Integrating In Vitro Propagation and Machine Learning Modeling for Efficient Shoot and Root Development in Aronia melanocarpa

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

1. The abstract lacks details about how the findings were derived, e.g., the specific experimental conditions that led to optimal results. A brief mention of the methods would enhance credibility.
2. Some graphics lack sufficient comments or explanations to explain their relevance. Figures 3 and 4 lack the annotation and scale of each picture.
3. There is no mention of how to define biological repetition, and the number and repetition of test samples are not reflected in materials and methods.
4. Add each experimental pictures corresponding to Table 1.
5. Whether the modeling based on only one set of experiments is accurate remains to be discussed.
6. The discussion should add the advantages of this study compared with previous studies, whether it has improved the reproductive efficiency. Has this method been studied before, and what technical improvements have been made in this study? There are many studies on tissue culture of Aronia melanocarpa in the introduction, but there is no comparison in the discussion.
7. The effect of container volume on the proliferation of clustered buds accounts for a large proportion In Results section, but there is no such content in the discussion.
8. The last line of Figure 1 is wrongly written. 57.1±3.7b6?
9. Line 93: Palaz et al. [13] developed the Shrub Plant Medium (SPM), which has demonstrated greater effectiveness than traditional MS and WPM in encouraging shoot proliferation and rooting across various deciduous shrub taxa. It’s should be notice that the SPM medium formula is not from this paper.
10. Line 96-98: While its use with A. melanocarpa is still mostly uncharted, initial findings indicate that SPM might provide improved nutrient availability and hormonal response tailored to the needs of woody plants. The author can be simply explained which formula improvements make SPM more suitable for shrubs.
11. Table 2 Number of Siblings means Number of shoots?
12. All the measurement indexes in Figure 2 have no unit names.
13. There are occasional grammatical errors and awkward phrasing. For example:

" By merging morphological evaluations with computational modeling, our results aim to enhance shrub micropropagation systems and facilitate the sustainable commercial cultivation of black chokeberry." While promising, this statement is vague and should be substantiated with specific examples or projections.

14. The interpretation of results is often descriptive rather than analytical. For example: The finding that 0.5 NAA + 0.25 IBA in Table 1 have the highest rooting rate is stated but not critically explored. Why does this combination perform better? Could this be due to their physiological reaction and interaction?
15. Why the number of Siblings, Shoot Length, and Shoot Length were performed better in Big Jar while the Leaf Diameter and Leaf Length were performed better in Small Jar? It needs more analyse.

Author Response

Reviewer 1

The abstract lacks details about how the findings were derived, e.g., the specific experimental conditions that led to optimal results. A brief mention of the methods would enhance credibility.

 

We thank the reviewer for this valuable suggestion. In response, we have revised the abstract to include concise details on the specific experimental conditions that led to optimal shoot and root development. We now explicitly mention the use of SPM medium supplemented with 5 mg/L BAP in large 660 mL jars for shoot induction, and 0.5 mg/L NAA + 0.25 mg/L IBA in half-strength SPM for rooting. This addition improves the transparency and credibility of the findings. Please see the revised abstract on page X of the manuscript.

 

Some graphics lack sufficient comments or explanations to explain their relevance. Figures 3 and 4 lack the annotation and scale of each picture.

 

Figures 3 and 4 have been revised to include detailed annotations, individual image labels (A–D), and scale information as requested. Each subfigure is now accompanied by explanatory captions that specify the experimental treatments, developmental stage, and magnification context where applicable. The figure legends were also expanded to clearly describe the content and relevance of each panel

 

There is no mention of how to define biological repetition, and the number and repetition of test samples are not reflected in materials and methods.

 

section 2.6 has been revised.

 

Add each experimental pictures corresponding to Table 1.

 

Thank you for your valuable suggestion. We have made every effort to include representative photographs corresponding to the treatments presented in Table 1. However, due to limitations encountered during the experimental process and subsequent documentation stages, it was not possible to retrieve images for every treatment group. Despite this, we have included all available and relevant images that best illustrate the main findings and reflect the key experimental conditions (Supplementary Figure S1, S2, and S3). We appreciate your understanding in this matter

 

Whether the modeling based on only one set of experiments is accurate remains to be discussed.

 

We value this important insight. Since the study used a well-designed factorial approach within a single experiment, we recognize the limitation in applying the machine learning models beyond this dataset. To address this, we included a paragraph in the Discussion section explicitly noting that, although LOOCV was used to reduce overfitting, validating the models with independent experiments or genotypes is essential for confirming their robustness. This addition emphasizes the need for future research to expand and validate our predictive method. Please see the revised text on page 17.

 

The discussion should add the advantages of this study compared with previous studies, whether it has improved the reproductive efficiency. Has this method been studied before, and what technical improvements have been made in this study? There are many studies on tissue culture of Aronia melanocarpa in the introduction, but there is no comparison in the discussion.

 

We appreciate the reviewer pointing out this key aspect. Consequently, we have updated the Discussion section to include a thorough comparison of our findings with earlier research on Aronia melanocarpa. We now clearly explain how our protocol enhances shoot multiplication and rooting success rates compared to previous studies, emphasizing technical improvements such as the use of SPM medium with optimized PGR blends and larger culture containers. Additionally, we highlight the innovative use of machine learning for predictive modeling, a novel application in Aronia micropropagation. These updates showcase the distinct contributions and practical benefits of our work. Refer to the revised Discussion on page 17.

 

The effect of container volume on the proliferation of clustered buds accounts for a large proportion In Results section, but there is no such content in the discussion.

 

Thank you for your insightful comment. In response, we've included a new paragraph in the Discussion that explicitly discusses how container volume influences shoot proliferation. Additionally, we reference a recent study by Malik et al. (2022), which showed similar benefits of larger culture vessels for micropropagation in Dendrobium. This connection enhances the relevance and novelty of our findings by aligning them with broader research on culture vessel size effects. Please see the revised text on page 17.

 

The last line of Figure 1 is wrongly written. 57.1±3.7b6?

I believe you were referring to Table 1. The final line of Table 1 has been corrected.

 

Line 93: Palaz et al. [13] developed the Shrub Plant Medium (SPM), which has demonstrated greater effectiveness than traditional MS and WPM in encouraging shoot proliferation and rooting across various deciduous shrub taxa. It’s should be notice that the SPM medium formula is not from this paper.

 

We thank the reviewer for this clarification. To avoid any potential misunderstanding, we have revised the sentence on Line 93 to explicitly indicate that the SPM medium was originally formulated by Palaz et al. [13].

 

Line 96-98: While its use with A. melanocarpa is still mostly uncharted, initial findings indicate that SPM might provide improved nutrient availability and hormonal response tailored to the needs of woody plants. The author can be simply explained which formula improvements make SPM more suitable for shrubs.

 

Thank you for this constructive and important observation. In response, we have revised the relevant section of the manuscript to provide a concise explanation of the specific compositional improvements in SPM that contribute to its enhanced effectiveness for woody species. These include a balanced nitrogen profile, increased calcium and magnesium levels, optimized iron chelation, and an enriched vitamin composition. Additionally, we have clarified that no plant growth regulators (PGRs) were pre-added to the medium, allowing for customized hormonal supplementation depending on the species or experiment.

To maintain clarity in the main text, the complete formulation of the SPM medium has been provided in Supplementary Table S1, and a reference to this table has been added in Section 2.3.

 

Table 2 Number of Siblings means Number of shoots?

 

Thank you for pointing out this ambiguity. We have updated Table 2 by replacing “Number of Siblings” with the clearer and more suitable term “Number of Shoots,” to align with standard micropropagation terminology.

 

All the measurement indexes in Figure 2 have no unit names.

 

We appreciate the reviewer highlighting this oversight. Consequently, we have revised Table 2 to display the correct unit names on all measurement axes.

 

There are occasional grammatical errors and awkward phrasing. For example:

" By merging morphological evaluations with computational modeling, our results aim to enhance shrub micropropagation systems and facilitate the sustainable commercial cultivation of black chokeberry." While promising, this statement is vague and should be substantiated with specific examples or projections.

We acknowledge that the original statement was too broad. Therefore, we revised it to emphasize how combining morphological data and machine learning in this study creates a predictive framework—achieving an R² > 0.95 for key traits—that can significantly cut down experimental iterations and improve protocol accuracy.

The interpretation of results is often descriptive rather than analytical. For example: The finding that 0.5 NAA + 0.25 IBA in Table 1 have the highest rooting rate is stated but not critically explored. Why does this combination perform better? Could this be due to their physiological reaction and interaction?

 

Thank you for highlighting the importance of a more analytical interpretation. In response, we have broadened the Discussion to include a physiological explanation for why the 0.5 mg/L NAA + 0.25 mg/L IBA treatment resulted in the highest rooting rates.

 

Why the number of Siblings, Shoot Length, and Shoot Length were performed better in Big Jar while the Leaf Diameter and Leaf Length were performed better in Small Jar? It needs more analyse.

 

Thank you for raising this insightful point, if we explain why large jars supported higher shoot numbers, shoot length, and number of leaves, while smaller jars resulted in greater leaf diameter and length. We interpret this as a physiological adjustment: larger containers improve gas exchange and space availability, promoting shoot proliferation, whereas smaller containers induce a mild constraint that leads to individual leaf expansion as a compensatory adaptation. This addition provides a more nuanced interpretation of our container volume findings.

Reviewer 2 Report

Comments and Suggestions for Authors

The authors conducted a study on Integrating In Vitro Propagation and Machine Learning Modeling for Efficient Shoot and Root Development in Aronia melanocarpa, which has certain value, but there are also several issues:

 

Lines 34–35: What is the optimal concentration specifically? Readers cannot obtain this information from the abstract.

 

Lines 79–80: The citation of Bayhan and Yücesan is too general and lacks specific information. What is the nature of the relationship mentioned? Moreover, the citation format is inappropriate and should be placed at the end of the sentence. Similar issues should also be corrected throughout the text.

 

Lines 81–82: The same issue applies to Kaya and Sarıyer, what is the relationship being referenced?

 

Lines 93–100: The text states that “SPM was more effective than traditional MS and WPM,” but does not provide the basal nutritional composition, micronutrients, pre-added hormones, or carbon source concentration of SPM.

 

Line 123: The Latin name of a plant should be abbreviated after its first appearance.

 

Lines 173–176: On what basis were these hormone combinations chosen?

 

Line 221: What was the total number of samples used in LOOCV? Where did the training data come from, and what type of samples were used?

 

Lines 225–229: The authors seem to have used only a few input variables. According to the description, only two input variables were used to predict six morphological traits. Does this present a risk of underfitting due to low dimensionality and limited modeling capacity?

 

Figure 3: The figure lacks a legend, and under the title "under different auxins," only three samples are shown.

 

Line 260: Aronia should be italicized.

 

Line 272: “Observed in the 0.5 IBA treatment alone (8.7 ± 8.7 mm)”, the standard deviation is as large as the mean. Can such highly dispersed data still be considered “significantly higher than other groups”?

 

Line 293: Under 0.5 IBA treatment, 8.70 ± 8.7a? A standard deviation equal to the mean? And still showing significant differences from other groups? What was the sample size used? Furthermore, with so many comparisons, using LSD is inappropriate, as LSD is prone to Type I errors under large-scale multiple comparisons.

 

Also, in the table, “57.1 ± 3.7b6”, what does “b6” mean?

 

Lines 298–302: Is this a table note? It suddenly appears in the main text without any contextual connection.

 

 

Lines 353–356: Although the combination of large jars and high BAP concentration significantly promoted shoot number and length, it should be noted that this effect may not solely result from hormone concentration but is closely related to the microenvironment formed by the culture vessels. Larger vessels may offer better gas exchange, lower humidity buildup, and more uniform hormone distribution, thereby mitigating toxicity or vitrification effects caused by hormone overdose.

 

Figure 4: Missing figure legend.

 

Lines 364–374: Only Pearson correlation coefficients (r) are provided, without corresponding p-values or significance levels. Especially when correlation coefficients are moderate or weak, biological interpretations lack meaning if p-values are not significant.

 

Line 409: What was the standardization method used for PCA?

 

Lines 538–544: Did the authors actually include data on acclimatization? Although the Materials and Methods section mentions “2.5 Acclimatization,” I did not find any corresponding data in the text. What is its relationship to the main topic?

Author Response

Reviewer 2

The authors conducted a study on Integrating In Vitro Propagation and Machine Learning Modeling for Efficient Shoot and Root Development in Aronia melanocarpa, which has certain value, but there are also several issues:

Lines 34–35: What is the optimal concentration specifically? Readers cannot obtain this information from the abstract.

 We revised the abstract to explicitly specify the optimal concentrations: 5 mg/L BAP in large jars for maximum shoot proliferation, and 0.5 mg/L NAA combined with 0.25 mg/L IBA for complete rooting. This offers clear and actionable guidance for readers.

Lines 79–80: The citation of Bayhan and Yücesan is too general and lacks specific information. What is the nature of the relationship mentioned? Moreover, the citation format is inappropriate and should be placed at the end of the sentence. Similar issues should also be corrected throughout the text.

 Thank you for highlighting this point. We have revised the sentence

Lines 81–82: The same issue applies to Kaya and Sarıyer, what is the relationship being referenced?

 Thank you for highlighting this point. We have revised the sentence

Lines 93–100: The text states that “SPM was more effective than traditional MS and WPM,” but does not provide the basal nutritional composition, micronutrients, pre-added hormones, or carbon source concentration of SPM.

Thank you for this constructive and important observation. In response, we have revised the relevant section of the manuscript to provide a concise explanation of the specific compositional improvements in SPM that contribute to its enhanced effectiveness for woody species. These include a balanced nitrogen profile, increased calcium and magnesium levels, optimized iron chelation, and an enriched vitamin composition. Additionally, we have clarified that no plant growth regulators (PGRs) were pre-added to the medium, allowing for customized hormonal supplementation depending on the species or experiment.

To maintain clarity in the main text, the complete formulation of the SPM medium has been provided in Supplementary Table S1, and a reference to this table has been added in Section 2.3.

Line 123: The Latin name of a plant should be abbreviated after its first appearance.

 Thanks, we have revised it

Lines 173–176: On what basis were these hormone combinations chosen?

Thank you for this insightful question. The auxin combinations used in the root induction phase were selected based on previously reported rooting protocols in woody shrubs, particularly those related to Aronia melanocarpa and similar taxa such as Rhus coriaria, Vaccinium spp., and Rubus spp. (Rusea et al., 2022; Polat & Eskimez, 2022).

Specifically, the use of IBA, NAA, and IAA either singly or in binary combinations reflects a commonly adopted strategy to explore potential synergistic effects of auxins on root initiation and elongation. Prior studies have shown that IBA enhances root elongation, while NAA improves root number and callus-free induction. We designed our combinations to evaluate both individual and interactive effects, optimizing the rooting response while minimizing undesired outcomes such as callus formation or root thickening.

This rationale has now been briefly added to the Materials and Methods section (Lines 211–218) for clarity.

Line 221: What was the total number of samples used in LOOCV? Where did the training data come from, and what type of samples were used?

Thank you for seeking this important clarification. We have now clearly indicated that the total dataset

Lines 225–229: The authors seem to have used only a few input variables. According to the description, only two input variables were used to predict six morphological traits. Does this present a risk of underfitting due to low dimensionality and limited modeling capacity?

 Thank you for this insightful comment. Our models used two input variables—PGR concentration and container volume—that align directly with the factorial experimental design of the study. Although the model complexity was low, the high predictive accuracy shown by LOOCV (with R² > 0.95 for most traits) indicates that underfitting was unlikely within this parameter space.

Figure 3: The figure lacks a legend, and under the title "under different auxins," only three samples are shown.

 Fig. 3 was revised.

Line 260: Aronia should be italicized.

 Corrected

Line 272: “Observed in the 0.5 IBA treatment alone (8.7 ± 8.7 mm)”, the standard deviation is as large as the mean. Can such highly dispersed data still be considered “significantly higher than other groups”?

Thank you for this important statistical observation. We have reviewed the post hoc analysis results, which confirmed that the 0.5 IBA treatment was significantly different from other groups despite the high standard deviation. However, we agree that the large relative variability warrants careful interpretation.

Line 293: Under 0.5 IBA treatment, 8.70 ± 8.7a? A standard deviation equal to the mean? And still showing significant differences from other groups? What was the sample size used? Furthermore, with so many comparisons, using LSD is inappropriate, as LSD is prone to Type I errors under large-scale multiple comparisons.

Regarding the high standard deviation: Although the LSD post hoc test indicated a significant difference for the 0.5 IBA treatment, we acknowledge that the standard deviation was equal to the mean (8.7 ± 8.7 mm), indicating substantial variability. We have revised the text to explicitly state this and to advise caution in interpreting the biological relevance of this difference.

Regarding use of LSD: We agree that LSD is more liberal and prone to Type I errors under multiple comparisons. In future studies, we intend to employ more conservative tests (e.g., Tukey HSD or Bonferroni) to better control experiment-wise error rates.

Also, in the table, “57.1 ± 3.7b6”, what does “b6” mean?

This was a typo; it has now been corrected.

Lines 298–302: Is this a table note? It suddenly appears in the main text without any contextual connection.

Thank you for highlighting the sudden placement. To enhance clarity, we've incorporated this statement into the Results section, explicitly linking it to Table 1.

Lines 353–356: Although the combination of large jars and high BAP concentration significantly promoted shoot number and length, it should be noted that this effect may not solely result from hormone concentration but is closely related to the microenvironment formed by the culture vessels. Larger vessels may offer better gas exchange, lower humidity buildup, and more uniform hormone distribution, thereby mitigating toxicity or vitrification effects caused by hormone overdose.

Thank you for your insightful comment. We agree that the improvements in shoot proliferation seen in large jars with higher BAP levels are probably due not just to hormone concentrations but also to the microenvironment created by the bigger containers. We have updated the Discussion to highlight this, pointing out that larger vessels improve gas exchange, decrease humidity buildup, and facilitate more even hormone distribution, helping to reduce potential toxicity or vitrification effects.

Figure 4: Missing figure legend.

Fig. 3 was revised.

Lines 364–374: Only Pearson correlation coefficients (r) are provided, without corresponding p-values or significance levels. Especially when correlation coefficients are moderate or weak, biological interpretations lack meaning if p-values are not significant.

Thank you for this valuable observation. We acknowledge that our correlation analysis reported only the Pearson correlation coefficients (r) without accompanying p-values. Our primary aim was to explore general linear relationships among morphological traits to complement the ANOVA findings, and thus we focused on the strength and direction of correlations as descriptive indicators. However, we agree that including p-values would provide a clearer understanding of statistical significance, particularly for moderate or weak correlations.

Line 409: What was the standardization method used for PCA?

Thank you for requesting this clarification. We confirm that before performing PCA, the data were standardized by centering each variable to a zero mean and scaling to unit variance (z-scores) using the prcomp function in R with center = TRUE and scale. = TRUE. This step ensures all morphological variables have equal influence on the PCA, regardless of their original units. We have now explicitly included this detail in the Methods section.

Lines 538–544: Did the authors actually include data on acclimatization? Although the Materials and Methods section mentions “2.5 Acclimatization,” I did not find any corresponding data in the text. What is its relationship to the main topic?

3.6 Acclimatization Success: Thank you for this helpful observation. Yes, acclimatization was performed following the rooting phase, and survival rates were recorded to assess the practical applicability of the in vitro protocol. In response to your comment, we have now included a brief summary of the acclimatization results in the Results section.

Specifically, survival rates varied from 60% in the control group to a maximum of 85% in the treatment combining 0.5 mg/L NAA + 0.25 mg/L IBA. NAA-based treatments generally yielded higher acclimatization success, indicating the downstream benefits of optimized auxin combinations during rooting. This addition helps demonstrate the full pipeline from in vitro induction to successful ex vitro establishment, reinforcing the practical relevance of the developed protocol.

Reviewer 3 Report

Comments and Suggestions for Authors

REVIEWER COMMENTS

Aronia melanocarpa (black chokeberry) is a highly nutritious and medicinal shrub, prized for its rich antioxidant and polyphenol content. Micropropagation is a powerful technique for the rapid, large-scale production of genetically uniform plants, particularly for species with low propagation efficiency or high genetic variability. However, optimizing tissue culture protocols often involves extensive trial-and-error experimentation due to the complex interactions between growth media, hormones, and environmental conditions. Recent advances in artificial intelligence (AI), particularly machine learning, offer a transformative approach by analyzing large datasets to predict optimal culture conditions, reducing time and resource expenditure. The integration of AI with micropropagation can enhance reproducibility, scalability, and efficiency, making it especially valuable for recalcitrant or commercially significant plant species. This synergy between biotechnology and computational modeling represents a promising frontier in plant propagation and biotechnology.

In this study, the authors aimed to enhance the efficiency and precision of Aronia melanocarpa micropropagation by integrating biotechnological methods with artificial intelligence. Specifically, the authors developed a callus-free micropropagation and rooting protocol using a modified SPM medium supplemented with varying concentrations of BAP and IBA, achieving high-frequency shoot proliferation and rooting without vitrification or callus formation. In parallel, supervised machine learning models—including Random Forest, XGBoost, Gaussian Process, and Multilayer Perceptron—were employed to predict key morphogenic traits based on culture conditions, with XGBoost and RF demonstrating superior performance.

TITLE:
No comment.

ABSTRACT:
The abstract requires minor revisions to improve clarity. Specifically:

• In the sentence, “High-frequency shoot proliferation and rooting were achieved without vitrification or callus formation under optimal PGR combinations.”, the authors omitted a summary of the quantitative findings. Including numerical data here would enhance the informativeness of the abstract.
• Additionally, the term “in vitro” and all similar Latin expressions should be italicized consistently throughout the manuscript.

KEYWORDS:
The current keywords could be optimized for better discoverability and specificity. I recommend the following alternatives: “Aronia melanocarpa; Micropropagation; Plant growth regulators; Artificial intelligence; Machine learning; Tissue culture optimization.”

INTRODUCTORY SECTION: The introduction is generally adequate, though minor revisions are needed.

• In the statement (lines 70–73), “Initial investigations concentrated on utilizing standard media, including Murashige and Skoog (MS) and Woody Plant Medium (WPM), augmented with cytokinins such as benzylaminopurine (BAP), kinetin (KIN), and thidiazuron (TDZ), which have demonstrated differential effects on shoot initiation and multiplication [7].”, the authors discuss multiple investigations but provide only a single reference. Please ensure citations are appropriately matched to the scope of the claims.
• In the phrase (line 77), “Recent research has addressed these issues by optimizing culture media and…”, the term “issues” is used in plural, yet the preceding paragraph explicitly mentions only one (the genotype-dependent response). Please clarify or elaborate on the other issues being referenced.
• Improve the specificity of the statement (line 117): “our results aim to enhance shrub micropropagation systems”. Is the developed protocol broadly applicable to all shrubs, or is it specific to Aronia melanocarpa? Clarification is needed.

MATERIALS AND METHODS:

• Update the heading “2.1 Plant Material” to: “2.1 Plant Material and Reagents”.
• The description of plant material should be streamlined and avoid first-person pronouns (e.g., “we”). The authors may consider the following revision:

“This study was conducted at the Tissue Culture Laboratories of the East Mediterranean Transitional Zone Agricultural Research Institute to establish a reliable micropropagation protocol for Aronia melanocarpa var. Nero (black chokeberry). Actively growing axillary buds were carefully collected from 3–4-year-old donor plants maintained under controlled greenhouse conditions in the institute’s experimental orchard. Using sterile instruments, the buds were precisely excised from lateral branches and immediately transferred to the laboratory to initiate the in vitro culture process.”

• Please include complete information on all reagents used, including product names and vendor details. This information should ideally be provided in a separate paragraph following the plant material description.
• In line 131, the sentence “the collected shoots underwent a cleansing process” is unclear and inconsistent with earlier references to axillary buds as the explants. If “shoots” and “buds” are being used interchangeably, please standardize terminology. Otherwise, clarify the difference and revise the paragraph for coherence.
• The sentence (line 134), “The initial disinfection step consisted of immersing the explants in 70% ethanol for 30 seconds to remove surface contaminants.” is imprecise. Since ethanol is only part of the disinfection process, stating that it removes contaminants might be misleading. Please revise this sentence to reflect its role in the broader sterilization protocol.
• In line 146, please provide a detailed composition of the newly formulated Shrub Plant Medium (SPM). This information should be included in the supplementary data for transparency and reproducibility.
• Regarding line 148: “To achieve consistent gelling, the medium was briefly boiled before being dispensed.” — this requires further explanation. Was autoclaving insufficient for gelling? This step is not described in subsequent procedures (lines 168–169), suggesting inconsistency. Please clarify why this step was necessary.
• In line 153, correct the formatting issue by ensuring there is a space between “1 mg/L” and “BAP”.
• In the caption of Figure 1, use “mL” instead of “cc” for consistency with the rest of the manuscript.
• For clarity and accessibility, the treatment combinations described in lines 173–175 should be presented in a table format.
• In the sentence (lines 183–185): “The acclimation process commenced by gradually loosening the lids of the containers to permit incremental exposure to ambient humidity.”, the duration and specific parameters of the acclimatization process are not provided. Were humidity, temperature, or survival rate monitored? Please elaborate if these were relevant.
• Subsections 2.3 to 2.6 do not mention the number of biological replicates used. This is a significant omission. If no replicates were used, please explain how the statistical analysis was performed. Were the experiments repeated independently? This must be clearly described to validate the robustness of the study design.
• Several areas for improvement were identified in the study's machine learning methodology. First, the data pre-processing steps lacked detail, particularly regarding normalization or scaling—crucial for models like SVM and MLP—as well as the handling of outliers and categorical variables such as auxin combinations. Second, the use of leave-one-out cross-validation (LOOCV) raises concerns due to its high computational cost and potential for high-variance performance estimates; a more efficient alternative like 10-fold CV may offer a better balance. Third, the process for hyperparameter tuning was unclear—specifics on methods (e.g., grid or random search) for optimizing parameters like RF’s mtry or MLP’s architecture were not provided. Fourth, model interpretability could be enhanced by analyzing feature importance in tree-based models to extract biological insights, such as identifying key factors influencing morphogenesis. Finally, it was unclear whether interactions between paired auxins were explicitly modeled, which could be important for capturing synergistic or antagonistic effects in morphogenic responses.

RESULTS:

Under the Results section, it is evident that all subheadings are currently structured around statistical analyses rather than the core findings of the study. This approach is unconventional and detracts from the narrative clarity. The authors are encouraged to revise the subheadings to reflect the key experimental findings. Additionally, restructuring the results with thematically appropriate subheadings will significantly improve readability and coherence (see the recommended structure in this study: https://doi.org/10.1371/journal.pone.0307823).

To maintain logical flow and facilitate comprehension, the results should follow the same sequence as described in the Methods section—namely, beginning with shooting, followed by rooting, and finally acclimatization. Please revise the section accordingly, and ensure that all figure and table numberings are updated to reflect this new structure.

The caption for Figure 3 is insufficient as it does not specify the treatments involved, thereby limiting interpretability. Please revise the figure caption to include this critical information.

The following analyses currently found in lines 307–312 and 322–324 should be moved to the Discussion section:

• “The significant two-way interaction between BAP concentration and jar size indicates that the proliferation response is dependent on hormones and heavily influenced by the microenvironment in the culture vessel.”
• “Shoot elongation peaked in large jars with 5 mg/L BAP (75.6 ± 3.6 mm), reflecting a beneficial interaction between high cytokinin concentration and better gas exchange or more available space.”
• “This suggests that elevated cytokinin levels in larger volumes may favor proliferation at the cost of individual organ growth.” These interpretive statements are not appropriate for the Results section, which should be limited to objective data presentation. Please review the entire section to eliminate similar instances.

Regarding the correlation analysis, the focus should have been confined to examining the relationships between the primary independent variables (vessel size and PGR treatments) and the morphological traits. This would have aligned better with the study’s stated objectives. The current inclusion of PCA appears to unnecessarily inflate the analysis and is not justified by the study’s goals.

The supervised machine learning results could benefit from several refinements:

1. Model comparisons lack statistical validation. Performance differences between models (e.g., RF and MLP vs. GBM) should be substantiated using ANOVA or pairwise comparisons (e.g., “RF and MLP significantly outperformed GBM, p < 0.05, Tukey’s HSD”).
2. Exceptionally high R² values (≥0.90) suggest potential overfitting. To demonstrate robustness, consider reporting LOOCV results and providing train–test RMSE or MAE ratios (e.g., “consistent train–test RMSE ratios ≤ 1.2”).
3. Feature-importance analyses were underutilized. For instance, “RF feature-importance ranked BAP concentration as the top predictor for shoot proliferation (relative importance = 0.85).”
4. The underperformance of GBM warrants further inspection. Examine whether its hyperparameters were properly tuned (e.g., tree depth, learning rate), as “GBM’s higher RMSE may reflect suboptimal tuning or sensitivity to noisy traits.”
5. The low R² values for root thickness (0.19–0.56) could be attributed to biological or measurement variability. This may stem from auxin-distribution heterogeneity or subjectivity in measurement, which should be explored in more detail.

Overall Assessment of the Results Section: This section is disorganized and requires substantial restructuring in accordance with academic writing standards. The information should be grouped and presented under logical and descriptive subheadings that reflect the experimental outcomes. Many critical elements are missing or unclear, as noted in the comments above. Importantly, this section should focus exclusively on presenting the results without interpretation.

DISCUSSION & CONCLUSION:

The Discussion section should mirror the structure established in the Methods and Results sections to maintain logical consistency. The current lack of alignment disrupts the narrative and compromises interpretability. This organizational issue extends into the Conclusion section and must be comprehensively addressed.

The authors refer to findings on acclimatization, yet these are not presented in the Results section. This indicates a disconnect between data presentation and interpretation, further reinforcing the need for better structure and transparency.

Given the extensive revisions required in the Results section, it is clear that both the Discussion and Conclusion sections must also be revised accordingly. These sections must reflect the corrected analyses and interpretations.

Furthermore, the Discussion should adopt a systematic approach to comparing and contrasting the study’s findings with existing literature, thereby situating the work within the broader research landscape. The contribution of this study should be contextualized clearly.

Lastly, the limitations of the study—of which there are several—should be explicitly acknowledged. A thoughtful discussion of these limitations, along with recommendations for future research, is essential and should not be omitted. This is an imperative for any rigorous scientific manuscript.

Comments for author File: Comments.pdf

Comments on the Quality of English Language

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Author Response

Reviewer 3

Aronia melanocarpa (black chokeberry) is a highly nutritious and medicinal shrub, prized for its rich antioxidant and polyphenol content. Micropropagation is a powerful technique for the rapid, large-scale production of genetically uniform plants, particularly for species with low propagation efficiency or high genetic variability. However, optimizing tissue culture protocols often involves extensive trial-and-error experimentation due to the complex interactions between growth media, hormones, and environmental conditions. Recent advances in artificial intelligence (AI), particularly machine learning, offer a transformative approach by analyzing large datasets to predict optimal culture conditions, reducing time and resource expenditure. The integration of AI with micropropagation can enhance reproducibility, scalability, and efficiency, making it especially valuable for recalcitrant or commercially significant plant species. This synergy between biotechnology and computational modeling represents a promising frontier in plant propagation and biotechnology.

In this study, the authors aimed to enhance the efficiency and precision of Aronia melanocarpa micropropagation by integrating biotechnological methods with artificial intelligence. Specifically, the authors developed a callus-free micropropagation and rooting protocol using a modified SPM medium supplemented with varying concentrations of BAP and IBA, achieving high-frequency shoot proliferation and rooting without vitrification or callus formation. In parallel, supervised machine learning models—including Random Forest, XGBoost, Gaussian Process, and Multilayer Perceptron—were employed to predict key morphogenic traits based on culture conditions, with XGBoost and RF demonstrating superior performance.

TITLE:
No comment.

ABSTRACT:
The abstract requires minor revisions to improve clarity. Specifically:

In the sentence, “High-frequency shoot proliferation and rooting were achieved without vitrification or callus formation under optimal PGR combinations.”, the authors omitted a summary of the quantitative findings. Including numerical data here would enhance the informativeness of the abstract.

 

We revised the abstract to explicitly specify the optimal concentrations: 5 mg/L BAP in large jars for maximum shoot proliferation, and 0.5 mg/L NAA combined with 0.25 mg/L IBA for complete rooting.

 

Additionally, the term “in vitro” and all similar Latin expressions should be italicized consistently throughout the manuscript.

Corrected

KEYWORDS:
The current keywords could be optimized for better discoverability and specificity. I recommend the following alternatives: “Aronia melanocarpa; Micropropagation; Plant growth regulators; Artificial intelligence; Machine learning; Tissue culture optimization.”

Thank you for this helpful suggestion. We concur that the proposed keywords enhance our article's precision and visibility. Therefore, we have revised the keywords section in our manuscript.

INTRODUCTORY SECTION: The introduction is generally adequate, though minor revisions are needed.

In the statement (lines 70–73), “Initial investigations concentrated on utilizing standard media, including Murashige and Skoog (MS) and Woody Plant Medium (WPM), augmented with cytokinins such as benzylaminopurine (BAP), kinetin (KIN), and thidiazuron (TDZ), which have demonstrated differential effects on shoot initiation and multiplication [7].”, the authors discuss multiple investigations but provide only a single reference. Please ensure citations are appropriately matched to the scope of the claims.

Thanks for your thoughtful comment. We recognize the importance of ensuring citations accurately reflect the scope of claims. Reference [7] thoroughly covers the use of MS and WPM media, along with various cytokinins like BAP, KIN, and TDZ, in micropropagation protocols. As such, we believe this single reference sufficiently supports the statement. However, we have carefully reviewed the sentence to ensure its scope aligns precisely with the content of Reference [7].

 

In the phrase (line 77), “Recent research has addressed these issues by optimizing culture media and…”, the term “issues” is used in plural, yet the preceding paragraph explicitly mentions only one (the genotype-dependent response). Please clarify or elaborate on the other issues being referenced.

 

Thank you for highlighting this point. We agree that the original wording could confuse. To clarify, we have revised the sentence.

 

Improve the specificity of the statement (line 117): “our results aim to enhance shrub micropropagation systems”. Is the developed protocol broadly applicable to all shrubs, or is it specific to Aronia melanocarpa? Clarification is needed.

Thank you for highlighting this important point. Our results mainly improve micropropagation protocols for Aronia melanocarpa, though the methodological approach could also be adapted for other woody shrubs.

MATERIALS AND METHODS:

Update the heading “2.1 Plant Material” to: “2.1 Plant Material and Reagents”.

Corrected

 

The description of plant material should be streamlined and avoid first-person pronouns (e.g., “we”). The authors may consider the following revision:

“This study was conducted at the Tissue Culture Laboratories of the East Mediterranean Transitional Zone Agricultural Research Institute to establish a reliable micropropagation protocol for Aronia melanocarpa var. Nero (black chokeberry). Actively growing axillary buds were carefully collected from 3–4-year-old donor plants maintained under controlled greenhouse conditions in the institute’s experimental orchard. Using sterile instruments, the buds were precisely excised from lateral branches and immediately transferred to the laboratory to initiate the in vitro culture process.”

Please include complete information on all reagents used, including product names and vendor details. This information should ideally be provided in a separate paragraph following the plant material description.

 

Sentences Revised

 

In line 131, the sentence “the collected shoots underwent a cleansing process” is unclear and inconsistent with earlier references to axillary buds as the explants. If “shoots” and “buds” are being used interchangeably, please standardize terminology. Otherwise, clarify the difference and revise the paragraph for coherence.

Thank you for this helpful observation. We realize that our initial wording inconsistently used terms like “shoots” and “axillary buds,” which could lead to confusion about the exact nature of the explants used. To clarify and ensure consistency, we have updated the text.

 

The sentence (line 134), “The initial disinfection step consisted of immersing the explants in 70% ethanol for 30 seconds to remove surface contaminants.” is imprecise. Since ethanol is only part of the disinfection process, stating that it removes contaminants might be misleading. Please revise this sentence to reflect its role in the broader sterilization protocol.

 

Thank you for pointing this out. We agree that the original statement possibly exaggerated ethanol's role. We have updated the sentence.e

 

In line 146, please provide a detailed composition of the newly formulated Shrub Plant Medium (SPM). This information should be included in the supplementary data for transparency and reproducibility.

 

To maintain clarity in the main text, the complete formulation of the SPM medium has been provided in Supplementary Table S1, and a reference to this table has been added in Section 2.3.

 

Regarding line 148: “To achieve consistent gelling, the medium was briefly boiled before being dispensed.” — This requires further explanation. Was autoclaving insufficient for gelling? This step is not described in subsequent procedures (lines 168–169), suggesting an inconsistency. Please clarify why this step was necessary.

Thank you for this important question. The brief boiling step was included to ensure complete dissolution of agar and other medium components before autoclaving. This step is especially useful for achieving uniform gelling, which is essential for woody plant tissue culture media that might otherwise gel unevenly. We have updated the sentence in the text accordingly.

 

In line 153, correct the formatting issue by ensuring there is a space between “1 mg/L” and “BAP”.

Thank you for bringing this formatting issue to our attention. We have corrected the spacing

 

In the caption of Figure 1, use “mL” instead of “cc” for consistency with the rest of the manuscript.

 

Revised

 

For clarity and accessibility, the treatment combinations described in lines 173–175 should be presented in a table format.

Thank you for suggesting improvements in clarity. We have now standardized the container volume descriptions to milliliters (mL), consistently referring to them as 425 mL (small jar) and 600 mL (large jar) throughout the manuscript.

In the sentence (lines 183–185): “The acclimation process commenced by gradually loosening the lids of the containers to permit incremental exposure to ambient humidity.”, the duration and specific parameters of the acclimatization process are not provided. Were humidity, temperature, or survival rate monitored? Please elaborate if these were relevant.

 

2.5 Acclimatization: Thank you for your valuable comment. We have now expanded the description of the acclimatization procedure.

 

Subsections 2.3 to 2.6 do not mention the number of biological replicates used. This is a significant omission. If no replicates were used, please explain how the statistical analysis was performed. Were the experiments repeated independently? This must be clearly described to validate the robustness of the study design.

 

section 2.6 has been revised.

 

Several areas for improvement were identified in the study's machine learning methodology. First, the data pre-processing steps lacked detail, particularly regarding normalization or scaling—crucial for models like SVM and MLP—as well as the handling of outliers and categorical variables such as auxin combinations. Second, the use of leave-one-out cross-validation (LOOCV) raises concerns due to its high computational cost and potential for high-variance performance estimates; a more efficient alternative like 10-fold CV may offer a better balance. Third, the process for hyperparameter tuning was unclear—specifics on methods (e.g., grid or random search) for optimizing parameters like RF’s mtry or MLP’s architecture were not provided. Fourth, model interpretability could be enhanced by analyzing feature importance in tree-based models to extract biological insights, such as identifying key factors influencing morphogenesis. Finally, it was unclear whether interactions between paired auxins were explicitly modeled, which could be important for capturing synergistic or antagonistic effects in morphogenic responses.

Thank you for your comprehensive and thoughtful evaluation of our machine learning methodology. We address each of your concerns as follows. Regarding data pre-processing, we performed z-score standardization (centering to zero mean and scaling to unit variance) across all numerical predictors, which is particularly important for models sensitive to scaling, such as SVM and MLP; we have now clarified this in the Methods section. Outlier inspection was conducted using boxplots, and no extreme values outside biological plausibility were identified, while categorical variables such as auxin combinations were treated as factors. Concerning the use of leave-one-out cross-validation (LOOCV), given our relatively small datasets, we chose LOOCV to maximize training data in each iteration; however, we acknowledge that k-fold cross-validation (e.g., 10-fold) may offer a better balance of variance and computational efficiency and have noted this as a future improvement. We also confirm that we did not conduct explicit hyperparameter tuning (e.g., via grid or random search) in this exploratory study, instead employing standard default settings to establish a comparative baseline; we recognize this as a limitation and plan to incorporate systematic optimization in subsequent research. Similarly, while we did not perform detailed interpretability analyses such as extracting feature importance from tree-based models, we agree that this could yield valuable biological insights and have identified it as an important direction for future work. Finally, although our factorial design inherently included auxin combinations, explicit interaction modeling was not implemented in the machine learning framework, and we acknowledge this as another opportunity for methodological enhancement.

RESULTS:

Under the Results section, it is evident that all subheadings are currently structured around statistical analyses rather than the core findings of the study. This approach is unconventional and detracts from the narrative clarity. The authors are encouraged to revise the subheadings to reflect the key experimental findings. Additionally, restructuring the results with thematically appropriate subheadings will significantly improve readability and coherence (see the recommended structure in this study: https://doi.org/10.1371/journal.pone.0307823).

Thank you for this valuable suggestion. We concur that focusing the Results section on the main experimental findings, rather than on statistical methods, greatly enhances clarity and readability. Accordingly, we have reorganized the Results section with new subheadings based on themes.

To maintain logical flow and facilitate comprehension, the results should follow the same sequence as described in the Methods section—namely, beginning with shooting, followed by rooting, and finally acclimatization. Please revise the section accordingly, and ensure that all figure and table numberings are updated to reflect this new structure.

Thank you for your helpful suggestion. We have thoroughly revised the Results section to align with the logical order outlined in the Methods.

The caption for Figure 3 is insufficient as it does not specify the treatments involved, thereby limiting interpretability. Please revise the figure caption to include this critical information.

Figures are Revised

The following analyses currently found in lines 307–312 and 322–324 should be moved to the Discussion section:

• “The significant two-way interaction between BAP concentration and jar size indicates that the proliferation response is dependent on hormones and heavily influenced by the microenvironment in the culture vessel.”
• “Shoot elongation peaked in large jars with 5 mg/L BAP (75.6 ± 3.6 mm), reflecting a beneficial interaction between high cytokinin concentration and better gas exchange or more available space.”
• “This suggests that elevated cytokinin levels in larger volumes may favor proliferation at the cost of individual organ growth.” These interpretive statements are not appropriate for the Results section, which should be limited to objective data presentation. Please review the entire section to eliminate similar instances.

Thank you for guiding us on relocating interpretive statements from the Results to the Discussion properly. We have revised accordingly.

Regarding the correlation analysis, the focus should have been confined to examining the relationships between the primary independent variables (vessel size and PGR treatments) and the morphological traits. This would have aligned better with the study’s stated objectives. The current inclusion of PCA appears to unnecessarily inflate the analysis and is not justified by the study’s goals.

Thank you for your insightful observation. We agree that analyzing correlations directly between the primary independent variables—vessel size and PGR treatments—and the morphological traits would better align with our study’s goals. However, because vessel size and PGR treatments are categorical factors examined through ANOVA and factorial design, calculating correlations in this context would not be statistically appropriate. Instead, our correlation and PCA analyses were designed to examine how morphological traits varied together across different treatment combinations, offering insights into potential trade-offs important for optimizing protocols. We recognize that PCA extends beyond our immediate objectives. In future studies, we plan to incorporate multivariate methods more directly linked to treatment effects to enhance biological understanding.

The supervised machine learning results could benefit from several refinements:

1. Model comparisons lack statistical validation. Performance differences between models (e.g., RF and MLP vs. GBM) should be substantiated using ANOVA or pairwise comparisons (e.g., “RF and MLP significantly outperformed GBM, p < 0.05, Tukey’s HSD”).

 

We agree that formal statistical comparisons (for example, ANOVA or pairwise Tukey’s HSD on RMSE/MAE across models) would offer stronger evidence for differences in predictive performance. Because of the structure of our analysis (LOOCV on relatively small datasets), we did not initially conduct such tests, but we recognize their significance and have noted this as a methodological limitation in the revised Discussion, highlighting it as a priority for future research.

 

1. Exceptionally high R² values (≥0.90) suggest potential overfitting. To demonstrate robustness, consider reporting LOOCV results and providing train–test RMSE or MAE ratios (e.g., “consistent train–test RMSE ratios ≤ 1.2”).

 

We observe that the consistently high R² values (often above 0.90) might indicate potential overfitting. However, since these were obtained from LOOCV—which inherently tests on unseen data—this somewhat reduces the concern. To strengthen this, we have now explicitly included the LOOCV-based RMSE and MAE, which serve as cross-validated performance measures, and emphasized the importance of systematically examining train–test RMSE ratios in future datasets.

 

1. Feature-importance analyses were underutilized. For instance, “RF feature-importance ranked BAP concentration as the top predictor for shoot proliferation (relative importance = 0.85).”

 

We agree that extracting feature importance, especially from RF models, would provide valuable biological insights. Although not explored in the current exploratory study, we have included a statement in the Discussion acknowledging this as a key future direction, which could reveal, for example, the relative influence of BAP concentration or vessel volume on shoot multiplication.

 

1. The underperformance of GBM warrants further inspection. Examine whether its hyperparameters were properly tuned (e.g., tree depth, learning rate), as “GBM’s higher RMSE may reflect suboptimal tuning or sensitivity to noisy traits.”

 

The higher RMSE observed for GBM might be attributed to the default parameter settings, since we did not perform explicit hyperparameter tuning, such as adjusting tree depth or learning rates, in this initial study. We recognize this as a limitation and intend to include systematic tuning methods, like grid or random search, in future work to enhance GBM calibration and performance.

 

1. The low R² values for root thickness (0.19–0.56) could be attributed to biological or measurement variability. This may stem from auxin-distribution heterogeneity or subjectivity in measurement, which should be explored in more detail.

 

We agree that the limited predictive power for root thickness (R² between 0.19 and 0.56) probably results from inherent biological variability, such as uneven auxin distribution and microenvironment influences, as well as potential subjective measurement differences. This is now clearly addressed in the revised manuscript to explain the prediction difficulties related to this trait.

 

Overall Assessment of the Results Section: This section is disorganized and requires substantial restructuring in accordance with academic writing standards. The information should be grouped and presented under logical and descriptive subheadings that reflect the experimental outcomes. Many critical elements are missing or unclear, as noted in the comments above. Importantly, this section should focus exclusively on presenting the results without interpretation.

Thank you for your valuable feedback on the Results section. In response, we have extensively reorganized this section to meet academic standards and enhance clarity and logical flow. The results are now organized under descriptive subheadings that directly correspond to the main experimental findings, following the same order as the Methods: (1) effects of BAP concentration and container volume on shoot multiplication, (2) impact of auxin treatments on rooting responses, (3) acclimatization outcomes, (4) correlations among morphological traits, (5) multivariate trait patterns via PCA, and (6) machine learning-based predictive models. We also thoroughly reviewed the entire Results section to remove interpretive comments and moved them to the Discussion, ensuring a clear distinction between objective data presentation and biological interpretation. Furthermore, we incorporated clarifications from previous comments. We believe these revisions greatly improve the organization, clarity, and scientific rigor of the Results section.

 

DISCUSSION & CONCLUSION:

The Discussion section should mirror the structure established in the Methods and Results sections to maintain logical consistency. The current lack of alignment disrupts the narrative and compromises interpretability. This organizational issue extends into the Conclusion section and must be comprehensively addressed.

Thank you for this valuable overall observation. We fully recognize the importance of maintaining a consistent structure across the Methods, Results, Discussion, and Conclusion to aid reader comprehension. While completely reorganizing the Discussion to exactly match the Methods and Results was not feasible in this revision, we carefully reviewed and adjusted it to follow the same general progression of key findings—starting with shoot proliferation, then rooting responses, trait relationships, and ending with multivariate and predictive modeling insights. We aligned interpretations more closely with the order in the Results to improve narrative flow. This approach maintains logical consistency while allowing the Discussion to incorporate comparisons with existing literature and broader biological context, which often span multiple sections. We also ensured that the Conclusion reinforces this logical sequence clearly. We appreciate your guidance and plan to adopt a more explicitly parallel structure in future manuscripts to enhance clarity.

The authors refer to findings on acclimatization, yet these are not presented in the Results section. This indicates a disconnect between data presentation and interpretation, further reinforcing the need for better structure and transparency.

3.6 Acclimatization Success: Thank you for this helpful observation. Yes, acclimatization was performed following the rooting phase, and survival rates were recorded to assess the practical applicability of the in vitro protocol. In response to your comment, we have now included a brief summary of the acclimatization results in the Results section.

Given the extensive revisions required in the Results section, it is clear that both the Discussion and Conclusion sections must also be revised accordingly. These sections must reflect the corrected analyses and interpretations.

Thank you for your valuable feedback on the Results section. In response, we have extensively reorganized this section to meet academic standards and enhance clarity and logical flow. The results and discussion are now revised accordingly.

Furthermore, the Discussion should adopt a systematic approach to comparing and contrasting the study’s findings with existing literature, thereby situating the work within the broader research landscape. The contribution of this study should be contextualized clearly.

We have revised and edited the discussion

Lastly, the limitations of the study—of which there are several—should be explicitly acknowledged. A thoughtful discussion of these limitations, along with recommendations for future research, is essential and should not be omitted. This is an imperative for any rigorous scientific manuscript.

Thank you for highlighting this important point. We have now explicitly included a discussion of the study’s limitations and outlined specific directions for future research to address these issues, which enhances the manuscript’s rigor and transparency.

 

 

 

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

Some of the figures in the manuscript need to add scale bars, such as Fig. 2, Fig. 3, and Fig. 4.

Author Response

Reviewer 1

Some of the figures in the manuscript need to add scale bars, such as Fig. 2, Fig. 3, and Fig. 4.

Thank you for your valuable suggestion. We have revised the figures accordingly and added appropriate scale bars to Fig. 2, Fig. 3, and Fig. 4 to enhance clarity and accuracy.

Reviewer 2 Report

Comments and Suggestions for Authors

 I have no further comments. However, I suggest that the authors further optimize the figure layout, as the revised figures remain visually unsatisfactory.

Author Response

Reviewer 2

I have no further comments. However, I suggest that the authors further optimize the figure layout, as the revised figures remain visually unsatisfactory.

Thank you for your feedback. We appreciate your suggestion regarding the figure layout. Accordingly, we have further optimized the layout of the revised figures to improve their visual quality and overall presentation.

Reviewer 3 Report

Comments and Suggestions for Authors

REVIEWER COMMENTS

The authors have done a good job addressing most of the major concerns highlighted in my previous comments. However, minor issues remain to be addressed:

Firstly, in the title, the last part of the scientific name needs to be italicized for uniformity with the first part.

Secondly, relocate the paragraph, “All reagents and culture media components used in this study were of analytical grade. Plant growth regulators, including IAA (Indole-3-acetic acid), IBA (Indole-3-butyric acid), and NAA (1-Naphthaleneacetic acid), were purchased from Sigma-Aldrich (St. Louis, MO, USA). Macronutrients and micronutrients used in the SPM formulation were also obtained from Sigma-Aldrich (St. Louis, MO, USA). Vitamins (thiamine HCl, nicotinic acid, pyridoxine HCl, and myo-inositol) and gelling agents (agar) were sourced from Duchefa Biochemie (Haarlem, The Netherlands). Ethanol (≥96%) and sodium hypochlorite were acquired from Sigma-Aldrich (St. Louis, MO, USA). All solutions were prepared using sterile distilled water, and pH was adjusted using 1N NaOH or HCl as needed.” and place it as the second paragraph under the subsection “2.1 Plant Material and Reagents”.

Thirdly, most of the content in the supplementary data file has not been referenced in the main text. Please revise accordingly.

Fourthly, in subheading 3.1, correct “Ausxin” to “Auxin”.

Lastly, there is still lingering interpretation of results in the Results section. The authors should pay attention to words like “indicate”, “suggest”, which are markers of interpretation. Please double-check the Results section and streamline accordingly. Following this streamlining, the content that needs to be relocated to the Discussion should be integrated seamlessly.

Comments on the Quality of English Language

See my comments!

Author Response

Reviewer 3

The authors have done a good job addressing most of the major concerns highlighted in my previous comments. However, minor issues remain to be addressed:

Firstly, in the title, the last part of the scientific name needs to be italicized for uniformity with the first part.

We thank the reviewer for this helpful observation. The title has been corrected so that the entire scientific name (Aronia melanocarpa) is now consistently italicized.

Secondly, relocate the paragraph, “All reagents and culture media components used in this study were of analytical grade. Plant growth regulators, including IAA (Indole-3-acetic acid), IBA (Indole-3-butyric acid), and NAA (1-Naphthaleneacetic acid), were purchased from Sigma-Aldrich (St. Louis, MO, USA). Macronutrients and micronutrients used in the SPM formulation were also obtained from Sigma-Aldrich (St. Louis, MO, USA). Vitamins (thiamine HCl, nicotinic acid, pyridoxine HCl, and myo-inositol) and gelling agents (agar) were sourced from Duchefa Biochemie (Haarlem, The Netherlands). Ethanol (≥96%) and sodium hypochlorite were acquired from Sigma-Aldrich (St. Louis, MO, USA). All solutions were prepared using sterile distilled water, and pH was adjusted using 1N NaOH or HCl as needed.” and place it as the second paragraph under the subsection “2.1 Plant Material and Reagents”.

Thank you for your valuable suggestion, we placed in the subsection 2.1 Plant Material and Reagents

Thirdly, most of the content in the supplementary data file has not been referenced in the main text. Please revise accordingly.

We thank the reviewer for this important observation. We have now thoroughly revised the manuscript to ensure that all supplementary tables and figures are properly referenced and integrated into the main text.

Fourthly, in subheading 3.1, correct “Ausxin” to “Auxin”.

Thank you, it is corrected

Lastly, there is still lingering interpretation of results in the Results section. The authors should pay attention to words like “indicate”, “suggest”, which are markers of interpretation. Please double-check the Results section and streamline accordingly. Following this streamlining, the content that needs to be relocated to the Discussion should be integrated seamlessly.

We appreciate the reviewer’s insightful comment. We have carefully re-examined the Results section, removed interpretive language (e.g., “indicate,” “suggest”), and relocated such content to the Discussion to ensure a clear separation between factual findings and interpretation.

 

Also, The language of the article has been edited and improved.