Effects of Spectral Quality and Light Quantity of LEDs on In Vitro Shoot Development and Proliferation of Ananas comosus L. Merr
, Giovanni Avola 1,*, Giancarlo Fascella 2, Alessandra Pellegrino 1 and Anita Ierna 1Round 1
Reviewer 1 Report
The MS "Effects of spectral quality and light quantity of LEDs on in vitro shoot development and proliferation of Ananas comosus L. Merr.' is very timely as theoretically LED lighting offers possibility to better control spectral composition and light intensity in closed systems, including in vitro plant propagation. Indeed there are a lot of contrasing results in the literature and the causes of it are not clear so far. The introduction is well written and the aim of the resrarch is clear. At the same time the MS has some shortcomings.
1. English needs to be improved to avoid sucj awkward phrases as (line 23) - 'the number of shoots significantly exceded that of fluorescence',
(line 112) - "the leaf content of .... was extracted" et al.
2. Fig. 2 Why don't you provide results obtained for different light intensities (like on fig. 1), but provide "in the average of the light intensities"
3. How do you explain that the cover index is half lower under RL61?
4. How do you explaine lower chl content under light intensity 100?
Author Response
We appreciate the time and effort that you have dedicated to providing your valuable feedback on our manuscript. We have carefully considered your comments, and we have been able to incorporate changes to reflect most of the suggestions provided.
Please see below for a point-by-point response and in the MS the changes are highlighted in red.
R1: The MS … is very timely as theoretically LED lighting offers possibility to better control spectral composition and light intensity in closed systems, including in vitro plant propagation... The introduction is well written and the aim of the research is clear.
A: Thank you for the positive comment.
R1: English needs to be improved to avoid such awkward phrases as (line 23) - 'the number of shoots significantly exceeded that of fluorescence', (line 112) - "the leaf content of .... was extracted" et al.
A:. Thank you for pointing this out. We revised all the text trying to eliminate awkward phrases and make more concise sentences. You will find all the revisions in the text.
R1_Fig. 2: Why don't you provide results obtained for different light intensities (like on fig. 1), but provide "in the average of the light intensities".
A: Differently from what is shown in Fig. 1 for the secondary shoot number, the results of the ANOVA revealed that the dry weight of in vitro secondary shoots was significantly influenced only by the light spectrum (F3,64=5.32, p<0.001), whereas neither Light Intensity nor the interactions affected this parameter. We believe that the graphical representation we chosen (showing the values in the average of the light intensities) is more consistent with the results obtained. By the way, we modified the text to better clarify the results and to easily interpret the figure.
R1: How do you explain that the cover index is half lower under RL61?
A: We have not a explanation based on the literature, however, we think it may be attributed to the negative interaction light intensity x red enriched light. Cybularz-Urban et al. (2007) found that in Cattleya plantlets grown in vitro yet at 20 μmol m-2s-1, RL caused the collapse of some of the mesophyll cells and a reduction of leaf blades. In Prunus domestica it was demonstrated that RL was already effective at 37 μmol m−2 s−1 and at 9 μmol m−2 s−1(Baraldi er al., 1988). Therefore, 100 μmol m-2s-1 has proven to be in this species a very high intensity to be used with this light.
R1: How do you explain lower Chl content under light intensity 100?
A: This is a good question, thank you for pointing this out. We inserted a explanation of the effects of the higher (100 micromoles) light intensity tested on photosynthetic pigments in the discussion, rows 270-275 in the new version of manuscript.
Author Response File: 
Author Response.docx
Reviewer 2 Report
Please, the first time an abbreviation appears, it is necessary to write it out in full. Maintain the same standard and consistent nomenclature throughout the manuscript, particularly when describing the qualities of light.
-Check citation of references; numbers; names; check Author Instructions;
Introduction section is good.
Material and methods need improve descriptions. In its current form, someone who is not in the field or trying to reproduce the work will not be able to do so. A high level of detail is needed for the step-by-step process, reagents, and concentrations. The entire section should be rewritten for clarification, and not just state that it was carried out according to the author, XXXX.
Results need correction in sentences or clearance. The discussion needs to be compared with references from the literature and to more clearly demonstrate the findings, based on the objectives stated in the introduction.
Minor comments:
-in vitro -> italic
L17. Changes: WL to …”white light”; “far-red”; “UV”….
L19 and 94. 16/8 h photoperiod
-Keywords in alphabetic order;
L44-45. What is observed?
L87. in vitro; italic;
L94 abbreviations;
-L131. I did not understand. What did you use the library for? You open the package.
-Change term content for concentration; L192 chlorophyll concentration;
-Table 1, Figures 1, 2, 3, and 5 need to have the standard deviation or standard error of the mean added. Legends are need improve and better descriptions.
Figure 1, correct “.” Dot not “,” common;
Figure 4. y-axis, need add concentration;
Why high and low intensity? 100 or 80 umol m-2 s-1 are both low! I suggesting change or 80 and 100 umol m-2 s-1. Therefore, changes in text ae necessary.
Why were these proportions/ratios of light qualities chosen?
Check od references and change if necessary;
Best regards
Author Response
Dear Reviewer,
We appreciate the time and effort that you have dedicated to providing your valuable feedback on our manuscript. We have carefully considered your comments, and we have been able to incorporate changes to reflect most of the suggestions provided.
Please see below for a point-by-point response and in the MS the changes are highlighted in red.
R2: Please, the first time an abbreviation appears, it is necessary to write it out in full. Maintain the same standard and consistent nomenclature throughout the manuscript, particularly when describing the qualities of light.
A: Thank you for pointing this out. According to these constructive remarks, we have amended the manuscript to ensure the same nomenclature and abbreviations consistently throughout the manuscript.
R2: Check citation of references; numbers; names; check Author Instructions
A: Thank you for pointing this out. We checked the References and modified when necessary following the Journal instruction.
R2: Material and methods need improve descriptions. In its current form, someone who is not in the field or trying to reproduce the work will not be able to do so. A high level of detail is needed for the step-by-step process, reagents, and concentrations. The entire section should be rewritten for clarification, and not just state that it was carried out according to the author xxx
A: In response to the reviewer's advice, the Material and Methods section underwent significant revision to include a comprehensive description of all the processes and methodologies utilized in the study.
R2: Results need correction in sentences or clearance. The discussion needs to be compared with references from the literature and to more clearly demonstrate the findings, based on the objectives stated in the introduction.
A: Thank you for pointing this out. The Discussion section was extensively revised as suggested by the reviewer.
R2 in vitro -> italic
A: According to this constructive remark, we changed all “in vitro” in italic.
R2_L17: Changes: WL to …”white light”; “far-red”; “UV”….
A: In the abstract, we aimed to combine the need for conciseness with the need for clarity. For this reason, we preferred to report the same information that will be found later in the text.
R2_L19 and 94: 16/8 h photoperiod
A: We have now modified the text accordingly.
R2: Keywords in alphabetic order
A: Thank you for pointing this out. We have now modified the keywords in alphabetic order.
R2:_L44-45: What is observed?
A: According to this constructive remark, we added information in rows 44-46 of the new manuscript.
R2:_L94: abbreviations
A: According to this constructive remark, we added requested information.
R2:_L131: I did not understand. What did you use the library for? You open the package.
A: R packages are extensions to the R statistical programming language and represent the fundamental units of reproducible R code. They include reusable R functions, code, data, documentation and sample data. We did not modify any package, but we just report the specific R packages we used to allow the reader to follow the statistical process applied.
R2: Change term content for concentration; L192 chlorophyll concentration
A: Thank you for pointing this out. We have now modified the text accordingly.
R2: Table 1, Figures 1, 2, 3, and 5 need to have the standard deviation or standard error of the mean added. Legends are need improve and better descriptions.
A: Thank you for pointing this out. All the figures were modified to meet the reviewer’s advice. Moreover, a more detailed description of studied factors was added in the their caption.
Regarding to the Table 1, the insert of the standard error of the mean resulted in complexity in the table formatting capable of reducing its readability, so we preferred to relegate this information only to the figures.
R2: Figure 1, correct “.” Dot not “,” common;
A: Thank you for pointing this out.
R2: Figure 4. y-axis, need add concentration;
A: According to this constructive remark, we added requested information.
R2: Why high and low intensity? 100 or 80 umol m-2 s-1 are both low! I suggesting change or 80 and 100 umol m-2 s-1. Therefore, changes in text are necessary.
A: Thank you for pointing this out. We changed the text and figures as suggested.
R2: Why were these proportions/ratios of light qualities chosen?
A: Plant photoreceptors responsible for plant development and photosynthesis are known to be primarily and most significantly stimulated by red (RL) and blue (BL) regions of the light spectrum. The different light fixtures were chosen to have a better evaluation of the role of red and blue spectra to ensure a good and stable proliferation in the LED Lamps. The different proportions are described in the materials and method section. The wavelengths corrispondono to red grows from 38% in the white lamps (blue 20%; green 36%; red 38%; far red 6%) to 63% in the prevalently red lamps in which also blue green and far red are still present (blue 10% green 19%; far red 8%). In the blue/red lights the percentage of red is almost the same of the prevalently red lamps, but the percentage of blue raised to 33 % and the other lights spectra are eliminated. In this way the effects of the different light spectra and in particular of the red one are more evident as have been debated in the discussion section.
Author Response File: Author Response.docx
Reviewer 3 Report
Dear Authors,
I have a few suggestions to improve your manuscript:
Materials and Methods:
The first paragraph should be section 2.1 (For example... In vitro culture and light treatments or as you wish).
The duration of a culture cycle from the initiation of the in vitro culture to the performance of the measurements is not clearly presented. Ten to twelve week old in vitro pineapple plants grown under fluorescent light at 80 μmol m-2s-1 were subjected to four different light spectra for another 60 days? The duration of a culture cycle was ten weeks + 60 days?
Please, clarify.
Results
In the results section you should present several values obtained from the measurements. In section 3.4. Chlorophyll content, you do not present any value.
Table 1 - if possible, add ± SE or SD
Figures -1,2,3,5 - add error bars
Explain the abreviations (FL, WL, RL61% , RL/BL) in Table and figures
Discussion
“Our study” is repeated too much.
Conclusions
In conclusion, you state that:
“Moreover, even if WL is less effective as RL in overcoming apical dominance, high and stable proliferation rates are reported to be obtained by the addition of cytokinin to the medium. However, in species that are particularly difficult to regenerate in vitro and/or show a high sensibility to higher concentration of citokinins, a few days exposure to red enriched light may exert a useful initial stimulatory effect on shoot proliferation whereas WL may ensure the subsequent plantlet growth.”
What is the basis for this conclusion? The only plant growth regulator in the medium you used, was 0.5 mg/L BA.
Adjust the conclusions with the research objectives.
References
The cited references are relevant but not up to date. It's better if the author has to add new references within 5 years and omit the old references.
Also, they must be written according to the author's guide.
Author Response
Dear Reviewer,
We appreciate the time and effort that you have dedicated to providing your valuable feedback on our manuscript. We have carefully considered your comments, and we have been able to incorporate changes to reflect most of the suggestions provided.
Please see below for a point-by-point response and in the MS the changes are highlighted in red.
R3: The first paragraph (of M&M) should be section 2.1 (For example... In vitro culture and light treatments or as you wish).
A: According to this constructive remark, we have now modified the title of paragraph accordingly.
R3: The duration of a culture cycle from the initiation of the in vitro culture to the performance of the measurements is not clearly presented. Ten to twelve week old in vitro pineapple plants grown under fluorescent light at 80 μmol m-2s-1 were subjected to four different light spectra for another 60 days? The duration of a culture cycle was ten weeks + 60 days? Please, clarify.
A: According to your suggestion, we described more clearly all the steps of the process in the paragraph 2.1 of the Materials and Methods.
R3: In the results section you should present several values obtained from the measurements. In section 3.4. Chlorophyll content, you do not present any value.
A: According to this constructive remark, we have now modified the text accordingly, inserting some relevant values and inserted the reference to table 1.
R3: Table 1 - if possible, add ± SE or SD - Figures -1,2,3,5 - add error bars Explain the abreviations (FL, WL, RL61% , RL/BL) in Table and figures
A: Thank you for pointing this out. All the figures were modified to meet the reviewer’s advice. Moreover, a more detailed description of studied factors was added in the their caption.
Regarding to Table 1, the insert of the standard error of the mean resulted in complexity in the table formatting capable of reducing its readability, so we preferred to relegate this information only to the figures.
Author Response File: Author Response.docx
Round 2
Reviewer 1 Report
English gramma still needs to be improved.
Reviewer 2 Report
I consider that the authors have made the necessary improvements to the manuscript, and I recommend it for publication.
Reviewer 3 Report
The article has been greatly improved and many thanks to the authors for the answers.
I have one more comments:
Line 26: Keywords: rewritten in lower case (according to instructions for authors)
