Efficiency of a Double-Phase Medium in Micropropagation of Serviceberry (Amelanchier sp.)

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

Manuscript entitled “Efficiency of a double-phase medium in micropropagation of serviceberry (Amelanchier sp.) written by Wojciech Litwińczuk, focuses on establishment of in vitro cultures of three serviceberry cultivars through application of 1F and 2F media. While the study produced some results, the presentation of the results and preparation of the manuscript can be much improved. There were many demerits as listed followings:

• Line 29 and 55: Rosaceae and Pomoideae are family name and should not be italic format.
• Line 89: The freezing temperature is -26 ℃. It is confusing here. Is it 26℃?
• Line 95: mikronutrients is misspelling.
• Table 1: What is the chemical of FeEDDHA?
• Lin2 112-115: The chemicals names are not written properly.
• Line 135: change “1,5 cm” into “1.5 cm”.
• Table 2: 1) numer is misspelling. 2) What is meaning of *, ** and ***. The same is the following tables.
• Table 4 and Table 6: numer is misspelling.
• Table 3: Significant difference in pH was detected among the three serviceberry cultivars. Why? How many agar were added in those medium?
• Line 183-184 and line 223-224: There are three SL but they stand for different contents.
• Figure 2: the pictures are not well organized.
• It seems that the medium is not suitable for rooting of shoots since there are many callus around the roots.
• Is any mutation detected in micropropagation studies of the three serviceberry cultivarsusing molecular marker?
• Line 261: the references are chaotic.
• Line 285: change “((” into “(” .
• Line 316: “(Malus – 22,28,33).” is confusing.
• Line 324-325: the sentence “unknown physiological disorder?” is confusing.

Author Response

Manuscript entitled “Efficiency of a double-phase medium in micropropagation of serviceberry (Amelanchier sp.) written by Wojciech Litwińczuk, focuses on establishment of in vitro cultures of three serviceberry cultivars through application of 1F and 2F media. While the study produced some results, the presentation of the results and preparation of the manuscript can be much improved. There were many demerits as listed followings: Thank You for review, comments and suggestions. I hope we have made satisfactory corrections.

• Line 29 and 55: Rosaceae and Pomoideae are family name and should not be italic format. It has been corrected.
• Line 89: The freezing temperature is -26 ℃. It is confusing here. Is it 26℃? No, the jars containing the used culture medium were frozen and then thawed to room temperature to obtain the liquid for conductivity and pH measurement. Therefore, the freezing temperature was -26°C.
• Line 95: mikronutrients is misspelling. - It has been corrected.
• Table 1: What is the chemical of FeEDDHA? The name has been added
• Lin2 112-115: The chemicals names are not written properly. I'm sorry, but I don't know what the problem is!? Please clarify.
• Line 135: change “1,5 cm” into “1.5 cm”. It has been corrected.
• Table 2: 1) numer is misspelling. It has been corrected.
•  2) What is meaning of *, ** and ***. The same is the following tables. Such information was added under tables
• Table 4 and Table 6: numer is misspelling. It has been corrected.
• Table 3: Significant difference in pH was detected among the three serviceberry cultivars. Why? How many agar were added in those medium? Agar was added at 7g/L (Table 1). I suppose the differences resulted from differential cation (and anion) uptake depending on the studied clones.  I have provided the correct values in Tables 3, 5, and 7, along with additional information about the parameters of unused, autoclaved media under the tables and 2 other media traits.
• Line 183-184 and line 223-224: There are three SL but they stand for different contents. I have completed the information
• Figure 2: the pictures are not well organized. It has been corrected (I believe).
• It seems that the medium is not suitable for rooting of shoots since there are many callus around the roots. I agree. In earlier preliminary experiments, callus formation wasn't as abundant, so we used this medium. Fortunately, the callus grew rather after roots appeared, grew over them, and didn't disrupt the connection between shoots and roots. Nevertheless, we still need to work on optimizing shoot rooting (perhaps a two-step rooting process or a riboflavin supplement will help). I expressed this in the discussion.
• Is any mutation detected in micropropagation studies of the three serviceberry cultivarsusing molecular marker? No, we did not conduct molecular marker analyses. However, we did not find any off-type variants among the young acclimated plants.
• Line 261: the references are chaotic. It has been corrected.
• Line 285: change “((” into “(” . It has been corrected.
• Line 316: “(Malus – 22,28,33).” is confusing. It has been corrected.
• Line 324-325: the sentence “unknown physiological disorder?” is confusing. I changed that sentence.

Reviewer 2 Report

Comments and Suggestions for Authors

This study investigated the efficiency of a double-phase medium in the micropropagation of three cultivars of serviceberry. The topic is interesting, and the study could provide a scientific basis for improving the micropropagation of serviceberry. However, I have a few comments on the manuscript, which are outlined below.

Line 35-61: The authors should clearly state a hypothesis to be tested in this study, based on the relevant previous literature.

Line 134-135: “The ‘SnCl’ clone produced significantly more short (<1,5cm) and long (>1,5 cm) shoots than ‘Ball’ clone.” What do “<1,5cm” and “>1,5 cm” mean in this sentence? Should they be ‘<15mm’ and ‘>15mm’ as indicated in Table 2?

Table 2: The values in Table 2 should be presented as mean ± SE.

Table 2: “No. of cultures with vitrified shoots [%]”and “No. of cultures with shoot tip necrosis [%]”. Since the unit is “%”, “No.” should be revised to “Percentage”.

Table 3: The values in Table 3 should be presented as mean ± SE.

Table 3: Notes 1 and 2 should be indicated in Table 3.

Table 4: The values in Table 4 should be presented as mean ± SE.

Table 4: “No. of cultures with vitrified shoots [%]” and “No. of cultures with shoot tip necrosis [%]”. Since the unit is “%”, “No.” should be revised to “Percentage”.

Table 4: Notes 1 and 2 should be indicated in Table 4.

Table 5: The values in Table 5 should be presented as mean ± SE.

Table 5: Notes 1 and 2 should be indicated in Table 5.

Table 6: The values in Table 6 should be presented as mean ± SE.

Table 6: “No. of cultures with vitrified shoots [%]” and “No. of cultures with shoot tip necrosis [%]”. Since the unit is “%”, “No.” should be revised to “Percentage”.

Table 6: Notes 3 and 4 do not correspond to the indications in Table 6.

Table 7: The values in Table 7 should be presented as mean ± SE.

Table 7: Notes 1 and 2 should be indicated in Table 7.  Note 3 does not correspond to the indications in Table 7.

Table 8: The values in Table 8 should be presented as mean ± SE.

Table 8: “No. of rooted shoots [%]” and “No. of acclimated plantlets [%]”. Since the unit is “%”, “No.” should be revised to “Percentage”.

Table 8: Notes 1 and 2 should be indicated in Table 8.

Table 9: The values in Table 9 should be presented as mean ± SE.

Table 9: “No. of rooted shoots [%]” and “No. of acclimated plantlets [%]”. Since the unit is “%”, “No.” should be revised to “Percentage”.

Table 9: Notes 1 and 2 should be indicated in Table 9.

Table 10: The values in Table 10 should be presented as mean ± SE.

Table 10: “No. of rooted shoots [%]” and “No. of acclimated plantlets [%]”. Since the unit is “%”, “No.” should be revised to “Percentage”.

Table 10: Notes 1 and 2 should be indicated in Table 10. There are two indications for “3”, but no indication for “4” in Table 10.

Table 11: The values in Table 11 should be presented as mean ± SE.

Table 11: “No. of rooted shoots [%]” and “No. of acclimated plantlets [%]”. Since the unit is “%”, “No.” should be revised to “Percentage”.

Table 11: In the row “Micropropagation efficiency [%]”, what do “190,9” and “65,6” mean?

Table 11: Notes 1 and 2 should be indicated in Table 11.

Figure 2: I suggest revising the subpanel to match the style of Figure 1, as this would make the entire figure appear neater and more consistent.

Line 261-262: This sentence repeats information already presented in the introduction.

Line 266-267: What do “>1,5 cm” and “>3,0 cm” mean in the sentence?

Line 285: There's an extra parenthesis in '((Tables 4, 6)'.

Line 309: “data not presented” should be revised to “data are not presented”.

Line 316: “On the other hand (6) informed that…”. What is the meaning of this sentence? Why does 'On the other hand' require citation of reference [6]?

 

 

Author Response

This study investigated the efficiency of a double-phase medium in the micropropagation of three cultivars of serviceberry. The topic is interesting, and the study could provide a scientific basis for improving the micropropagation of serviceberry. However, I have a few comments on the manuscript, which are outlined below. Thank You for positive opinion, comments and suggestions. I hope we have made satisfactory corrections.

Line 35-61: The authors should clearly state a hypothesis to be tested in this study, based on the relevant previous literature. The justification for undertaking the research and its purpose were provided.

Line 134-135: “The ‘SnCl’ clone produced significantly more short (<1,5cm) and long (>1,5 cm) shoots than ‘Ball’ clone.” What do “<1,5cm” and “>1,5 cm” mean in this sentence? Should they be ‘<15mm’ and ‘>15mm’ as indicated in Table 2? It has been corrected.

Table 2: The values in Table 2 should be presented as mean ± SE. SE values have been added except results presented as percentages (binomial distribution).

Table 2: “No. of cultures with vitrified shoots [%]”and “No. of cultures with shoot tip necrosis [%]”. Since the unit is “%”, “No.” should be revised to “Percentage”. Not necessarily as the numbers can also be expressed as a percentage. Percentage [%] - this smacks of tautology… However, we have changed it as suggested.

Table 3: The values in Table 3 should be presented as mean ± SE. SE values have been added except results presented as percentages (binomial distribution).

Table 3: Notes 1 and 2 should be indicated in Table 3. The table has been supplemented with references. 

Table 4: The values in Table 4 should be presented as mean ± SE. SE values have been added except results presented as percentages (binomial distribution).

Table 4: “No. of cultures with vitrified shoots [%]” and “No. of cultures with shoot tip necrosis [%]”. Since the unit is “%”, “No.” should be revised to “Percentage”. It has been changed.

Table 4: Notes 1 and 2 should be indicated in Table 4. The table has been supplemented with references. 

Table 5: The values in Table 5 should be presented as mean ± SE. SE values have been added except results presented as percentages (binomial distribution).

Table 5: Notes 1 and 2 should be indicated in Table 5. The table has been supplemented with references. 

Table 6: The values in Table 6 should be presented as mean ± SE. SE values have been added except results presented as percentages (binomial distribution).

Table 6: “No. of cultures with vitrified shoots [%]” and “No. of cultures with shoot tip necrosis [%]”. Since the unit is “%”, “No.” should be revised to “Percentage”. It has been changed.

Table 6: Notes 3 and 4 do not correspond to the indications in Table 6. The table has been supplemented with proper references. 

Table 7: The values in Table 7 should be presented as mean ± SE. SE values have been added except results presented as percentages (binomial distribution).

Table 7: Notes 1 and 2 should be indicated in Table 7.  Note 3 does not correspond to the indications in Table 7. The table has been supplemented with proper references. 

Table 8: The values in Table 8 should be presented as mean ± SE. SE values have been added except results presented as percentages (binomial distribution).

Table 8: “No. of rooted shoots [%]” and “No. of acclimated plantlets [%]”. Since the unit is “%”, “No.” should be revised to “Percentage”. It has been changed.

Table 8: Notes 1 and 2 should be indicated in Table 8. The table has been supplemented with references. 

Table 9: The values in Table 9 should be presented as mean ± SE. SE values have been added except results presented as percentages (binomial distribution).

Table 9: “No. of rooted shoots [%]” and “No. of acclimated plantlets [%]”. Since the unit is “%”, “No.” should be revised to “Percentage”. It has been changed.

Table 9: Notes 1 and 2 should be indicated in Table 9. The table has been supplemented with references. 

Table 10: The values in Table 10 should be presented as mean ± SE. SE values have been added except results presented as percentages (binomial distribution).

Table 10: “No. of rooted shoots [%]” and “No. of acclimated plantlets [%]”. Since the unit is “%”, “No.” should be revised to “Percentage”. It has been changed.

Table 10: Notes 1 and 2 should be indicated in Table 10. There are two indications for “3”, but no indication for “4” in Table 10. The table has been supplemented with proper references. 

Table 11: The values in Table 11 should be presented as mean ± SE. SE values have been added except results presented as percentages (binomial distribution).

Table 11: “No. of rooted shoots [%]” and “No. of acclimated plantlets [%]”. Since the unit is “%”, “No.” should be revised to “Percentage”. It was changed.

Table 11: In the row “Micropropagation efficiency [%]”, what do “190,9” and “65,6” mean?  I have provided an explanation below the table.

Table 11: Notes 1 and 2 should be indicated in Table 11. The table has been supplemented with references. 

Figure 2: I suggest revising the subpanel to match the style of Figure 1, as this would make the entire figure appear neater and more consistent. The layout of Fig 2 'fell apart' when formatting to the MDPI pattern. I hope the layout is correct now.

Line 261-262: This sentence repeats information already presented in the introduction. The sentence has been changed but its meaning remained the same. :/ This is an introduction to the next paragraph...

Line 266-267: What do “>1,5 cm” and “>3,0 cm” mean in the sentence? It has been changed into 15mm, etc.

Line 285: There's an extra parenthesis in '((Tables 4, 6)'. It has been corrected.

Line 309: “data not presented” should be revised to “data are not presented”. It has been corrected.

Line 316: “On the other hand (6) informed that…”. What is the meaning of this sentence? Why does 'On the other hand' require citation of reference [6]? It has been corrected. 

Reviewer 3 Report

Comments and Suggestions for Authors

1. The abstract should explicitly state which three cultivars were used in the experiment. Furthermore, the opening of the abstract should briefly introduce the background of this study, and the conclusion should also point out the significance of the research.

 

2. In the introduction, the description of the application of the "double-phase medium technique" is somewhat general. While it is noted that this technique has been known and used for many years (refs 18-22) and its beneficial effects have been proven for many woody plants (refs 20-29), the specific aspects of its application are unclear. In what detailed areas has research been conducted in plants, and what results were obtained? It is recommended to supplement this section with recent relevant studies to highlight the novelty of the present work.

 

3. In the Materials and Methods section, the methodology for measuring the culture medium pH and mS should be described. Furthermore, what physiological states do these two measurement indicators reflect?

 

4. The authors mentionedin the methods that each treatment used 6 jars, which constitutes the biological replicates in the experiment. However, in the results section and statistical analysis, it must be clearly stated that the ANOVA and multiple comparisons were performed based on the means per jar (n=6). Please explicitly state the unit of data processing and statistical analysis to ensure the validity of the statistical inference. Additionally, the specific numerical values in the tables in the results section should be presented as Mean±Standard Deviation, based on the analysis of the established biological replicates. Performing ANOVA and multiple comparisons without biological replicates, relying solely on technical replicates, is scientifically invalid, and any significant conclusions drawn are unreliable.

 

5. In the discussion section, the physiological mechanisms behind why the "2F medium was beneficial for multiplication but detrimental for rooting and acclimation" should be discussed in depth. This could include aspects such as hormone carry-over effects and carbon source utilization, etc.

 

6. The discussion of the "clone-specific response" in this paper should be analyzed in conjunction with the cultivars' genetic backgrounds or physiological characteristics. Firstly, clarify the species differences. Secondly, perform correlation analysis based on physiological traits and phenotypes, including: the relationship between multiplication vigor and rooting ability (the "multiplication-rooting" trade-off phenomenon); the relationship between callus and rooting (e.g., excessive callusing is often associated with poor rooting and impaired vascular connection); and the relationship between medium pH and mS values (this dataset is key evidence for revealing physiological differences but was not fully utilized in the original text).

 

7. The conclusion should more clearly state the applicability limitations of the double-phase medium and propose directions for further optimization.

 

8. The full text contains several grammatical and spelling errors, such as "clone-depended" and "numer" (should be "number"). It is recommended to perform professional language editing and proofreading for the entire manuscript.

 

9. The figures of results mentioned in the main text should be placed in their corresponding positions below the relevant text to facilitate matching with the textual information. Furthermore, the images should have appropriate scale bars annotated.

 

10. The reference format is not unified. It is recommended to standardize them according to the reference format requirements of the target journal.

Author Response

Thank You for very insightful review. Many of the comments are more suited to work in the field of basic sciences rather than applied ones. Nevertheless, we also presented more results of analyses of the medium used and analysis of correlations between the tested characteristics of the media and cultures. We also have tried to introduce corrections that will at least minimally satisfy the Reviewer.

1. The abstract should explicitly state which three cultivars were used in the experiment. Furthermore, the opening of the abstract should briefly introduce the background of this study, and the conclusion should also point out the significance of the research. Some information has been added.
2. In the introduction, the description of the application of the "double-phase medium technique" is somewhat general. While it is noted that this technique has been known and used for many years (refs 18-22) and its beneficial effects have been proven for many woody plants (refs 20-29), the specific aspects of its application are unclear. In what detailed areas has research been conducted in plants, and what results were obtained? It is recommended to supplement this section with recent relevant studies to highlight the novelty of the present work. More details are given in the Discussion. However, I provided more information in the Introduction. However, I am afraid that another Reviewer will make a comment about repeating the information in the introduction and discussion. I've already come across this objection... :/
3. In the Materials and Methods section, the methodology for measuring the culture medium pH and mS should be described. Furthermore, what physiological states do these two measurement indicators reflect? I have expanded the description. Although not very precise, these indicators are related to the nutrition of cultures and physiological diseases.
4. The authors mentionedin the methods that each treatment used 6 jars, which constitutes the biological replicates in the experiment. However, in the results section and statistical analysis, it must be clearly stated that the ANOVA and multiple comparisons were performed based on the means per jar (n=6). Please explicitly state the unit of data processing and statistical analysis to ensure the validity of the statistical inference. Additionally, the specific numerical values in the tables in the results section should be presented as Mean±Standard Deviation, based on the analysis of the established biological replicates. Performing ANOVA and multiple comparisons without biological replicates, relying solely on technical replicates(?), is scientifically invalid, and any significant conclusions drawn are unreliable. Technical replicates - where does this suspicion come from? No, even the jars were not biological replicates in the experiment. They were used to randomly assign the jar's position on the shelf/fluorescent lamp in each block (randomised block design). Analysis of variance (ANOVA) and multiple comparisons were performed not on means per jar, but on individual cultures! Each culture was removed from the jar and analyzed/measured separately! The data was then subjected to statistical analyses (n=32 ÷ 180, depending on the independent variable)! The exception was the analyses of media, which were performed on individual jars (n=4 ÷ 12, depending on the independent variable). Information about sample sizes for statistical analyses will be provided below the relevant tables. I've given the results in the tables as mean ± SE, as requested by the second Reviewer. I hope this will be accepted.
5. In the discussion section, the physiological mechanisms behind why the "2F medium was beneficial for multiplication but detrimental for rooting and acclimation" should be discussed in depth. This could include aspects such as hormone carry-over effects and carbon source utilization, etc. We performed additional media analyses and correlation analyses. The results are described and used in the discussion.
6. The discussion of the "clone-specific response" in this paper should be analyzed in conjunction with the cultivars' genetic backgrounds or physiological characteristics. Firstly, clarify the species differences. Secondly, perform correlation analysis based on physiological traits and phenotypes, including: the relationship between multiplication vigor and rooting ability (the "multiplication-rooting" trade-off phenomenon); the relationship between callus and rooting (e.g., excessive callusing is often associated with poor rooting and impaired vascular connection); and the relationship between medium pH and mS values (this dataset is key evidence for revealing physiological differences but was not fully utilized in the original text). The two cultivars studied: 'Autumn Brilliance' (AtBr) and 'Ballerina' (Ball) are classified as species A. × grandiflora Rehd., while 'Snowcloud' (SnCl)  as  A. laevis Wieg. According to their descriptions, shrubs of the former species grow slightly less vigorously than those of the latter. However, the 'AtBr' in vitro cultures resembled the 'SnCl' cultures much more closely than the 'Ball' cultures. Therefore, it is difficult to establish a relationship between the cultivars' species affiliation and their in vitro performance. Statistical analyses revealed no significant relationship between shoot and root base callus formation and shoot rooting and acclimation. Callus grew rather after roots appeared, grew over them, and didn't disrupt the connection between shoots and roots. Interestingly, a significant negative relationship was found between callus formation in the previous stage (culture multiplication) and and shoot rooting and acclimation. We performed additional media analyses and correlation analyses. The results are described and used in the discussion.
7. The conclusion should more clearly state the applicability limitations of the double-phase medium and propose directions for further optimization. We placed some suggestions in Discussion.
8. The full text contains several grammatical and spelling errors, such as "clone-depended" and "numer" (should be "number"). It is recommended to perform professional language editing and proofreading for the entire manuscript. OK
9. The figures of results mentioned in the main text should be placed in their corresponding positions below the relevant text to facilitate matching with the textual information. Furthermore, the images should have appropriate scale bars annotated. Unfortunately, we didn't have enough time to put scale bars before the deadline. Instead, I provided information about the size of the jars. If the manuscript is accepted, we may make such a change if the Reviewer still considers it necessary.
10. The reference format is not unified. It is recommended to standardize them according to the reference format requirements of the target journal. OK

 

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

The authors have addressed most of my previous comments. However, its current presentation and style as well as English language expression can be much improved since there were demerits as listed following:

1. Line 145-147:  Change "CaCl₂ × 2 H₂O" into "CaCl_2•2H₂O",  "MgSO₄ × 7 H₂O" into "MgSO_4•7H₂O", "MnSO₄ × 4 H₂O" into "MnSO_4•4H₂O", "ZnSO₄ × 7 H₂O" into "ZnSO_4•7H₂O", "Na₂MoO₄× 2 H₂O" into "Na₂MoO_4•2H₂O", "CuSO₄ × 5 H₂O" into "ZnSO_4•5H₂O", "CoCl₂ × 6 H₂O" into "CoCl_2 •6H₂O".

2. Line 320:  Change "compared to" into "compared with".

3. Figure 2:  Remove the pink background from all pictures.

Author Response

Thank You for positive decision. I will make corrections soon except for the color of the figures.

I have to ask a specialist for correction here because I am partially daltonist...

 

Kind regards, Wojciech Litwińczuk

Reviewer 2 Report

Comments and Suggestions for Authors

The authors corrected all the points I suggested, and the manuscript has been improved.

Author Response

Thank You for your positive decision! :)  

Reviewer 3 Report

Comments and Suggestions for Authors

This version of the manuscript has been refined on the basis of the first draft. However, it still contains a considerable number of issues in terms of language expression, grammatical consistency, terminology unification, and formatting standardization, which require thorough linguistic polishing and proofreading before publication.

Author Response

Thank You for positive decision and effort in preparing such a thorough and good first review!

Kind regards, WL

P.S. Now it's time to improve our English...