An Efficient System for Mesophyll Protoplast Isolation, Purification, and Transformation in Loquat: Studies on Fluorescent Marker Analysis and Subcellular Localization

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

In this manuscript, Shuming Wang and colleagues established an efficient system for mesophyll protoplast isolation, purification, and transformation in Loquat. I have following comments:

1, For the title, I suggest to employ “An Efficient System for Mesophyll Protoplast Isolation, Purification, and Transformation in Loquat (Eriobotrya japonica Lindl.)”.

2, For the Abstract, details like “The parameters of this system were as follows: protoplast concentration of 2 × 105 cells·mL-1, exogenous DNA concentration of 5 μg/100 μL, final concentration of PEG-4000 at 20% w/v, mannitol concentration 0.4 M and transformation duration of 5 min.” should be removed.

3, For the key words, “protoplast isolation” and “protoplast purification” appeared in the title should be rephrased.

4, For the introduction, required citation should be provided. For instance, please provide the citation for the sentence “... protein-protein interaction and transgenic studies. (Line 44)”

5, For the results, scale bars should be included in Figure 1a, b, c,d, Figure 3, and Figure 5. The value for all scale bars should be described in Figure legend. For the Figure 5, what does the arrow indicate? and error value should be provided in Table 1. Sizes of protoplasts are distinct between 7a and 7C, but the scale bars are the same, please explain.

6, For the materials and methods, genotypes of Loquat examined in this study should be described. Biological and technical replicates, as well as randomization methods should be clearly stated. Plant growth conditions and sampling size like leaf numbers should be included.

7, For the discussion, I would like to see the discussion section was divided into subsections with appropriate titles.

8, A separate conclusion section should be included.

9, A figure depicting the workflow of mesophyll protoplast isolation, purification, and transformation in Loquat should be provided.

Author Response

Comment 1. For the title, I suggest to employ “An Efficient System for Mesophyll Protoplast Isolation, Purification, and Transformation in Loquat (Eriobotrya japonica Lindl.)”.

Response 1: We thank this reviewer for your positive comments. As suggested, we have modifying the title to ‘An Efficient System for Mesophyll Protoplast Isolation, Purification, and Transformation in Loquat (Eriobotrya japonica Lindl.).

Comment 2. For the Abstract, details like “The parameters of this system were as follows: protoplast concentration of 2 × 105 cells·mL-1, exogenous DNA concentration of 5 μg/100 μL, final concentration of PEG-4000 at 20% w/v, mannitol concentration 0.4 M and transformation duration of 5 min.” should be removed.

Response 2: Thank you for your comments. As suggested, we made a revision to clarify the picking period of the bud, please find in Row 83-84

Comment 3. For the key words, “protoplast isolation” and “protoplast purification” appeared in the title should be rephrased.

Response 3: Thank you for your comments. As suggested, we have rephrased the key words.

Comment 4. For the introduction, required citation should be provided. For instance, please provide the citation for the sentence “... protein-protein interaction and transgenic studies. (Line 44)”

Response 4: Thank you. According to your suggestions, we have added references in introduction section (Row 38, Row 47-50, Row 268, Row 270, Row 273, Row 298).

Comment 5. For the results, scale bars should be included in Figure 1a, b, c,d, Figure 3, and Figure 5. The value for all scale bars should be described in Figure legend. For the Figure 5, what does the arrow indicate? and error value should be provided in Table 1. Sizes of protoplasts are distinct between 7a and 7C, but the scale bars are the same, please explain.

Response 5: Thanks for pointing this out. We have added the value for all scale bars and the annotation of arrows in the corresponding figures.

Comment 6. For the materials and methods, genotypes of Loquat examined in this study should be described. Biological and technical replicates, as well as randomization methods should be clearly stated. Plant growth conditions and sampling size like leaf numbers should be included.

Response 6: Thanks so much. We have added the genotypes of Loquat, the young leaf size examined in this study to the materials and methods section (Row 327-337), and Biological and technical replicates were stated in Data analysis section (Row 399-401).

Comment 7. For the discussion, I would like to see the discussion section was divided into subsections with appropriate titles.

Response 7: Thanks. We have divided the discussion section into three parts.

Comment 8. A separate conclusion section should be included.

Response 8: Thanks. We have added the separate conclusion section in the end of this manuscript.

Comment 9. A figure depicting the workflow of mesophyll protoplast isolation, purification, and transformation in Loquat should be provided.

Response 9: Thank you. As you suggested, we have supplemented the Flow chart as Figure 9 (Row 338).

Reviewer 2 Report

Comments and Suggestions for Authors

The manuscript on the preparation of loquat protoplasts shows some methodological insights. However, there are significant shortcomings in the data collection process, which limit the overall impact and value of the study.

Major Comments:

1. Introduction:

   • The authors mention the production of DNA-free genome-edited individuals by introducing RNPs into protoplasts. This led to the expectation that results on protoplast culture would be presented. However, no such experiments were conducted, raising concerns about how effectively the current results alone can capture the reader's interest.

2. Table 1:

   • There is no mention of protoplast yield in Table 1. The microscopic observations suggest that the protoplasts were deformed, but were all the observed protoplasts deformed? It seems unlikely that the protoplast yield was zero for the other conditions. More detailed data should be provided.

3. Figure 3:

   • The yield of protoplasts should vary depending on the initial tissue used, but the manuscript does not address this. Clarification is needed to understand the significance of this experiment.

4. Figure 4:

   • The initial amount of protoplasts used in the experiment should be known, allowing for the calculation of the remaining protoplasts after the purification process. However, these results are not provided. Additionally, no subsequent experiments requiring purification are presented, making it unclear how significant this process is. Would the protoplasts shown in Figure 1e not already be sufficiently pure?

5. Figure 8:

   • The data presents a localization of a gene, but would not a test using protoplasts from other plant species, such as Arabidopsis, be sufficient? The usefulness of loquat protoplasts is not convincingly demonstrated, especially given that even a rice gene is accurately transferred to the chloroplast.

Minor Comments:

1. Introduction:

   • Lines 42–43: Why is Lin et al. (2021) (https://www.tandfonline.com/doi/full/10.1080/14620316.2020.1814876) on E. japonica transformation not cited?

   • Lines 53–54: The phrase "still challenging" might be misleading, as there are multiple successful examples of woody plant transformations, such as poplar and pine.

2. Materials and Methods:

   • Provide detailed growing conditions to allow reproducibility. For example, the term "artificial soil mixture" should specify the proportions of the three ingredients used. Additionally, clarify whether the greenhouse was outdoors and include details on temperature, humidity, and light conditions.

3. Figures:

   • A scale bar should be added to each photo, with its size clearly indicated in the figure legend.

   • Figures 2 and 6: The meaning of letters (a, b, c, etc.) should be explained in the legend.

   • Figure 5: The significance of the red and blue arrows should be explicitly stated.

4. General Typos:

   • The manuscript contains numerous typographical errors. For example:

     • Line 145: "EMS" should be "MES."

     • Figure 2: "cellulose RS" should be "Cellulase RS."

     • Line 360: "mess" should be "mesh."

   • A thorough proofreading is recommended to improve readability and accuracy.

Overall, while the study provides some methodological contributions, addressing these issues will significantly enhance the quality and impact of the manuscript.

Comments on the Quality of English Language

A thorough proofreading is recommended to improve readability and accuracy.

Author Response

Comment 1. Introduction:

The authors mention the production of DNA-free genome-edited individuals by introducing RNPs into protoplasts. This led to the expectation that results on protoplast culture would be presented. However, no such experiments were conducted, raising concerns about how effectively the current results alone can capture the reader's interest.

Response 1: We sincerely appreciate this reviewer for the positive comments. Currently, DNA-free genome editing technologies indeed represent one of the most transformative applications of protoplast transformation systems in modern plant biotechnology. That's why we introduced this technology in the introduction section. In fact, we are now conducting experiments on applying genome - editing within the system developed in this study. We are confident that we will achieve promising results in the future.

Comment 2. Table 1:

There is no mention of protoplast yield in Table 1. The microscopic observations suggest that the protoplasts were deformed, but were all the observed protoplasts deformed? It seems unlikely that the protoplast yield was zero for the other conditions. More detailed data should be provided.

Response 2: Thank you for your comments. As you suggested, we supplemented the figures in the supplementary materials (Figure 2S). Under the higher osmotic pressure of mannitol in the digestion solution, most protoplasts were deformed. As can be clearly seen from the figures, the protoplast yield decreased significantly. Therefore, we did not calculate the quantity of protoplasts.

Comment 3. Figure 3:

The yield of protoplasts should vary depending on the initial tissue used, but the manuscript does not address this. Clarification is needed to understand the significance of this experiment.

Response 3: Thank you for your comments. As you suggested, we have improved the description of this part and added the data to the supplementary materials (Figure 3S).

Comment 4. Figure 4:

The initial amount of protoplasts used in the experiment should be known, allowing for the calculation of the remaining protoplasts after the purification process. However, these results are not provided. Additionally, no subsequent experiments requiring purification are presented, making it unclear how significant this process is. Would the protoplasts shown in Figure 1e not already be sufficiently pure?

Response 4: Thank you for your comments. While the omission of protoplast quantification in the purification section represents a technical limitation, this parameter does not compromise the validity of the results illustrated in Figures 4 and 5.

Comment 5. Figure 8:

The data presents a localization of a gene, but would not a test using protoplasts from other plant species, such as Arabidopsis, be sufficient? The usefulness of loquat protoplasts is not convincingly demonstrated, especially given that even a rice gene is accurately transferred to the chloroplast.

Response 5: Thank you. We conducted transient expression assays of EjDELLA, EjbHLH79 proteins using our optimized transient transformation method, which has been previously reported in loquat. OsPHT4 serving as a marker gene across different species, was employed in our experimental system. Collectively, these results demonstrate the spatial resolution and cross - species applicability of our transformation platform.

Minor Comments:

Comment 1. Introduction:

Lines 42–43:Why is Lin et al. (2021) (https://www.tandfonline.com/doi/full/10.1080/14620316.2020.1814876) on E. japonica transformation not cited?

Response 1: Thanks for pointing this out. We have cited this paper in the introduction section, specifically at line 38.

Comment 2. Lines 53–54:The phrase "still challenging" might be misleading, as there are multiple successful examples of woody plant transformations, such as poplar and pine.

Response 2: We have refined the wording to make it more precise.

Comment 3. Materials and Methods:

Provide detailed growing conditions to allow reproducibility. For example, the term "artificial soil mixture" should specify the proportions of the three ingredients used. Additionally, clarify whether the greenhouse was outdoors and include details on temperature, humidity, and light conditions.

Response3. : Thanks for pointing this out. We have added correlating parameters

(Row 336-352).

Comment 4. Figures:

A scale bar should be added to each photo, with its size clearly indicated in the figure legend.

Figures 2 and 6: The meaning of letters (a, b, c, etc.) should be explained in the legend.

Figure 5: The significance of the red and blue arrows should be explicitly stated.

Response 4: Thanks for pointing this out. We have added them.

Comment 5. General Typos:

The manuscript contains numerous typographical errors. For example:

Line 145:"EMS" should be "MES."

Figure 2:"cellulose RS" should be "Cellulase RS."

Line 360:"mess" should be "mesh."

Response 5: Thanks for pointing this out. We have corrected them.

Reviewer 3 Report

Comments and Suggestions for Authors

Re:

Wang et al. presented a research article entitled “An Efficient System for Mesophyll Protoplast Isolation, Purification, and Transformation in Loquat: Applications in Fluorescent Marker Analysis and Subcellular Localization Studies” for publication consideration in IJMS.

The authors reported on loquat (Eriobotrya japonica Lindl.), one of the most important subtropical evergreen fruit trees, focusing on its gene transformation platforms. In this study, they developed an efficient protocol for protoplast isolation and purification, achieving high protoplast yields. Additionally, they established a transient protoplast gene expression system with approximately 40% efficiency using a GFP reporter gene. They claim this is the first report on an efficient protoplast isolation, purification, and transformation system in loquat mesophyll. The system described here is expected to facilitate advancements in breeding, genetic transformation, and molecular research.

The article is scientifically well presented. I would be grateful if the authors could consider modifying the title to “An Efficient System for Mesophyll Protoplast Isolation, Purification, and Transformation in Loquat: Studies on Fluorescent Marker Analysis and Subcellular Localization”.

Please ensure the readiness and flow of the manuscript before publication.

Comments on the Quality of English Language

Could be improved. 

Author Response

Comment: The article is scientifically well presented. I would be grateful if the authors could consider modifying the title to “An Efficient System for Mesophyll Protoplast Isolation, Purification, and Transformation in Loquat: Studies on Fluorescent Marker Analysis and Subcellular Localization”.

Response: We thank this reviewer for your positive comments. As suggested, we have modifying the title to ‘An Efficient System for Mesophyll Protoplast Isolation, Purification, and Transformation in Loquat (Eriobotrya japonica Lindl.).

Reviewer 4 Report

Comments and Suggestions for Authors

The objective of the paper by Wang et al. is to develop the efficient protoplast isolation, purification and transformation protocols in one of the most important subtropical evergreen fruit trees  (Eriobotrya japonica Lindl.). The argument is interesting; it is important to identify efficient protocols in a recalcitrant species that can promote knowledge and application of techniques for genetic improvement, but although the approach is correct, there are some important gaps and/or unclear aspects.

First of all the authors should better explain the experimental planning, including a clear description about
the plant material used for experiments: young leaves, seeding, flowers and callus? For callus and other explants are the protocols used the same as those for young leaves? This aspect in the manuscript needs to be revised.

The authors at line 145 refer to EMS, did they use a mutagenic agent? or did they use MES?

Line 147 (Fig 1 e, f) change with (Fig. 1 e and f).

Therefore, in summary, the manuscript is of interest, but It is suggested to review the manuscript

Author Response

Comment 1. First of all the authors should better explain the experimental planning, including a clear description about the plant material used for experiments: young leaves, seeding, flowers and callus? For callus and other explants are the protocols used the same as those for young leaves? This aspect in the manuscript needs to be revised.

Response 1: Thanks for your comments. We have re-described the experimental material section (Row 336-352).

Comment 1. The authors at line 145 refer to EMS, did they use a mutagenic agent? or did they use MES?

Line 147 (Fig 1 e, f) change with (Fig. 1 e and f).

Response 2: Thanks for pointing this out. We have corrected them.

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

Authors have positively responded to my concerns in the revision.