Multiplication of Axillary Shoots of Adult Quercus robur L. Trees in RITA^® Bioreactors

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The article refers to a study of micropropagation of Quercus robur by liquid culture in TIS, using RITA bioreactors. Considering the difficulty of micropropagating forest species, in general, and species of the Genus Quercus, in particular, the Authors have succeeded, through a series of coordinated experimental trials, in finally producing an effective protocol of in vitro propagation of pedunculate oak of high scientific-applicative value. The study is well articulated in the development of its experimental design, the ‘Results’ are well analyzed and described, the ‘Discussion’ part is balanced and appropriate in its development. 

• Try to insert each Figure with its relative captions all in one page, if necessary reducing the size of the Figure. This would make it much easier to understand and interpret the Figures themselves.
• Figure 2 does not present an image (e) that, instead, is cited in the text (line 153).
• line 297-300: it is stated that “(in CM) 6 immersions per day produced better results", but this is not true in general; indeed, it is only true in the "Apical+Cubes" and this fact should be better highlighted.
• the acronym 'SL' is used both for the SL clone and to indicate shoot length: this can create confusion in the reader.
• significance must always be expressed as ‘p≤0.05’
• line 432-433: it is stated that “apical sections of RUS could grow succesfully in both media". But from Figure 7 it clearly emerges that the WPM medium is decidedly more performing than the GD in every parameter evaluated.

Author Response

Please find our answers in bold font.

The article refers to a study of micropropagation of Quercus robur by liquid culture in TIS, using RITA bioreactors. Considering the difficulty of micropropagating forest species, in general, and species of the Genus Quercus, in particular, the Authors have succeeded, through a series of coordinated experimental trials, in finally producing an effective protocol of in vitro propagation of pedunculate oak of high scientific-applicative value. The study is well articulated in the development of its experimental design, the ‘Results’ are well analyzed and described, the ‘Discussion’ part is balanced and appropriate in its development. 

We appreciate the comments of the Reviewer 1 about the manuscript and the suggestions for its improvement.

We have modified the manuscript accordingly with the reviewers and the editor comments. We have added more references, included more tables and figures and substituted some figures for tables. As we are conscious that the version with the visible changes maybe complicated to understand, to facilitate the process we provide the final version in 2 documents: a word file (text.doc) in which all the changes are visible, and a pdf file in which only the final version is visible. We hope this is acceptable and make the evaluation easier.

• Try to insert each Figure with its relative captions all in one page, if necessary reducing the size of the Figure. This would make it much easier to understand and interpret the Figures themselves.

We agree with the Rev 1, the figures were difficult to understand in the original size. We tried to reduce the size but the visual effect was not satisfactory. Instead, data previously presented as Fig 4, 5 and 6 have been moved to Tables (Tables 4 and 6), that fit in less space, with the legend in the same page.

• Figure 2 does not present an image (e) that, instead, is cited in the text (line 153).

Thank you for noticing the mistake, it has been corrected.

• line 297-300: it is stated that “(in CM) 6 immersions per day produced better results", but this is not true in general; indeed, it is only true in the "Apical+Cubes" and this fact should be better highlighted.

Thank you for noticing the mistake, it has been corrected.

• the acronym 'SL' is used both for the SL clone and to indicate shoot length: this can create confusion in the reader.

We have modified SL to SLo in the case of the clone San Lourenzo

• significance must always be expressed as ‘p≤0.05’

Done

• line 432-433: it is stated that “apical sections of RUS could grow succesfully in both media". But from Figure 7 it clearly emerges that the WPM medium is decidedly more performing than the GD in every parameter evaluated.

  Rev 1 is right, WPM is better for apical sections and we have modified the text accordingly.

Reviewer 2 Report

Comments and Suggestions for Authors

The manuscript entitled “Proliferation of axillary shoots of adult Quercus robur trees in RITA® bioreactors” investigates the application of RITA® temporary immersion systems (TIS) for the micropropagation of mature oak trees. Five genotypes of Quercus robur, derived from in vitro-established cultures of trees aged 60 to 800 years and collected from Spain and Poland, were used. The authors examined key factors influencing culture success, including immersion frequency, culture medium, explant type, and shoot support (rockwool cubes). The results demonstrate that all five genotypes could be propagated using TIS, producing shoots that were successfully rooted and acclimated. This represents a notable advance towards large-scale clonal production of selected mature Q. robur genotypes.

 

The objective is ambitious, and the authors have achieved promising results by identifying key variables influencing the successful micropropagation of mature oaks of diverse origin.

 

However, since not all factors were tested uniformly across all genotypes, the experimental design remains somewhat complex to follow. For greater clarity and reader accessibility, I recommend the authors prepare a schematic diagram or table summarizing the overall experimental layout. Such a scheme would facilitate understanding of the Methods section, improve the structure of the Results section, and clarify the figure captions, where the authors currently tend to provide detailed experimental descriptions—likely due to the lack of an overarching visual summary. This would also support a more structured discussion, as some experiments covered more genotypes than others, allowing the authors to contextualise generalisations accordingly.

 

Regarding terminology, the manuscript consistently uses the term “proliferation” to describe shoot multiplication. While “proliferation” is widely accepted in biological contexts, it is more appropriate for cell-level processes such as callus growth or the expansion of pro-embryogenic masses. For shoot production, especially in organogenesis, the term “multiplication” is more accurate and commonly used. It specifically refers to the increase in shoot number through axillary or adventitious shoot formation, which is the case here.

 

A clarification is needed in the Introduction. The sentence: “As a result, oaks are generally considered suitable for propagation only after reaching maturity, following the juvenile-to-adult phase transition” may misrepresent the situation. I believe the authors intended to highlight the mismatch between the optimal stage for genotype selection (mature trees) and the more favorable juvenile stage for propagation. The following sentences in the manuscript appear to support this interpretation.

From a formal perspective, I noted several minor issues:

• The term in vitro should be italicised (e.g., lines 103, 111, 115, and 127).
• In figure captions (e.g., Figure 3), the species name Quercus robur should be mentioned where relevant.
• In Table 1, inconsistencies exist between the tree characteristics and line abbreviations. Only the first two entries from Chaian (ChBS1 and ChBS2) show a clear link between origin and abbreviation. In other cases (CM, SL, RUS), the origin of the material is not clearly reflected in the abbreviation. Also, “origin” is misspelled in the table header.

When comparing media (GD vs. MS), especially in relation to nitrate concentrations, it would be more scientifically sound to report and compare the ionic concentrations directly, rather than simply stating full vs. half-strength, since these two media differ significantly in composition.

Additionally, I recommend revising the figure captions as follows:

• Figure 6: Change to “Effect of tested culture medium on multiplication characteristics (shoot length, multiplication coefficient, and number of rootable shoots) of Quercus robur clone CM.”
• Figure 7: Change to “Effect of tested culture medium on multiplication characteristics (shoot length, multiplication coefficient, and number of rootable shoots) of Quercus robur clone RUS.”
• Figure 8: Correct the chemical formula; AgNO₃ should be properly formatted.

Lastly, in the “Author Contributions” section, please use author initials rather than full names for consistency with standard academic conventions.

Recommendation: Major Revision (Revisions Needed)

Author Response

Please find our answers in bold font.

The manuscript entitled “Proliferation of axillary shoots of adult Quercus robur trees in RITA® bioreactors” investigates the application of RITA® temporary immersion systems (TIS) for the micropropagation of mature oak trees. Five genotypes of Quercus robur, derived from in vitro-established cultures of trees aged 60 to 800 years and collected from Spain and Poland, were used. The authors examined key factors influencing culture success, including immersion frequency, culture medium, explant type, and shoot support (rockwool cubes). The results demonstrate that all five genotypes could be propagated using TIS, producing shoots that were successfully rooted and acclimated. This represents a notable advance towards large-scale clonal production of selected mature Q. robur genotypes.

The objective is ambitious, and the authors have achieved promising results by identifying key variables influencing the successful micropropagation of mature oaks of diverse origin.

However, since not all factors were tested uniformly across all genotypes, the experimental design remains somewhat complex to follow. For greater clarity and reader accessibility, I recommend the authors prepare a schematic diagram or table summarizing the overall experimental layout. Such a scheme would facilitate understanding of the Methods section, improve the structure of the Results section, and clarify the figure captions, where the authors currently tend to provide detailed experimental descriptions—likely due to the lack of an overarching visual summary. This would also support a more structured discussion, as some experiments covered more genotypes than others, allowing the authors to contextualise generalisations accordingly.

We have modified the manuscript accordingly with the reviewers and the editor comments. We have added more references, included more tables and figures and substituted some figures for tables. As we are conscious that the version with the visible changes maybe complicated to understand, to facilitate the process we provide the final version in 2 documents: a word file (text.doc) in which all the changes are visible, and a pdf file in which only the final version is visible. We hope this is acceptable and make the evaluation easier.

We appreciate the comments of Reviewer 2 for improving the manuscript. We have added a Table with the experimental layout as suggested (Table 2) and have modified the order of the results and discussion sections.

Regarding terminology, the manuscript consistently uses the term “proliferation” to describe shoot multiplication. While “proliferation” is widely accepted in biological contexts, it is more appropriate for cell-level processes such as callus growth or the expansion of pro-embryogenic masses. For shoot production, especially in organogenesis, the term “multiplication” is more accurate and commonly used. It specifically refers to the increase in shoot number through axillary or adventitious shoot formation, which is the case here.

We have substituted the term proliferation with multiplication in most of the cases, including the title.

A clarification is needed in the Introduction. The sentence: “As a result, oaks are generally considered suitable for propagation only after reaching maturity, following the juvenile-to-adult phase transition” may misrepresent the situation. I believe the authors intended to highlight the mismatch between the optimal stage for genotype selection (mature trees) and the more favorable juvenile stage for propagation. The following sentences in the manuscript appear to support this interpretation.

We appreciate that you have noticed the mistake, in the sentence we have substituted “propagation” with “selection”.

From a formal perspective, I noted several minor issues:

• The term in vitro should be italicised (e.g., lines 103, 111, 115, and 127).

We agree with you completely, but it seems that in mdpi’s opinion is that now this term has been fully accepted in English, meaning they will accept only the non-italicised version.

• In figure captions (e.g., Figure 3), the species name Quercus robur should be mentioned where relevant.

Now it has been added

• In Table 1, inconsistencies exist between the tree characteristics and line abbreviations. Only the first two entries from Chaian (ChBS1 and ChBS2) show a clear link between origin and abbreviation. In other cases (CM, SL, RUS), the origin of the material is not clearly reflected in the abbreviation. Also, “origin” is misspelled in the table header.

We have corrected Origin in the table and modified the abbreviation for clone San Lourenzo. However, the others have not been changed as the abbreviations of the table have already been used in previous articles dealing with propagation in semisolid medium.

When comparing media (GD vs. MS), especially in relation to nitrate concentrations, it would be more scientifically sound to report and compare the ionic concentrations directly, rather than simply stating full vs. half-strength, since these two media differ significantly in composition.

We have added a supplementary table with the detailed composition of the media that we used in the study.

Additionally, I recommend revising the figure captions as follows:

• Figure 6: Change to “Effect of tested culture medium on multiplication characteristics (shoot length, multiplication coefficient, and number of rootable shoots) of Quercus robur clone CM.”
• Figure 7: Change to “Effect of tested culture medium on multiplication characteristics (shoot length, multiplication coefficient, and number of rootable shoots) of Quercus robur clone RUS.”

We have modified the legends of tables and figures trying to make them more informative.

• Figure 8: Correct the chemical formula; AgNO₃ should be properly formatted.

We agree on that. We tried to modify it in the graphic, but unfortunately, we couldn’t make the subscript visible there. Instead, we substituted the formula by the abbreviation of the name (SN, Silver nitrate).

Lastly, in the “Author Contributions” section, please use author initials rather than full names for consistency with standard academic conventions.

                Done

Reviewer 3 Report

Comments and Suggestions for Authors

The manuscript used a commercial bioreactor to regenerate oak shoots from old trees. It would be a very good reference for the propagation of other oak trees as well. However, there are some minor issues to be addressed.

1. The abbreviation "TIS" should be spelled out as "Temporary Immersion System" when first mentioned in the text.

2. In line 153, the reference to "(figure 2 d, e)" is incorrect since figure 2 does not contain a panel "e".

3. The legend for Figure 2 should include specifications of the rockwool blocks (1 cm³) and the insertion depth of explants.

4. For Figure 4, it is recommended to use different colors for the bar charts in panels (a) and (b) to better distinguish them from panels (c)-(h) in terms of legend interpretation.

5. The "Results" section would benefit from subheadings to improve clarity. For example, lines 212-241 could be grouped under a subheading for the first major result.

6. The term "hyperhydricity" should be defined when first introduced in the abstract.

7. Figures should be friendly to color-blind people, and should be modified to keep the size of photos the same (better to add a scale bar).

Author Response

Please find our answers in bold font.

We acknowledge the comments of Reviewer 3 to improve the manuscript.

We have modified the manuscript accordingly with the reviewers and the editor comments. We have added more references, included more tables and figures and substituted some figures for tables. As we are conscious that the version with the visible changes maybe complicated to understand, to facilitate the process we provide the final version without the change tracking in the document called cover letter.

The manuscript used a commercial bioreactor to regenerate oak shoots from old trees. It would be a very good reference for the propagation of other oak trees as well. However, there are some minor issues to be addressed.

1. The abbreviation "TIS" should be spelled out as "Temporary Immersion System" when first mentioned in the text.

                Done

2. In line 153, the reference to "(figure 2 d, e)" is incorrect since figure 2 does not contain a panel "e".

                Corrected

3. The legend for Figure 2 should include specifications of the rockwool blocks (1 cm³) and the insertion depth of explants.

We added the size of the cubes as required. Regarding the second request, there is no insertion depth as the explants were placed between the cubes, not inserted in them. There is now explained in the text with more detail.

4. For Figure 4, it is recommended to use different colors for the bar charts in panels (a) and (b) to better distinguish them from panels (c)-(h) in terms of legend interpretation.

We appreciate the comment. Finally, and following the indications of other reviewer we substituted this Figure for a Table.

5. The "Results" section would benefit from subheadings to improve clarity. For example, lines 212-241 could be grouped under a subheading for the first major result.

We have included subheadings.

6. The term "hyperhydricity" should be defined when first introduced in the abstract.

Thank you for the comment, we weren’t conscious that it had not been defined. Now it is defined in the introduction as in the abstract we didn’t have the required space (only 200 words are allowed).

7. Figures should be friendly to color-blind people, and should be modified to keep the size of photos the same (better to add a scale bar).

We have added a scale bar, substituted complicated figures for tables and in the remaining tables we modified the colors following your indication.

Round 2

Reviewer 2 Report

Comments and Suggestions for Authors

The authors have significantly improved the quality of the manuscript and have sufficiently addressed the main suggestions provided. I recommend the manuscript for publication.