 2
Reviewer 1 Report
Comments and Suggestions for AuthorsThe authors have addressed all previous suggestions, significantly improving the manuscript's quality. However, a minor revision is still required for Figure 2. Specifically, the labels (a), (b), (c), and (d) must be clearly defined in the figure legend.
Author Response
Comments 1：The authors have addressed all previous suggestions, significantly improving the manuscript's quality. However, a minor revision is still required for Figure 2. Specifically, the labels (a), (b), (c), and (d) must be clearly defined in the figure legend.
Response 1：Thank you for your valuable suggestions. In this revised manuscript, the labels (a), (b), (c), and (d) have been clearly defined in the legend of Figure 2.
Author Response File: Author Response.pdf
Reviewer 3 Report
Comments and Suggestions for Authors
The authors has indeed tried hard and improved significally the manuscript. Thus, in my opinion, the manuscript is suitable for publication after minor revision. Bellow my specific remarks
173 'the contamination is all fungal' how do you know that for sure? Use the word possibly except if you have make the neccesary checks with the proper methods
Also, for a complete tissue culture protocol, the acclimatization stage should be added in the text. Thus, before the acception of the manuscript for publication, this section should be added in the text (both material and method, results and maybe, discussion). There are only photos...
Author Response
Comments 1：173 'the contamination is all fungal' how do you know that for sure? Use the word possibly except if you have make the neccesary checks with the proper methods.
Response 1：Thank you for your question. In plant tissue culture, fungal contamination is typically characterized by fluffy, cottony, or cobweb-like colonies, which can appear in various colors such as white, black, green, or yellow, often forming visible mold spots on the surface of the medium or near the explants. In this study, the observed contamination manifested as white and black fluffy fungal hyphae attached near the bud points of the explants, along with similar fungal spots on the medium surface. Given that these morphological features closely align with typical fungal contamination, this study suggests that the contamination type is likely fungal. To ensure a more precise expression, as per your suggestion, we have used the word "possibly" in this revised manuscript.
Comments 2：Also, for a complete tissue culture protocol, the acclimatization stage should be added in the text. Thus, before the acception of the manuscript for publication, this section should be added in the text (both material and method, results and maybe, discussion). There are only photos...
Response 2：Thank you for your suggestion. We have supplemented the corresponding content of "Plantlets acclimatization and transplanting" in the text (both Materials and Methods, and Results sections).
Author Response File: Author Response.pdf
