The Cell Wall Regeneration of Tobacco Protoplasts Based on Microfluidic System

Round 1

Reviewer 1 Report

 

Overall, this is a valuable contribution to microanalytics in cell biology using a novel crossed channel microfluidic system that holds substantial promise for the simultaneous microscopic observation of cell processes in several channels (such as the controlled experiment shown here with plant protoplasts) and the capacity to collect individual cells for further analyses using modern single-cell analytic techniques such as molecular genetic, proteomic, and transcriptomic studies. I believe that the authors provide convincing evidence of the applicability of their invention. As an experimental biologist, I have focused mainly on the biological research aspects, I cannot comment authoritatively on the full aspects of engineering design and application, but it appears the authors have been successful in their objectives.

 

I have only a few minor recommendations for improving the text. Although, in general, the manuscript is nicely organized.

Line     recommended change

32        “—and represents one of the most intricate network structures in nature [1].”

33        “--- and acts as a first tier within the defending system in response to external damage.”

38        “---of a protoplast is a precondition for cell division.”

40-41   This sentence is not clearly written in English. I have attempted to improve it as follows:

            “As a matter of fact, many studies have proposed that there are some major factors that influence the regeneration of cell walls in various plants [8,9].

42        “--- we found that the use of culture media is the most common method---”

69        “---materials, Co., Ltd. 1 mL syringes were bought ---”

81-82   “After baking, three holes were punched on the back of the glue using corresponding punchers with diameters of 1, 1.5 and 2.5 mm, which produced the ports for injection of the protoplasts that were suspended in solution (1), injection of the treatment solution (2), and outlet for the solution (3), respectively (Scheme 1C).

104      “---- Sun’s method was employed [20].

110-112           “After that, the solution containing protoplasts was centrifuged for 6 min at 150 rcf. Then the precipitated protoplasts were resuspended by using the modified W5 solution ----”

126-127           “--- protoplast solution, containing the 0.1% calcium fluoride white and the solution of the treatments, was injected---” The predicate should be ‘was’ because the subject of the sentence is ‘solution’.

143      “---- was calculated, and thus the regeneration ratio of the plant cell was obtained.”

155      “As shown in Figure 1, the protoplasts ------”

159      “It should be emphasized that, compared with the ----”

168      “As shown in Figures 2A and 2B, the protoplasts fluoresce red due to the irradiation of the chloroplasts with ultraviolet light.”

169-170           “The regeneration of the cell wall led to the appearance of a bluish white stain produced by the CFW stain of the cell wall (Figures 2d and 2f).”

171      “Compared with the control (Figure 2c), the ----”

173      “The cell wall of the protoplasts regenerated more rapidly when treated with BTH compared to the control, which suggested ----”

183      “Furthermore, the dynamic processes of protoplast cell wall regeneration within 7 hours after treatment with BTH were ----”

188      “A, C, and E show the cell wall regeneration of --------”

189      “----- under the white light: a, c and e show the cell wall---”

190      “----- B, D and H show ---”

191      “Also, b, d and f show the ----"

            When you refer to a figure or a table, etc. we use the present tense because the reader is being directed to look at the printed figure or the table at the present time.

202      “As shown in Figure 4, the ----”

204      “There was no significant variation in the amount and activation of the Pst DC3000 during the 24 h culture.”

206      “As shown in Figure 5, ---”

207-209           “In addition, constant moving, colliding and infecting of protoplasts by Pst DC3000 was observed in the microfluidic chip (Video S2).”

215      “Compared to the prior study [31], our results provided direct evidence for the Pst DC3000 infection of the plants as evidenced by the inhibition of cell wall regeneration, which is a novel discovery for the Pst DC3000 infection events at the cellular level.

 

Lines 220-221 Figure 4 caption. Authors: you have no A, B, C, or D labels on your Figure 4 panels. You need to place these alphabetic labels on each panel in the upper left, and move the hour labels to the right-hand side of each panel to be consistent with your other figures (such as Figure 5).

Line 221          “12 h (B), 24 h (C), the magnified image of Pst DC3000 at 24 h (D).

Line 224          “--- the magnified image (B).

229-230           “DC3000 under white light. b, d and f show the cell wall regeneration—”                There is no panel h in Figure 6.

234-235  “Therefore, the observed dynamics of the protoplast cell wall regeneration is- -—”

 

Author Response

Response 1: Thank you for your constructive suggestions. We have revised our manuscript as all these comments; for instance, some inaccurate descriptions and grammar mistakes have been revised. Besides, Figure 4 has also been modified.

Author Response File: Author Response.docx

Reviewer 2 Report

1. Please put the scales in figure 1.

2. The introduction does not reflect the concept and it should be precise and related to the manuscript content.

 

3. Authors are requested to explain in detail how the microfluidic channels with significantly larger diameters helped in better dispersion of protoplast with much lesser dimensions (page 4, lines 159-161).

Author Response

Point 1: Please put the scales in figure 1.

 

Response 1: Thanks for your suggestion. The scales have been put in Scheme 1.

 

Point 2: The introduction does not reflect the concept and it should be precise and related to the manuscript content.

 

Response 2: Thanks for your suggestion. We have added more description about the application of microfuidic device in animals and plants to make the introduction more precise.

 

Point 3: Authors are requested to explain in detail how the microfluidic channels with significantly larger diameters helped in better dispersion of protoplast with much lesser dimensions (page 4, lines 159-161).

 

Response 3: Actually, the most important thing helped in better dispersion of protoplast is not the crossed microchannel with significantly larger diameters, but the crossed microchannel provides more opportunities for protoplasts to choose. Therefore, when these protoplasts choose different channels and they will be dispersed. And this explanation has been included in our manuscript, as following: “It should be emphasized that, compared with the single channel, the crossed microchannel of our study can supply more opportunities for protoplasts to choose, which will help to separate and obtain the single protoplast.”.

Author Response File: Author Response.docx

Round 2

Reviewer 2 Report

The manuscript can be accepted.