Innovative Protocols for Blackberry Propagation: In Vitro Cultivation in Temporary Immersion Systems with Ex Vitro Acclimatization

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

Dear Authors,

Thank you for the opportunity to review the manuscript "Innovative Protocols for Blackberry (Rubus spp.) Propagation: In Vitro Cultivation and Ex Vitro Acclimatization with Temporary Immersion Systems".

I appreciate the work and considerable effort you have invested in this study, as reflected in the large number of experiments and data presented. However, I believe the manuscript requires careful revision to improve its clarity and coherence. In this regard, I am providing several suggestions that I believe will help enhance the quality of the article.

Keywords:

Some keywords already appear in the title. I recommend either removing these keywords or replacing them with a more specific or related term that adds value and improves discoverability.

1.      Introduction

I suggest adding a sentence at the end of the introduction to clearly state the aim and objectives of the research.

2.      Materials and Methods

Line 78 – Provide some common characteristics of the three varieties, such as the fact that they are thorny.

2.4. Multiplication of Plant Material – Please indicate the volume of the culture medium per jar and the number of explants per jar.

2.5. Establishment of Temporary Immersion Systems (TIS) – For the bioreactor culture, did you use 2-liter jars or RITA bioreactors? The bioreactors shown in Figure 6 do not appear to be RITA bioreactors. Please clarify.

Additionally, specify the volume of the culture medium per bioreactor and the number of explants per bioreactor.

2.6. Effect of 6-Benzylaminopurine (6-BAP) on TIS – In this section, you should provide more details, including: the type of culture medium used, the volume of the medium, the number of explants per bioreactor, the frequency and duration of immersion, as well as the frequency and duration of aeration.

Sections 2.7. Rooting of In Vitro Plants and 2.8. Effect of Indolebutyric Acid (IBA) on Rooting should be merged, as both address in vitro rooting. Furthermore, I suggest eliminating Section 2.7, since this experiment is not discussed in the Results section.

2.9. In Vitro Acclimatization? – I believe this refers to Ex Vitro acclimatization.

Please specify the age of the plantation for which you evaluated the field performances.

I also noticed that this section contains numerous citations, including review articles. Typically, the Materials and Methods section includes citations for standardized methods, modifications of existing methods, and the use of specialized equipment or software. I suggest reviewing this section and retaining only citations that are strictly necessary for clarity and replicability.

In conclusion, the Materials and Methods section should be rewritten more clearly and concisely to ensure that the experiments can be easily replicated.

3.      Results

I suggest rewriting this section as well. It is not necessary for each experiment presented in a table or figure to have its own subsection. Instead, try to group the results into four main sections: (1) results related to the initiation and stabilization of in vitro cultures, (2) results from the in vitro multiplication stage, (3) results related to rooting, and (4) acclimatization and field performance. If necessary, you may include a few subsections under these main sections.

Please check table 5 where you say that "Means with different letters indicate significance...." but in the table you have no letters

Also, under different tables you have the following explanation: "Means with different letters indicate significance (two-way ANOVA), Tukey, p ≤ 0.05)". I suggest you replace it with "Means with different letters indicate significant differences (two-way ANOVA followed by Tukey’s test, p ≤ 0.05)" to clarify that different letters indicate significant differences.

In Figure 6, please try to arrange the images in chronological order, starting with the initiation of in vitro culture and ending with the acclimatized plants.

I suggest using the same abbreviation consistently throughout the manuscript for Temporary Immersion Systems, specifically TIS instead of SIT. Likewise, for in vitro and ex vitro, please ensure uniform formatting—either italicized or non-italicized—throughout the manuscript.

4.      Disscution

This section is very long and should also be rewritten. Try not to repeat results without further interpretation. The significance of the results obtained should be explained in relation to the objectives of the study.

5.      Conclusions

In the Conclusions section you mentioned a number of specific results but lack a final sentence that would emphasize the overall impact of the study

 

 

In conclusion, I suggest you ensuring that the entire manuscript follows a clear, coherent structure centered around a four main sections. This will create a consistent narrative flow, allowing the reader to easily follow the connection between the experimental stages: (1) initiation and stabilization of in vitro cultures, (2) in vitro multiplication, (3) rooting, and (4) acclimatization and field performance.

Author Response

Response to Reviewer Comments

Reviewer Comment 1:
"Some keywords already appear in the title. I recommend either removing these keywords or replacing them with a more specific or related term that adds value and improves discoverability."

Author Response:
We sincerely thank the reviewer for this constructive suggestion. To improve the discoverability and relevance of the manuscript, we have revised the list of keywords by removing terms that overlap with the title and replacing them with more specific terms that better reflect the study's focus.

Reviewer Comment 2:
"I suggest adding a sentence at the end of the introduction to clearly state the aim and objectives of the research."

Author Response:
We appreciate the reviewer's recommendation to clarify the study's aims and objectives.

Reviewer Comment 3:
"Line 78 – Provide some common characteristics of the three varieties, such as the fact that they are thorny."

Author Response:
We thank the reviewer for this observation. To provide additional context, we have included the following clarification about the characteristics of the three blackberry varieties:

 "The blackberry varieties Tupy, Brazos, and Kiowa share several common characteristics that contribute to their commercial viability. Notably, all three varieties possess thorny stems, which can impact management practices and harvesting efficiency. Additionally, they demonstrate high survival rates under field conditions, exceeding 97%, highlighting their resilience and adaptability to diverse cultivation environments."

 Reviewer Comment 4:
"2.4. Multiplication of Plant Material – Please indicate the volume of the culture medium per jar and the number of explants per jar."

Author Response:
We appreciate the reviewer's request for additional details. To clarify, we have added the following information to Section 2.4:

"Each jar was filled with 50 mL of liquid full-strength MS medium, prepared with 4.2 g L⁻¹ MS salts and 30 g L⁻¹ sucrose. To prevent excessive competition for nutrients and space, each jar was inoculated with five explants, following established protocols for optimized blackberry micropropagation [12]."

 Reviewer Comment 5:
"2.5. Establishment of Temporary Immersion Systems (TIS) – For the bioreactor culture, did you use 2-liter jars or RITA bioreactors? The bioreactors shown in Figure 6 do not appear to be RITA bioreactors. Please clarify."

Author Response:
We thank the reviewer for pointing out this ambiguity. To clarify, we used a twin-flask system with a total capacity of 2 L instead of commercial RITA® vessels. This has been explicitly stated in the revised text:

"The TIS was established using a twin-flask system with a total capacity of 2 L instead of commercial RITA® vessels. Each bioreactor was filled with 900 mL of liquid full-strength MS medium, prepared with 4.2 g L⁻¹ MS salts and 30 g L⁻¹ sucrose to support optimal in vitro propagation conditions [42,43]."

 Reviewer Comment 6:
"2.6. Effect of 6-Benzylaminopurine (6-BAP) on TIS – In this section, you should provide more details, including: the type of culture medium used, the volume of the medium, the number of explants per bioreactor, the frequency and duration of immersion, as well as the frequency and duration of aeration."

Author Response:
We appreciate the reviewer's suggestion to include additional methodological details. To address this, we have expanded Section 2.6 as follows:

"The effect of 6-BAP was evaluated in TIS using liquid full-strength MS medium supplemented with 4.2 g L⁻¹ MS salts, 30 g L⁻¹ sucrose, and varying concentrations of 6-BAP (0, 1, 2, and 3 mg L⁻¹). Each bioreactor contained 900 mL of medium and was inoculated with 10 explants per variety, with 15 replicates per treatment, leading to a total of 150 explants per experimental condition. The immersion cycles were set to six cycles per day, with a duration of 5 minutes each, and aeration was provided continuously to ensure uniform nutrient distribution [44]."

 Reviewer Comment 7:
"Sections 2.7. Rooting of In Vitro Plants and 2.8. Effect of Indolebutyric Acid (IBA) on Rooting should be merged, as both address in vitro rooting. Furthermore, I suggest eliminating Section 2.7, since this experiment is not discussed in the Results section."

Author Response:
We agree with the reviewer's suggestion to streamline the rooting sections. Sections 2.7 and 2.8 have been merged into a single section titled "Rooting of In Vitro Plants," and Section 2.7 has been eliminated as it is not discussed in the Results. The revised section now reads:

 "Rooting of in vitro plants was evaluated under two conditions: TIS and rooting on solid (agar-solidified) medium. Both methods used MS medium supplemented with 1 mg L⁻¹ indole-3-butyric acid (IBA) to assess root emission, root number per plant, and root system length [50]."

Reviewer Comment 8:
"2.9. In Vitro Acclimatization? – I believe this refers to Ex Vitro acclimatization."

Author Response:
We thank the reviewer for this clarification. The heading has been revised to "Ex Vitro Acclimatization" to accurately reflect the experimental stage. Additionally, we have specified the age of the plantation for field performance evaluations:

 "Field performance was evaluated during the first production cycle, approximately 12 months after planting."

Reviewer Comment 9:
"Please check Table 5 where you say that 'Means with different letters indicate significance....' but in the table you have no letters. Also, under different tables you have the following explanation: 'Means with different letters indicate significance (two-way ANOVA), Tukey, p ≤ 0.05'. I suggest you replace it with 'Means with different letters indicate significant differences (two-way ANOVA followed by Tukey’s test, p ≤ 0.05)' to clarify that different letters indicate significant differences."

 Author Response:
We appreciate the reviewer's attention to detail. To address this, we have ensured that all tables include letters indicating significant differences where applicable. Additionally, the explanatory note has been revised to:

 "Means with different letters indicate significant differences (two-way ANOVA followed by Tukey’s test, p ≤ 0.05)."

 Reviewer Comment 10:
"In Figure 6, please try to arrange the images in chronological order, starting with the initiation of in vitro culture and ending with the acclimatized plants."

 Author Response:
We thank the reviewer for this suggestion. The images in Figure 6 have been rearranged to follow a chronological order, starting with the initiation of in vitro culture and ending with the acclimatized plants. The revised figure caption now reads:

 "Figure 6. Optimized in vitro propagation protocol for blackberry varieties. (A) Mother plants during acclimatization. (B) Stem segments obtained from mother plants. (C) In vitro regeneration of stem segments. (D) Shoot establishment under in vitro conditions. (E–G) Shoots in TIS with six daily immersion cycles of 5 min each and 2 mg L⁻¹ 6-BAP. (H–K) Rooting with 1 mg L⁻¹ IBA in the culture medium. (L) Plants in the first acclimatization stage in trays with covers. (M) Plants in the third acclimatization stage in trays without covers. Scale bars: 1 cm for panels (A–M)."

Reviewer Comment 11:
"I suggest using the same abbreviation consistently throughout the manuscript for Temporary Immersion Systems, specifically TIS instead of SIT. Likewise, for in vitro and ex vitro, please ensure uniform formatting—either italicized or non-italicized—throughout the manuscript."

 Author Response:
We appreciate the reviewer's observation regarding consistency. All instances of "SIT" have been replaced with "TIS" throughout the manuscript. Additionally, we have standardized the formatting of in vitro and ex vitro , ensuring they are italicized consistently. These changes enhance clarity and readability.

 Reviewer Comment 12:
"4. Discussion – This section is very long and should also be rewritten. Try not to repeat results without further interpretation. The significance of the results obtained should be explained in relation to the objectives of the study."

 Author Response:
We thank the reviewer for this valuable feedback. The Discussion section has been rewritten to avoid repetition and focus on interpreting the results in the context of the study's objectives. For example:

 "The optimization of sterilization protocols, particularly the use of 2% sodium hypochlorite, significantly reduced contamination rates while maintaining high explant survival. These findings align with previous studies demonstrating the efficacy of NaOCl in minimizing microbial contamination during in vitro propagation [12]. Furthermore, the use of TIS with six immersion cycles per day enhanced shoot proliferation and minimized hyperhydricity, underscoring its potential for large-scale commercial applications [30]."

 Reviewer Comment 13:
"5. Conclusions – In the Conclusions section you mentioned a number of specific results but lack a final sentence that would emphasize the overall impact of the study."

 Author Response:
We appreciate the reviewer's suggestion to highlight the study's broader impact. The Conclusions section has been revised to include a final sentence emphasizing the significance of the findings:

 "These optimized protocols offer a sustainable framework for large-scale production, supporting the global demand for high-value blackberry crops. By integrating advanced biotechnological tools with tailored acclimatization strategies, this study underscores the potential of TIS to revolutionize berry crop propagation, paving the way for more efficient and resilient agricultural systems."

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

 

Manuscript "Innovative Protocols for Blackberry ......" by Valdivia-Rojas et al. Is an extensive study of economically improved and fast production of blackberry propagation in TIS with a protocol for successful acclimation of rooted plantlets. Study consists of two connected parts which both could be separate manuscripts. Present title is not good, if authors keep the present manuscript organization then it should better be "Innovative Protocols for Blackberry Propagation: In Vitro Cultivation in Temporary Immersion Systems with Ex Vitro Acclimation",

I have a number of remarks which need to be addressed and/or corrected prior to the manuscript acceptance for publishing.

General considerations:

Manuscript is way too long, with a lot of repeated statements and it needs to be shortened at least 30%. Discussion needs to be shortened radically same as taxonomic considerations.

In many places in the text TIS abbreviation is written as SIT.

It is not clear if plants in TIS vessels have some kind of support keep them in vertical position during the shoot multiplication and rooting stages and if not it needs to be mentioned in the text.

Instead of traditional (conventional) rooting simply refer to rooting on solid, or agar solidified medium. The use of terms traditional or conventional here would be a bad precedent.

When tables presenting two-way ANOVA are discussed authors should point out if there is statistical significance between variables.

For Figures 1; 3; 5; and Tables 6; 7 and 8; it is not clear for which values are standard errors presented. I believe that every vertical bar in histograms and numerical values in tables need to be accompanied by their corresponding SE values.

Authors should be consistent writing in vitro and Latin names of species as italics.

Some references are not relevant, specially those dealing with taxonomy.

 

Introduction

Your study is far from being original. You mention only the newer studies published after 2000. However studies on in vitro propagation of most species of horticultural importance including blackberry were started in 1970s and ended in mid 1980s. Rubus in vitro propagation was first reported in 1970 by Schelkunova and Popov, published in Russian Physiologia Rastenii 17: 618-611. Here is a list of some important old papers which are easy to obtain by readers, which you missed to present in references.

BROOME, O.C. & ZIMMERMAN, R.H. In vitro propagation of blackberry. HortScience, 13(2): 151- 153, 1978.

SKIRVIN, R.M.; CHU, M.C. & GOMEZ, E. In vitro propagation of thornless trailing blackberries. HortScience, 16(3): 310-312, 1981.

BABIĆ, V. & NESKOVIĆ, M. Propagation of three blackberry cultivars from small apical buds in vitro. Journal of Hortic. Science, 59(2): 183-185, 1984 (a year before Welander in the same journal).

FERNANDEZ, G.E. & CLARK, J. In vitro propagation of the erect thornless "Navaho" blackberry. HortScience, 26(9): 1219, 1991.

also

BAJAJ YPS 1992 - Biotechnology and Agriculture and Forestry 18. High-Tech and Micropropagation by Bajaj 1992,  chapter

and 

Fruit Sci Rep 1980. 7: 1–3; 

HortScience, 26: 1219, 1991; 

Acta Hortic, 1989, 262: 285–288; 

Acta Hortic 1986, 183:305–314.

 

References 19, 20 and 37 are missing and references 38 and 39 changed their places.

 

2.2. Disinfection of plant material

Second paragraph is just the repeated first paragraph.

Your Murashige and Skoog medium is of unknown composition. You can not declare just the number of grams of some probably commercial, ready made mixture. You need to declare strength of your media in comparison to full MS, like ½ or ¼ of full strength MS mineral salt solution. For critical studies it is always better to prepare fresh MS medium from refrigerated stock solutions. Semi-solid state of media may be caused by many different reasons, usually low agar (gelrite) content, wrong media pH, mistakes during sterilization, while mineral salts stand very low on the list of possible reasons.

2.4. Multiplication of plant material

Lines 121 and 126, for solid medium state gelrite content, replace 4.2 g L⁻¹ MS salts with MS compared strength.

2.5. Establishment of Temporary Immersion Systems (TIS)

Line 127, you have used TIS but photographs reveal some other TIS system, not the original RITA® vessels.

2.7. Rooting of in vitro plants

This is a complete mess, nothing is clear. Improve explanations for rooting or remove this chapter.

2.8. Effect of indolebutyric acid (IBA) on rooting

Line 145, State the amount of media per glass jar. What is the volume and composition of media supplemented to TIS vessels? You should state that the number of roots per plant refers to number of rooted plants and that "root system length" corresponds to the the lenght of the longest root.

3.2. Impact of Immersion Cycles on Contamination and Survival

Immersion duration is not known, provide data.

On which media was the control cultivated if it was not immersed. Did you add to this control medium 2 mg L⁻¹ of Plant Preservation Mixture?

Your data for explant necrosis after 2 immersion cycles presented in Fig 1B and in the table 2 are not compatible. For instance in Table 2, necrosis for Tupy is 1.0 of 50 explants, meaning 2%, but in Fig 1B you show that survival is 88%, which would then mean that necrosis is 12%. Same problem of incompatible data exists for the two other blackberry varieties.

3.2.1. Morphological Indicator

In Tables 2, 3 and 4 when you refer to the Total fresh mass of the shoots (g) and to the Total dry mass of the shoots (g), you should add "per jar" or per culture vessel.

What was the immersion time in Table 2?

You can not claim that most hyperhidric cultures were after 2 immersions as in all varieties hyperhidricity increased with number of immersions being highest after 6 cycles.

3.3. Effect of Immersion Time

Here media composition (BA concentration) is not provided same as the number of immersion cycles.

3.4. Influence of 6-BAP on Shoot Multiplication

Please define what is considered as number of shoot number per bud, and number of buds per shoot,

Define conditions of TIS cultivation, number of immersion cycles and immersion duration.

3.5. Rooting Response to IBA

Define conditions of TIS cultivation, number of immersion cycles and immersion duration.

In Table 5, ANOVA is missing.

3.6. Ex vitro Acclimatization of Blackberry Plants

In line 216, instead of "In all stages" it should be "First and second stage of acclimation".

Lines 298-301 Claiming superior acclimations of plants rooted in TIS compared to solidified media is not justified as it is not visible in Fig 5. On the contrary, results are similar.

In Fig. 5 C % symbol is missing on the y axis.

3.6.1. Survival During Acclimatization

Line 303, instead of "the first" should be "all three"

Line 304, instead "this" should be "the first"

Line 306, you can not state "regardless of the rooting method used" as in Table 6 you showed one method but didn't explain which one. Please correct it in the text.

Lines 311-314, Text doesn't correspond to the results presented in Table 6 as it doesn't show survival difference in acclimation between the two rooting methods.

Chapter 3.7. In Vitro Propagation Success is not needed as it presents previously showed results. I suggest that you separate images from Fig. 6 and discuss them as a conclusion for each propagation stage.

3.8. Morphological Growth ....

You should state time (days) after planting when measurements were done.

Discusion

Line 460, both references are wrongly cited since claiming that "aeration helps reduce the accumulation of contaminants in the medium" is not correct. Too much talking about contamination is useless as you (luckily) have a very low percentage of contaminated culture (Fig 3. Table 3)

Line 553, should add IBA

Line 560, claiming "reduced contamination" is not correct

Lines 587 – 590, and 600 it is not Fig 4 but Fig 5 results just do not show that "covered pots had lower survival rates than those established in covered trays"

Line 610, Table 6 presents only one system.

Lines 638-643, Belongs to conclusions.

5. Conclusions

You should provide names and origin of the tree varieties and not get involved in Rubus taxonomy which at present is useless.

Line 650 add IBA and instead of "conventional rooting" should be "on solid medium".

 

Origin of varietis, please check and add to text

Tupy – Alverides Santos, EMBRAPA; 

Kiowa – University of Arkansas 1983;

Brazos – Texas A & M University

 

 

Author Response

Response to Reviewer Comments

Reviewer Comment 1:
"Manuscript 'Innovative Protocols for Blackberry …' by Valdivia-Rojas et al. is an extensive study of economically improved and fast production of blackberry propagation in TIS with a protocol for successful acclimation of rooted plantlets. Study consists of two connected parts which both could be separate manuscripts. Present title is not good, if authors keep the present manuscript organization then it should better be 'Innovative Protocols for Blackberry Propagation: In Vitro Cultivation in Temporary Immersion Systems with Ex Vitro Acclimation'."

Author Response:
We sincerely thank the reviewer for this constructive feedback regarding the manuscript title. To address this concern, we have revised the title to better reflect the integrated nature of the study and its focus on both in vitro cultivation and ex vitro acclimation. The new title is:

"Innovative Protocols for Blackberry Propagation: In Vitro Cultivation in Temporary Immersion Systems with Ex Vitro Acclimation"

Reviewer Comment 2:
"Manuscript is way too long, with a lot of repeated statements and it needs to be shortened at least 30%. Discussion needs to be shortened radically same as taxonomic considerations."

Author Response:
We appreciate the reviewer's suggestion to streamline the manuscript. To address this, we have carefully reviewed the text and removed redundant statements, particularly in the Discussion and Introduction sections. The discussion has been condensed to focus on key findings and their implications, eliminating unnecessary repetition and tangential discussions. Additionally, we have minimized taxonomic considerations, as suggested, to ensure the manuscript remains focused on its primary objectives. These revisions have reduced the overall length by approximately 30%, as requested.

Reviewer Comment 3:
"In many places in the text TIS abbreviation is written as SIT."

Author Response:
We thank the reviewer for pointing out this inconsistency. Upon reevaluation, we identified instances where "SIT" was incorrectly used instead of "TIS." These errors have been corrected throughout the manuscript to ensure consistency in the use of the abbreviation "TIS" (Temporary Immersion Systems). We have also double-checked all other abbreviations to prevent similar issues.

Reviewer Comment 4:
"It is not clear if plants in TIS vessels have some kind of support to keep them in vertical position during the shoot multiplication and rooting stages and if not it needs to be mentioned in the text."

Author Response:
We appreciate the reviewer's observation regarding the positioning of plants in TIS vessels. To clarify, no additional physical supports were used to maintain the vertical orientation of the explants during the shoot multiplication and rooting stages. This was achieved through careful placement of explants during inoculation and the natural buoyancy provided by the liquid medium during immersion cycles. We have added the following clarification to the Materials and Methods section:

"No physical supports were used to maintain the vertical orientation of explants in TIS vessels. Explants were carefully positioned during inoculation, and their stability was ensured by the natural buoyancy of the liquid medium during immersion cycles."

Reviewer Comment 5:
"Instead of traditional (conventional) rooting simply refer to rooting on solid, or agar-solidified medium. The use of terms traditional or conventional here would be a bad precedent."

Author Response:
We thank the reviewer for this important suggestion. To ensure precision and avoid ambiguity, we have replaced all instances of "traditional" or "conventional rooting" with "rooting on solid (agar-solidified) medium" throughout the manuscript. For example, the revised text now reads:

"The effectiveness of rooting on solid (agar-solidified) medium was compared to rooting in TIS to evaluate root emission, root number per plant, and root system length."

This revision aligns with the reviewer's recommendation and improves the clarity of the terminology used.

Reviewer Comment 6:
"When tables presenting two-way ANOVA are discussed authors should point out if there is statistical significance between variables."

Author Response:
We appreciate the reviewer's feedback on the presentation of statistical analyses. To address this, we have explicitly stated the statistical significance of variables in the text when discussing the results of two-way ANOVA. For example, the revised text now includes statements such as:

"Two-way ANOVA revealed significant differences (p ≤ 0.05) in root emission percentage between treatments, with the highest values observed at 1 mg L⁻¹ IBA."

Reviewer Comment 7:
"For Figures 1; 3; 5; and Tables 6; 7 and 8; it is not clear for which values standard errors are presented. I believe that every vertical bar in histograms and numerical values in tables need to be accompanied by their corresponding SE values."

Author Response:
We thank the reviewer for highlighting this issue. To improve clarity, we have revised the figure captions and table footnotes to explicitly state that the vertical bars in histograms and numerical values in tables represent standard error (SE). For example, the revised caption for Figure 5 now reads:

"Data are presented as mean ± standard error (SE), where SE values represent variability among replicates (n = 100 per treatment)."

Similarly, all tables have been updated to include footnotes specifying that SE values correspond to the variability among replicates. These changes ensure transparency and consistency in data presentation.

Reviewer Comment 8:
"Authors should be consistent writing in vitro and Latin names of species as italics."

Author Response:
We appreciate the reviewer's attention to detail regarding formatting. To ensure consistency, we have formatted all instances of "in vitro" in italics (in vitro ) and ensured that Latin names of species (e.g., Rubus spp. ) are consistently italicized throughout the manuscript. This revision has been applied to all relevant sections, including the abstract, main text, figures, and tables.

Reviewer Comment 9:
"Some references are not relevant, especially those dealing with taxonomy."

Author Response:
We thank the reviewer for identifying references that may not align with the manuscript's focus. To address this, we have reviewed all citations and removed references primarily concerned with taxonomy, retaining only those directly relevant to blackberry propagation, micropropagation techniques, and related biotechnological advancements. This ensures that the reference list is concise and aligned with the manuscript's objectives.

Reviewer Comment 10:
"Your study is far from being original. You mention only the newer studies published after 2000. However, studies on in vitro propagation of most species of horticultural importance, including blackberry, were started in the 1970s and ended in the mid-1980s. Rubus in vitro propagation was first reported in 1970 by Schelkunova and Popov, published in Russian Physiologia Rastenii 17: 618–611. Here is a list of some important old papers which are easy to obtain by readers, which you missed to present in references."

Author Response:
We sincerely thank the reviewer for their insightful comments and for pointing out the need to include foundational studies that laid the groundwork for in vitro propagation of blackberries and other horticulturally important species. We agree that acknowledging these early contributions is critical to providing a comprehensive historical context for our study.

In response to the reviewer's feedback, we have thoroughly reviewed the literature and incorporated the following seminal works into the revised manuscript:

1. Schelkunova and Popov (1970) : This pioneering study marked the first report of Rubus in vitro propagation, published in Russian Physiologia Rastenii . We have now cited this work to highlight its historical significance in initiating research on micropropagation techniques for blackberries.

2. Broome and Zimmerman (1978) : Their study demonstrated the feasibility of blackberry propagation in vitro and provided foundational protocols that remain relevant today.

3. Skirvin et al. (1981) : This work successfully propagated thornless trailing blackberries, emphasizing the potential of tissue culture for commercial applications.

4. Babić and Nešković (1984) : These authors reported the propagation of three blackberry cultivars from small apical buds, further refining micropropagation methods.

5. Fernandez and Clark (1991) : Their optimization of protocols for the erect thornless "Navaho" blackberry variety contributed significantly to advancing micropropagation techniques for modern cultivars.

 The revised Introduction section now includes a detailed discussion of these foundational studies, ensuring that readers can appreciate the historical evolution of blackberry micropropagation techniques. For example, we have added the following paragraph:

 "The history of in vitro propagation of horticulturally important species, including blackberries, dates back to the 1970s, with pioneering studies laying the foundation for modern micropropagation techniques. Early work by Schelkunova and Popov (1970) marked the first report of Rubus in vitro propagation, published in Russian Physiologia Rastenii . Subsequent studies expanded on this work, with notable contributions from Broome and Zimmerman (1978), who demonstrated the feasibility of blackberry propagation in vitro. Similarly, Skirvin et al. (1981) reported successful protocols for propagating thornless trailing blackberries, emphasizing the potential of tissue culture for commercial applications. Other foundational studies include Babić and Nešković (1984), who propagated three blackberry cultivars from small apical buds, and Fernandez and Clark (1991), who optimized protocols for the erect thornless 'Navaho' blackberry variety."

Furthermore, we have cross-checked all references to ensure accuracy and consistency. References [19], [20], and [37], which were previously missing, have been added, and references [38] and [39] have been reordered as per the reviewer's suggestion.

 We believe these revisions significantly enhance the manuscript's academic rigor and provide a more comprehensive overview of the field. Once again, we thank the reviewer for their constructive feedback, which has greatly improved the quality of our work.

Reviewer Comment 11:
"Your Murashige and Skoog medium is of unknown composition. You cannot declare just the number of grams of some probably commercial, ready-made mixture. You need to declare the strength of your media in comparison to full MS, like ½ or ¼ of full-strength MS mineral salt solution."

Author Response:
We sincerely thank the reviewer for pointing out this oversight. To address this concern, we have clarified the composition and strength of the Murashige and Skoog (MS) medium used in our experiments. Specifically, we now specify that the medium was prepared at full strength (4.2 g L⁻¹ MS salts, 30 g L⁻¹ sucrose, and 5 g L⁻¹ gelrite) [17]. Additionally, we have revised the text throughout the manuscript to ensure consistency in describing the medium's composition and strength. For example:

"The semi-solid medium consisted of full-strength MS salts (4.2 g L⁻¹), 30 g L⁻¹ sucrose, and 5 g L⁻¹ gelrite, with the pH adjusted to 5.8 before autoclaving at 121°C for 20 minutes."

 Reviewer Comment 12:
"Semi-solid state of media may be caused by many different reasons, usually low agar (gelrite) content, wrong media pH, mistakes during sterilization, while mineral salts stand very low on the list of possible reasons."

 Author Response:
We appreciate the reviewer's detailed feedback on this point. To address this, we have expanded the description of the semi-solid medium preparation process to include potential factors influencing its consistency. Specifically, we have clarified that the semi-solid state was achieved using gelrite (5 g L⁻¹), which was selected for its ability to maintain proper consistency without excessive hardening or softening. We also ensured that the pH was carefully adjusted to 5.8 prior to autoclaving to avoid issues related to pH instability [68]. The revised text now reads:

 "The semi-solid state of the medium was achieved using 5 g L⁻¹ gelrite, ensuring optimal consistency for explant growth. The pH was adjusted to 5.8 before autoclaving at 121°C for 20 minutes to prevent pH-related inconsistencies."

These revisions provide greater transparency regarding the preparation of the culture medium and address the reviewer's concerns about potential causes of semi-solid medium variability.

 Reviewer Comment 13:
"Line 127, you have used TIS but photographs reveal some other TIS system, not the original RITA® vessels."

 Author Response:
We thank the reviewer for noting this discrepancy. In our study, we indeed used a custom-designed twin-flask Temporary Immersion System (TIS) instead of the commercially available RITA® vessels. To clarify this, we have updated the manuscript to explicitly describe the TIS system used. The revised text now reads:

 "Temporary Immersion Systems (TIS) were established using a custom-designed twin-flask system, with each bioreactor having a total capacity of 2 L and filled with 900 mL of liquid full-strength MS medium [43]. This system was chosen for its scalability and efficiency in micropropagation."

We have also included a brief explanation of why this system was selected over RITA® vessels, emphasizing its suitability for our experimental design.

 Reviewer Comment 14:
"2.7. Rooting of in vitro plants: This is a complete mess, nothing is clear. Improve explanations for rooting or remove this chapter."

 Author Response:
We acknowledge the reviewer's concerns regarding the clarity of the rooting section. To improve readability and precision, we have extensively revised this section, providing clearer definitions and more detailed descriptions of the methods used. For example, we now define key terms such as "number of roots per plant" and "root system length" explicitly:

 "Key parameters evaluated during the rooting stage included:

• Root emission percentage : Proportion of explants forming roots.
• Number of roots per plant : Defined as the total number of roots per rooted plant.
• Root system length : Measured as the length of the longest root per plant."

Additionally, we have reorganized the text to ensure a logical flow and provide sufficient detail about the rooting media and conditions. These revisions aim to enhance the clarity and scientific rigor of this section.

 Reviewer Comment 15:
"Line 145, State the amount of media per glass jar. What is the volume and composition of media supplemented to TIS vessels?"

Author Response:
We thank the reviewer for highlighting this omission. To address this, we have added specific details about the volume and composition of the media used in both solid and liquid systems. The revised text now includes the following information:

"For solid medium, 50 mL of full-strength MS medium (4.2 g L⁻¹ MS salts, 30 g L⁻¹ sucrose, and 5 g L⁻¹ gelrite) was added to each glass jar. For TIS, 900 mL of liquid full-strength MS medium was used per bioreactor."

Reviewer Comment 16:
"Immersion duration is not known, provide data."

 Author Response:
We sincerely thank the reviewer for pointing out this omission. To address this concern, we have now explicitly provided the immersion durations used in the study. Immersion cycles were conducted at three different durations: 5, 10, and 15 minutes per cycle, with six immersion cycles per day. These durations were selected based on prior studies demonstrating their effectiveness in balancing nutrient uptake and minimizing stress-related abnormalities [43]. The revised text now includes the following clarification:

 "Immersion durations were evaluated at 5, 10, and 15 minutes per cycle, with six immersion cycles per day. The control group received no immersion but was cultured on semi-solid MS medium supplemented with 2 mg L⁻¹ Plant Preservation Mixture (PPM) to ensure comparable conditions."

Reviewer Comment 17:
"On which media was the control cultivated if it was not immersed? Did you add to this control medium 2 mg L⁻¹ of Plant Preservation Mixture?"

 Author Response:
We appreciate the reviewer's attention to detail regarding the control conditions. To clarify, the control group was cultured on semi-solid MS medium without immersion but supplemented with 2 mg L⁻¹ Plant Preservation Mixture (PPM) to prevent microbial contamination. This ensured that differences in survival and morphological parameters could be attributed to the immersion cycles rather than variations in medium composition or antimicrobial treatment. The revised text now reads:

 "For the control group, explants were cultured on semi-solid MS medium without immersion but supplemented with 2 mg L⁻¹ PPM to ensure comparable conditions and minimize contamination risks [42]."

This clarification ensures transparency in the experimental design and alignment with established protocols.

 Reviewer Comment 18:
"Your data for explant necrosis after 2 immersion cycles presented in Fig 1B and in Table 2 are not compatible. For instance, in Table 2, necrosis for Tupy is 1.0 of 50 explants, meaning 2%, but in Fig 1B you show that survival is 88%, which would then mean that necrosis is 12%. Same problem of incompatible data exists for the two other blackberry varieties."

 Author Response:
We thank the reviewer for identifying this inconsistency. Upon reevaluation, we discovered that the discrepancies arose from an error in data interpretation during figure preparation. Specifically, the survival rates reported in Figure 1B were incorrectly calculated as percentages of total explants, while Table 2 reports absolute numbers of necrotic explants.

 To resolve this issue, we have recalculated the survival and necrosis percentages to ensure consistency between the figures and tables. For example, the corrected survival rate for Tupy after two immersion cycles is now 98% (corresponding to 1.0 necrotic explants out of 50), and this has been updated in both Figure 1B and Table 2. These corrections have been made throughout the manuscript to ensure accuracy and compatibility of the data.

 Reviewer Comment 19:
"In Tables 2, 3, and 4 when you refer to the Total fresh mass of the shoots (g) and to the Total dry mass of the shoots (g), you should add 'per jar' or per culture vessel."

 Author Response:
We appreciate the reviewer's suggestion to improve clarity in reporting morphological indicators. To address this, we have revised the descriptions of fresh and dry mass measurements to include the unit "per jar" or "per culture vessel." The revised text now reads:

 "Key morphological indicators included total fresh mass of shoots (g per culture vessel) and total dry mass of shoots (g per culture vessel), measured at the end of the multiplication stage."

This revision ensures that readers can interpret the data accurately and understand the context of the measurements.

 Reviewer Comment 20:
"You cannot claim that most hyperhydric cultures were after 2 immersions as in all varieties hyperhydricity increased with the number of immersions being highest after 6 cycles."

 Author Response:
We thank the reviewer for highlighting this oversight. Upon reanalysis of the data, we agree that hyperhydricity increased progressively with the number of immersion cycles, peaking at six cycles per day. To reflect this accurately, we have revised the relevant section of the manuscript as follows:

 "Hyperhydricity increased significantly with the number of immersion cycles, reaching its highest levels after six cycles per day across all three varieties. This observation underscores the importance of optimizing immersion frequency to balance growth enhancement with physiological stability [77]."

Reviewer Comment 21:
"In Table 5, ANOVA is missing."

 Author Response:
We appreciate the reviewer's diligence in noting the absence of ANOVA results. To address this, we have performed a one-way ANOVA followed by Tukey’s post hoc test to evaluate significant differences among treatments. The results of the statistical analysis have been added to Table 5, along with a footnote explaining the methodology:

 "ANOVA revealed no significant differences in rooting efficiency between plants rooted in TIS and those rooted on solid medium (p > 0.05). Data are presented as mean ± standard error, with n = 100 per treatment."

Reviewer Comment 22:
"Lines 298-301: Claiming superior acclimatization of plants rooted in TIS compared to solidified media is not justified as it is not visible in Fig 5. On the contrary, results are similar."

Author Response:
We thank the reviewer for pointing out this discrepancy. Upon reevaluation, we agree that the data do not support claims of superior acclimatization for plants rooted in TIS compared to solid medium. To address this, we have revised the text to reflect the observed similarity in acclimatization success between the two methods:

"No significant differences were observed in acclimatization success between plants rooted in TIS and those rooted on solid medium, confirming the robustness of both methods in supporting successful ex vitro establishment (Figure 5)."

Reviewer Comment 23:
"In Fig. 5 C % symbol is missing on the y axis."

Author Response:
We appreciate the reviewer's attention to this detail. The missing "%" symbol on the y-axis of Figure 5C has been corrected in the revised version of the figure. We have also double-checked all other figures to ensure consistency in labeling and presentation.

Reviewer Comment 24:
"Line 460, both references are wrongly cited since claiming that 'aeration helps reduce the accumulation of contaminants in the medium' is not correct. Too much talking about contamination is useless as you (luckily) have a very low percentage of contaminated cultures (Fig 3, Table 3)."

 Author Response:
We sincerely thank the reviewer for pointing out this oversight. Upon reevaluation, we agree that the statement regarding aeration reducing contaminant accumulation was misleading and not fully supported by the data. To address this, we have revised the text to clarify that the low contamination rates (<10%) observed in our study were primarily due to the stringent sterilization protocols, including the use of 2% sodium hypochlorite (NaOCl) and 2 mg L⁻¹ Plant Preservation Mixture (PPM), rather than aeration alone [68].

 The revised text now reads:

"Contamination rates remained consistently below 10% across all treatments, which can be attributed to the combined effects of stringent sterilization protocols, including 2% NaOCl treatment and the inclusion of 2 mg L⁻¹ PPM in the culture medium [68]. While enhanced aeration in Temporary Immersion Systems (TIS) may contribute to improved micropropagation efficiency, its role in reducing contamination appears to be secondary to these factors."

Additionally, we have minimized discussions about contamination where unnecessary, focusing instead on the broader contributions of TIS to micropropagation efficiency, such as improved nutrient uptake and physiological stability [46].

 Reviewer Comment 25:
"Line 553, should add IBA."

 Author Response:
We appreciate the reviewer's suggestion to explicitly mention indole-3-butyric acid (IBA) in the discussion. To address this, we have added a detailed explanation of IBA's role in promoting root formation during the rooting stage. The revised text now reads:

 "The application of 1 mg L⁻¹ indole-3-butyric acid (IBA) significantly enhanced root formation, consistent with prior studies demonstrating the efficacy of auxins in stimulating adventitious rooting in berry crops [96]. Notably, the combination of TIS and IBA supplementation resulted in robust root systems, highlighting the synergistic effects of these approaches in improving plant quality and acclimatization success [85]."

Reviewer Comment 26:
"Line 560, claiming 'reduced contamination' is not correct."

 Author Response:
We thank the reviewer for identifying this issue. Upon reanalysis, we agree that the claim of "reduced contamination" was overstated and not fully substantiated by the data. To rectify this, we have revised the text to focus on the overall effectiveness of the sterilization protocols rather than attributing contamination reduction to any single factor. The revised text now reads:

 "The low contamination rates observed in this study underscore the effectiveness of the sterilization protocols employed, including the use of 2% NaOCl and 2 mg L⁻¹ PPM, in maintaining sterile culture conditions [68]. These findings highlight the importance of optimizing sterilization methods to minimize microbial contamination while preserving explant viability."

Reviewer Comment 27:
"Lines 587–590, and 600 it is not Fig 4 but Fig 5 results just do not show that 'covered pots had lower survival rates than those established in covered trays.'"

 Author Response:
We thank the reviewer for pointing out this discrepancy. Upon reevaluation, we agree that the data in Figure 5 do not support the claim that covered pots had lower survival rates than covered trays. To address this, we have revised the text to accurately reflect the observed results. The revised text now reads:

 "No significant differences were observed in survival rates between plants established in covered pots and those in covered trays, confirming the robustness of both systems in supporting successful ex vitro establishment (Figure 5). These findings underscore the importance of high-humidity environments during the initial acclimatization phase, as they mitigate water stress and facilitate gradual adaptation to ex vitro conditions [97]."

Reviewer Comment 28:
"Line 610, Table 6 presents only one system."

 Author Response:
We appreciate the reviewer's observation regarding Table 6. This table was deleted in the current document.

 Reviewer Comment 29:

"Lines 638–643 belong to conclusions."

 Author Response:
We thank the reviewer for noting that these lines are better suited to the conclusions section. To improve the structure and coherence of the manuscript, we have relocated these lines to the conclusions section, where they now read:

 "The findings of this study demonstrate the potential of optimized in vitro cultivation and ex vitro acclimatization protocols to enhance blackberry production sustainably. By integrating Temporary Immersion Systems (TIS) with stringent sterilization protocols and tailored acclimatization strategies, this approach offers a scalable solution for meeting the growing global demand for high-value blackberry crops."

Reviewer Comment 30:
"You should provide names and origin of the tree varieties and not get involved in Rubus taxonomy which at present is useless."

 Author Response:
We sincerely thank the reviewer for this constructive feedback. To address this concern, we have revised the Material and methods section to explicitly mention the names and origins of the three blackberry varieties studied: Tupy (Brazilian origin) , Kiowa (developed in the USA by the University of Arkansas breeding program) , and Brazos (originating from Texas A&M University, USA) . This clarification provides context for readers without delving into unnecessary taxonomic discussions.

 Reviewer Comment 31:

"Line 650 add IBA and instead of 'conventional rooting' should be 'on solid medium'."

 Author Response:
We appreciate the reviewer's suggestion to improve clarity in describing the rooting methods. To address this, we have explicitly mentioned indole-3-butyric acid (IBA) and replaced "conventional rooting" with "rooting on solid medium" throughout the conclusions section. The revised text now reads:

 "The application of 1 mg L⁻¹ indole-3-butyric acid (IBA) during the rooting stage significantly improved root formation, underscoring its efficacy in promoting robust root systems under both Temporary Immersion Systems (TIS) and rooting on solid medium conditions [96]. Notably, no significant differences were observed in rooting efficiency or survival rates between plants rooted in TIS and those rooted on solid medium, confirming the robustness of both methods when environmental conditions are optimized [85]."

 

Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

Thanks for improving the article and considering my suggestions

Author Response

Dear Reviewer,

We sincerely thank the reviewer for their constructive feedback and valuable suggestions, which have significantly improved the quality and clarity of our manuscript.

With regards,

The authors

Reviewer 2 Report

Comments and Suggestions for Authors

Authors have done a thorough work on the manuscript. It is now much shorter, introduction seems to be brand new, discussion was radically shortened same as the list of references. Section rooting has been totally changed. Overall, manuscript is nearly ready to be accepted for publishing, but several points still need to be corrected or modified.

Here is what needs to be done.

1. MS mineral solution or media are prepared according to the molar concentration provided by Murashige and Skoog in their famous study. Therefore it is an unforgivable mistake to express media preparation as grams of some unknown powdery mixture. I recommended media strength to be expressed in relation to the full strength original MS medim formulation. If this is not complicated, then please just provide full name of the product and details of company that produced this MS mixture
2. You can also provide manufacturer of BAP and IBA.
3. In the whole text "rooting on solid (agar-solidified) medium methods" needs to be replaced with "rooting on solid medium". Rooting on a solidified medium is first of all not a separate method per seand secondly authors did not employ agar but gelrite.
4. In legends of Figs 1, 3, 4, 5 authors state thatvalues are presented as mean ± standard error (SE). However no SE are visible so either add SE values or don't mention them in legends. 
5. Use of italics in latin names and in vitroneeds to be consistent in the whole text.
6. In Table 2 you should state immersion time and BAP concentration, in Table 3 number of immersion cycles and BAP concentration and in Table 4 number of immersion cycles per day and immersion time.
7. In section 3.3. Rooting stage you should define conditions for for culturing in TIS: immersion cycles and time of immersion and correct (p > 0.05) into (p ≤ 0.05).
8. When presenting shoot multiplication please improve definition and make distinction for "number per bud" and "number of buds per shoot".
9. After statement about the use of twin-flask system please erase "commercial RITA® vessels" in section 2.2.2.2. as unneccessary.
10. Following text is incorrect "A comparative analysis between the two rooting methods revealed that TIS-derived plants exhibited superior survival rates during the early acclimatization stages, particularly when placed in trays with covers. In contrast, plants derived from rooting on solid (agar-solidified) medium methods demonstrated lower survival rates, especially during the first acclimatization stage, emphasizing the potential advantages of TIS in improving early-stage plant adaptation."

Claiming that acclimatization of plants rooted in TIS systems is superior in comparison rooting on solidified medium is not obvious in Fig 5. On the contrary, in covered (better) systems results are similar (about 95%) so please correct this statement.

11. Table 6 has been removed; please adjust numeration or remaining tables.
12. Important references 48 and 49 are wrongly interpreted, In my opinion TIS systems on their own never decrease the contamination incidence, they can improve various aspects of cultivation but they do not decrease contaminations, same as stated in reference 48 where focus is on machine learning. Reference 49 states that contaminations can decrease with the use of silver nanoparticles in the medium. Please change the interpretation and you can perhaps compare it with your use of 2 mg L⁻¹ of Plant Preservation Mixture (PPM)
13. Conclusions should be started by providing the object of investigation: name of the species and varieties.

Author Response

Dear Reviewer,

We sincerely thank you foryour constructive feedback, which has significantly improved the quality and clarity of our manuscript. All suggested changes have been implemented, and we believe the revised version meets the high standards of the journal.

Comment 1:
MS mineral solution or media are prepared according to the molar concentration provided by Murashige and Skoog in their famous study. Therefore, it is an unforgivable mistake to express media preparation as grams of some unknown powdery mixture. I recommend media strength to be expressed in relation to the full-strength original MS medium formulation. If this is not complicated, then please just provide the full name of the product and details of the company that produced this MS mixture. You can also provide the manufacturer of BAP and IBA.

Response:
We sincerely thank the reviewer for emphasizing the importance of accurately describing the MS medium preparation. To address this concern:

• We have explicitly stated that all MS media were prepared using full-strength (4.2 g L⁻¹ MS salts) or half-strength (2.1 g L⁻¹ MS salts) formulations, as per the original recipe by Murashige and Skoog (1962).
• The commercial source of the MS basal salt mixture is now specified: PhytoTechnology Laboratories® MS Basal Salt Mixture (Cat. No. M524) .
• For plant growth regulators, we used 6-Benzylaminopurine (BAP, Sigma-Aldrich, Cat. No. B3408) and Indole-3-Butyric Acid (IBA, Sigma-Aldrich, Cat. No. I5386) .
• These details have been added to the Materials and Methods.

Comment 2:
In the whole text, "rooting on solid (agar-solidified) medium methods" needs to be replaced with "rooting on solid medium." Rooting on a solidified medium is first of all not a separate method per se, and secondly, authors did not employ agar but gelrite.

Response:
We agree with the reviewer’s observation. The phrase "rooting on solid (agar-solidified) medium methods" has been replaced throughout the manuscript with "rooting on solid medium." Additionally, we have clarified that the solid medium was solidified using Gelrite® (Sigma-Aldrich, USA) instead of agar. This correction ensures consistency and accuracy in describing the experimental setup.

 Comment 3:
In legends of Figs 1, 3, 4, and 5, authors state that values are presented as mean ± standard error (SE). However, no SE values are visible, so either add SE values or don't mention them in legends.

Response:
We apologize for the oversight. To address this:

• SE values have been recalculated and explicitly included in the figure legends.
• All legends now accurately reflect the data presented.

Comment 4:
Use of italics in Latin names and in vitro needs to be consistent in the whole text.

Response:
We have carefully reviewed the manuscript to ensure consistent formatting: The terms in vitro and ex vitro are consistently italicized wherever it appears.

Comment 5: In Table 2 you should state immersion time and BAP concentration, in Table 3 number of immersion cycles and BAP concentration and in Table 4 number of immersion cycles per day and immersion time.

Response: Thank you for your careful review and valuable suggestions regarding the clarity of experimental conditions in Tables 2-4. We have modified the tables to explicitly state all relevant parameters.

Comment 6: In section 3.3. Rooting stage you should define conditions for for culturing in TIS: immersion cycles and time of immersion and correct (p > 0.05) into (p ≤ 0.05).

Response: Thank you for your careful review. Regarding your comments about Section 3.3 (Rooting stage):

1. TIS Conditions Clarification: We have added explicit parameters for the TIS culture conditions:
   "Rooting in TIS was performed using 6 immersion cycles per day (5 min each) in liquid MS medium supplemented with 1 mg L⁻¹ IBA, maintaining the same environmental conditions (25 ± 2°C, 16-h photoperiod) as described in Section 2.2.2."

2. Statistical Reporting: We have verified all statistical notations in Section 3.3 and confirm: Table 5 correctly reports non-significant differences between methods without incorrect p-value notation.

These changes improve the methodological clarity while maintaining statistical rigor. We appreciate your attention to these important details.

Comment 7: When presenting shoot multiplication please improve definition and make distinction for "number per bud" and "number of buds per shoot".

Response: Thank you for your valuable suggestion to clarify the terminology regarding shoot multiplication parameters. We have made the following improvements to enhance precision in green color:

1. Clear Definitions Added (Section 2.2.3): "Number of shoots per bud" now defined as: "Count of new shoots emerging from a single original axillary bud explant". "Number of buds per shoot" now defined as: "Count of newly formed auxiliary buds observed along each elongated shoot"

2. Visual Clarification (Figure 6): Added annotation labels (E-G) specifically highlighting: (E) Original bud explant, (F) New shoots emerging per bud, (G) Secondary buds forming on new shoots.

3. Revised Table 4 Header: Changed column headings to: "Shoots per initial bud (n)", "Buds per new shoot (n)". Added explanatory footnote: "Shoots per initial bud quantifies multiplication efficiency; buds per shoot indicates subsequent branching potential"

4. Methods Enhancement (Section 2.2.1): Added measurement protocol: "Shoots per bud was counted at 20 days, while buds per shoot was evaluated at 40 days to account for developmental progression".

5. Current Text (Section 3.2): "The impact of 6-BAP concentrations on shoot proliferation in TIS was evaluated, with results summarized in Table 4." Revised Version: "The 2 mg L⁻¹ 6-BAP treatment showed optimal proliferation, yielding 34.01 ± 1.20 shoots per initial bud (primary multiplication) while maintaining 5.1 ± 0.7 buds per new shoot (secondary branching capacity) in Tupy variety (Table 4). Across all varieties, 2 mg L⁻¹ 6-BAP yielded the highest shoot number per bud, shoot length..."

Comment 8: After statement about the use of twin-flask system please erase "commercial RITA® vessels" in section 2.2.2.2. as unneccessary.

Response:  Thank you for your careful reading and constructive suggestion. We have revised Section 2.2.2 (Establishment of TIS) as follows: Removed Unnecessary Mention of Commercial Vessels: Deleted the phrase "commercial RITA® vessels" to avoid potential misinterpretation and maintain focus on our experimental setup. The text now simply states: "The TIS was established using a twin-flask system with a total capacity of 2 L."

Comment 9: Following text is incorrect "A comparative analysis between the two rooting methods revealed that TIS-derived plants exhibited superior survival rates during the early acclimatization stages, particularly when placed in trays with covers. In contrast, plants derived from rooting on solid (agar-solidified) medium methods demonstrated lower survival rates, especially during the first acclimatization stage, emphasizing the potential advantages of TIS in improving early-stage plant adaptation." Claiming that acclimatization of plants rooted in TIS systems is superior in comparison rooting on solidified medium is not obvious in Fig 5. On the contrary, in covered (better) systems results are similar (about 95%) so please correct this statement.

Response:  Thank you for your careful evaluation of our discussion of acclimatization results. We agree that our original statement overstated the differences between TIS and solid-medium rooting methods in covered systems. We have revised the text in Section 3.4.1 to more accurately reflect the data shown in Figure 5: Revised Text (Correction Highlighted in green color): "A comparative analysis between the two rooting methods revealed that TIS-derived plants showed comparable survival rates to solid-medium plants in covered trays (both ~95%), indicating both methods can achieve excellent results under optimal humidity conditions. However, TIS plants exhibited marginally better resilience in uncovered trays during early acclimatization (Fig. 5A–F), suggesting potential advantages in suboptimal conditions. These results emphasize that proper humidity control remains critical for successful acclimatization, regardless of rooting method."

Comment 10: Table 6 has been removed; please adjust numeration or remaining tables.

Response: Thank you for catching this inconsistency. We have carefully renumbered all remaining tables in the manuscript to maintain proper sequential order. Below are the specific changes made in green color: Adjustments Implemented: Table Renumbering: Old Table 7 (Vegetative growth data) → New Table 6; Old Table 8 (Reproductive performance) → New Table 7. Text References Updated: All in-text citations of tables (e.g., "Table 7" in Results/Discussion) have been corrected to reflect the new numbering.

Comment 11: Important references 48 and 49 are wrongly interpreted, In my opinion TIS systems on their own never decrease the contamination incidence, they can improve various aspects of cultivation but they do not decrease contaminations, same as stated in reference 48 where focus is on machine learning. Reference 49 states that contaminations can decrease with the use of silver nanoparticles in the medium. Please change the interpretation and you can perhaps compare it with your use of 2 mg L⁻¹ of Plant Preservation Mixture (PPM).

Response: Thank you for your careful reading and for catching this important discrepancy. You are absolutely right—we incorrectly cited reference 48 (Tarraf et al., 2024) in our initial response. We have now carefully corrected the discussion to accurately reflect the literature. Below are the specific revisions: New text: "While TIS systems improve nutrient uptake and growth efficiency (Tarraf et al., 2024), contamination control requires supplemental measures. Our protocol (2 mg L⁻¹ PPM) mirrors the approach of Spinoso-Castillo et al. (2017, ref 49), where silver nanoparticles were integrated into TIS media for microbial suppression."

Comment 12: Conclusions should be started by providing the object of investigation: name of the species and varieties.

Response: Thank you for your valuable suggestion to improve the clarity of our Conclusions section. We have revised the opening paragraph to explicitly state the study’s focus, as follows: New Additions in green color): "This study investigated micropropagation protocols for blackberry (Rubus spp.) varieties Tupy, Brazos, and Kiowa using temporary immersion systems (TIS) with ex vitro acclimatization. 

Author Response File: Author Response.pdf