Induction of Polyploidy in Citrus Rootstocks through In Vitro Colchicine Treatment of Seed-Derived Explants
Round 1
Reviewer 1 Report
Thank you for this valuable research the following points must be considered:
-The genus of plants through out the article must be italic
-More elaboration of the preparation and procedure of flowwcytometery
-Staining dye used for DNA must mention
-Must add control group for DMSO
- What is ment by except for control 50?
-What is f in seed f=16.7?
-What is the abbreviation CD in table 1 mean?
-In table 2 must mention unit of measurment
-Revise the title of table 3
- lethal dose mention in result not in materialand methods
-Figure 2 mention before figure 1
- Figure 5 has different magnification power
- Time and place of cultivation must mention
Author Response
Reviewer 1.
- The genus of plants throughout the article must be italic:
Incorporated in the document (changes with red lettering).
- More elaboration of the preparation and procedure of flow Cytometry
Ploidy analysis was carried out using a flow cytometer (Partec Gmbh, Munster, Germany). Flow Cytometry works by estimating the volume and florescence of isolated nuclei. The ploidy was presented in the form of a histogram of integral fluorescence with the peaks depicting the ploidy level of the respective sample.
The protocol consists a sequence of steps starting with excision of a 0.3-cm2 piece of emerging leaf tissue and placing in a Petri plate. The samples were prepared for analysis using a High-Resolution Staining Kit (Partec GmbH). The samples were chopped with a sharp blade in the presence of 500-800μl of Nuclei Extraction Buffer and the nuclei were filtered through a nylon screen 30-μm filter into a 3.5-mL tube and stained with 0.5- 1ml of Nuclei staining buffer (42, 6- diamidino-2-phenylindole dihydrochloride). After that, samples were used for flow Cytometry analysis. When the cells were labelled with fluorescent colouring due to the staining buffer passed through the measuring area one after the other, the individual cells or particles got illuminated by the excitation light and the fluorescent light intensity was proportional to DNA content. The samples were analyzed in a UV-LED Partec flow cytometer with light emission at 365 nm, adjusted to florescence optical detection to gain as per the control sample of cultivar or as per species. More than 5000 nuclei were assessed in each sample. Nuclear DNA histograms were constructed using CyView software (Partec Gmbh, Germany), which determines peak position and relative ploidy level of the tested samples.
- Staining dye used for DNA must mention:
We have used 4’,6-Diamidino-2-phenylindole dihydrochloride (DAPI) as DNA Staining dye in flow cytometry study
- Must add control group for DMSO:
In our experiment, we have used DMSO for initial dissolving of colchicine to improve the colchicine efficiency. As a control we have use without colchicine and DMSO where seedlings were simply emmersed in liquid MS medium at a specified exposure times (16 hrs & 24 hrs)
- What is meant by except for control 50
It is wrongly mentioned (typing mistake), 50 explants per treatment is common including control with four replications.
- What is f in seed f=16.7?
F test is used to determine whether the experiment is statistically significant or not in this experiment (under the heading 3 results), 16.776 is F calculated value of interaction effect of cultivar & treatment (sources of variation) at 14 degrees of freedom, which was found statistically significant both at 1% & 5% CD (critical difference value). The value 16.776 which is f calculated value of interaction effect of cultivar & treatment obtained through analysis of variance to calculate the CD values.
Actually it is not required / to be mentioned in the text portion as this is not reflected in the table, as CD values are already reflected at the bottom of table to know whether the treatment results are statistically significant or not.
- What is the abbreviation CD in table 1 mean?
CD (critical difference value) it is mentioned in every table.
The difference of two treatments mean is less than CD it can be concluded both the treatments are on par.
In our experiment the difference between any two treatments means is coming higher than the CD value both at 5% and 1 %. So, it means treatments effects are statistically and significantly apart.
- In table 2 must mention unit of measurement
Unit of measurement mentioned in table in red lettering
- Revise the title of table 3
Revised title: Effect of in vitro colchicine treatment on percentage of plant morphology variation of different citrus rootstocks.
- Lethal dose mention in result not in material and methods
LD 50 value is not mentioned in this present experiment, the main objective of this experiment to find out most efficient and safe method for induction of tetraploidy for handling large number of seeds and to reduce safety risks associated with the colchicine handling. So in this experiment we have demonstrated most safe, economically viable and technologically feasible method for induction of tetraploidy . So while conducting this experiment
The LD50 value was calculated based on seedling mortality / survival percentage compared with control treatment.
- Figure 2 mention before figure 1
Modification done in document
- Figure 5 has different magnification power
The stomata / guard cell were counted at 40 x magnification and stomata length was counted at 100 x.
- Time and place of cultivation must mention
Experiment was conducted at Tissue culture lab and nursery site of ICAR-CCRI, Nagpur, MS, India during 20-2022.
Author Response File: 
Author Response.docx
Reviewer 2 Report
The study deals with the induction of polyploidy in citrus rootstock. It presents relevance for the sector and contribution, although observing the potential of these polyploids as a rootstock in promoting increased production, among others, should be an important point.
Some suggestions were made
Q1. No abstract. Would it be interesting to mention the result of how many % was this reduction?? when using each concentration of colchicine.
Q2. Introduction. In the second paragraph, when the authors talk about susceptibility to cold stress, it seems that they apply to all rootstocks evaluated, but is this really the case?
Q3. Still in the second paragraph, the authors present text without proper citation. All this information needs to be based on some literature, therefore I recommend that the citation be carried out. Also, how can your study solve these problems in these rootstock species?.
Q4. Material and methods. in item 2.2 Was part of this methodology based on any previous study? Or did the authors develop the entire protocol from scratch? it is necessary to cite the bases that supported your research. The same goes for item 2.3.
Q5. In item 2.5 it is necessary to mention the Laica microscope model, as there may be variation in the quality of the images in relation to the type.
Q6. In item 2.6, what software did the authors use for data analysis?
Q7. Results. Only table two was called in the results, it is important that all tables presented in the results are properly mentioned and the results contained in them are presented. In addition, formatting is necessary to help in the interpretation of the tables, informing what the value in parentheses ( )) means in Table 1, the information contained in the isolated factors and interaction.
Q8. In the tables (A, T, I) It was not clear that these values correspond to the ANAVA F test ??? the ANAVA P value ?? need to describe in the footer of the tables =.
Q9. Many words throughout the text are together Ex "Table 3. Effect of invitrocolchicinetreatmenton" I don't know if it was a problem at the time of generating the PDF but this needs to be reviewed in detail.
Q10. In table 4 I am not seeing any correlation, the table call needs to be reformulated.
Q11. At the end of topic 3.2 there is a citation [21], I believe there was a formatting error.
Q12. In topic 3.3, it is more interesting to make a box plot with all the data in each treatment, demonstrating the variation of the stomata and presenting the p value in the same figure, instead of inserting the measurement of each stomata. This also applies to the other details of plant morphology.
Q13. Figure 9. It would be more interesting to present the height measurements of the plant of the thorns and the mosphology of the flowers and to record the variation by P value and insert the figures as a supplementary file.
Q14. Discussion. The discussion is shallow, without actually delving into what led to the reduction in survival with longer exposure to the product and concentration. It did not bring information on future studies, for example, using these rootstocks under commercial conditions to induce greater production in oranges. The discussion between the different rootstocks is vague and needs to be deepened. There was no recommendation on what would be the best treatment.
Q15. At the end of the discussion the authors present "Hence in the present study authors used a combination of cytology and flow cytometry techniques for confirmation of induced polyploidy
at field condition samples". Ok, but did this information bring greater contributions in relation to what is already available in the literature or in relation to morphological evaluations? This was not discussed in depth.
Q16. Conclusions. The authors are making a general summary of the study and in the conclusions it should be mentioned what was concluded after all the study and discussion of the results.
Author Response
Reviewer 2
Q1. No abstract. Would it be interesting to mention the result of how many % was this reduction?? when using each concentration of colchicine.
:Highest survival % obtained 70.140% at control treatment (without colchicine)
0.1% colchicine treatment for 24 hrs exposure time, resulted in mean cumulative survival percentage of 32.438% and 39.369 % at 16 hrs exposure.
Considering the safe dose of colchicine (0.1% at 16 hrs & 24 hrs exposures) where cumulative mean survival obtained as 35.90 % (average of 0.1% at 16 hrs & 24 hrs exposures time) of the survived seedlings, the survival rate compared to the cumulative mean survival obtained at control (16 hrs & 24 hrs) as 69.83%, hence the reduction in survival percentage is 48.58%.
Since these details are elaborative, hence not reflected in the abstract.
Q2. Introduction. In the second paragraph, when the authors talk about susceptibility to cold stress, it seems that they apply to all rootstocks evaluated, but is this really the case?
:In our study the main objective of this experiment is to find out most efficient and safe method for induction of tetraploidy for handling large number of seeds and to reduce safety risks associated with the colchicine handling. So in this experiment we have demonstrated most safe, economically viable and technologically feasible method for induction of tetraploidy and the future potential use of these produced or created tetraploids that can translate to better orchard performance in terms of abiotic stress (cold stress) and yield parameters indicated in the introduction paragraph.
Q3. Still in the second paragraph, the authors present text without proper citation. All this information needs to be based on some literature, therefore I recommend that the citation be carried out. Also, how can your study solve these problems in these rootstock species?.
As per requirement we have incorporated the citation in the document.
In our study the main objective of this experiment is to find out most efficient and safe method for induction of tetraploidy for handling large number of seeds and to reduce safety risks associated with the colchicine handling. So in this experiment we have demonstrated most safe, economically viable and technologically feasible method for induction of tetraploidy and the future potential use of these produced or created tetraploids that can translate to better orchard performance in terms of abiotic stress (cold stress) and yield parameters, as supported by the previously reported references / literature.
For confirmation kindly refer the articles on abiotic stress tolerances (Tan et al., 2015), drought (Allario et al., 2013), boron toxicity (Marta Ruiz et al., 2016), salinity stress (M. Ruiz et al., 2016), chromium toxicity (Balal et al., 2017), and chilling stress (Oustric et al., 2017) where they have reported increased yield and improved biotic and abiotic stress resistance in citrus, which are already mentioned in our references.
Q4. Material and methods. in item 2.2 Was part of this methodology based on any previous study? Or did the authors develop the entire protocol from scratch? it is necessary to cite the bases that supported your research. The same goes for item 2.3.
The method used in this present study for induction of tetraploidy in citrus rootstocks is based on the method employed by Kainth and Grosser, 2010 in Citrus grandis
Q5. In item 2.5 it is necessary to mention the Laica microscope model, as there may be variation in the quality of the images in relation to the type.
No, it is not necessary to mention the Leica microscope, there will not be any variation in the image in relation to type of any microscope with high resolution and quality.
Q6. In item 2.6, what software did the authors use for data analysis?
ICAR-GOA WASP 1.0 (Web Agri Stat Package)
Q7. Results. Only table two was called in the results, it is important that all tables presented in the results are properly mentioned and the results contained in them are presented. In addition, formatting is necessary to help in the interpretation of the tables, informing what the value in parentheses ( )) means in Table 1, the information contained in the isolated factors and interaction.
We have incorporated the adequate elaboration of the result with reference to each table which was indicated with red lettering, kindly refer line number & page number.
Values in parenthesis are transformed values
‘A’ factor is cultivar, ‘B’ factor is treatment (colchicine concentration & exposure time) & ‘A× B’ is the Interaction between A & B in all the presented tables.
Q8. In the tables (A, T, I) It was not clear that these values correspond to the ANAVA F test ??? the ANAVA P value ?? need to describe in the footer of the tables =
We have incorporated the P values at the footer of each tables and also A, T, I in bottom of the table
Q9. Many words throughout the text are together Ex "Table 3. Effect of invitrocolchicinetreatmenton" I don't know if it was a problem at the time of generating the PDF but this needs to be reviewed in detail.
We have properly drafted the document, but merging of words without space is happening while sending the documents through email.
Q10. In table 4 I am not seeing any correlation, the table call needs to be reformulated.
statistical analysis could not be performed keeping in view the limited number of confirmed field transferred polyploids, correlation details of the results presented in the document with red lettering.
Q11. At the end of topic 3.2 there is a citation [21], I believe there was a formatting error.
Incorporated in the document and shown in red lettering .
Q12. In topic 3.3, it is more interesting to make a box plot with all the data in each treatment, demonstrating the variation of the stomata and presenting the p value in the same figure, instead of inserting the measurement of each stomata. This also applies to the other details of plant morphology.
|
Sr. no. |
Tetraploid Stomata |
Diploid stomata |
|
Stomatal count |
11 |
10 |
|
Avg |
54.44 |
38.56 |
|
SD |
5.31 |
2.49 |
|
Variance |
28.23 |
6.24 |
|
T-Statistic |
8.614 |
|
|
T-Table (0.05) |
2.093 |
|
|
T-Table (0.01) |
2.861 |
|
Samples are significantly different at both 5% and 1% level of significance
Q13. Figure 9. It would be more interesting to present the height measurements of the plant of the thorns and the morphology of the flowers and to record the variation by P value and insert the figures as a supplementary file.
We have incorporated the tables with P values also in the document on plant morphology variation in colchicine induced polyplods of all the three studied rootstocks
Q14. Discussion. The discussion is shallow, without actually delving into what led to the reduction in survival with longer exposure to the product and concentration. It did not bring information on future studies, for example, using these rootstocks under commercial conditions to induce greater production in oranges. The discussion between the different rootstocks is vague and needs to be deepened. There was no recommendation on what would be the best treatment.
Detailed discussion incorporated in document for all the parameter and treatments studied, with red lettering.
Q15. At the end of the discussion the authors present "Hence in the present study authors used a combination of cytology and flow cytometry techniques for confirmation of induced polyploidy at field condition samples". Ok, but did this information bring greater contributions in relation to what is already available in the literature or in relation to morphological evaluations? This was not discussed in depth..
We have already detailed the reasons in our discussion for using the combination of the techniques for ploidy analysis by FCM, cytology and morphological evaluations. Kindly see the para mentioned below.
‘Plants were selected based on morphological features indicative of induced tetraploids (increased width-to-length leaf ratios, thicker stems, higher number of chloroplasts per guard cell, and larger stomata) in Lagerstroemia indica, and then validated by FCM [36]. Only half of the morphologically screened tetraploid plants were verified to be tetraploids [36]. FCM analysis can be performed upon various types of tissues and cell layers in order to rapidly determine ploidy in a large number of plants [37]. It allows the early analysis of polyploid plants, saving both effort and time [38, 39]. FCM is a rapid and efficient method to analyze a large number of samples, but it requires accurate samples and expensive equipment. Chromosome counting is the most accurate method, but it is time-consuming and cumbersome [40]. Hence in the present study authors used a combination of cytology and flow cytometry techniques for double confirmation of induced polyploidy of field transferred samples’.
Our main objective is not to find out the best ploidy analysis test for the verification of ploidy level of the generated citrus rootstock polyploids.
We have tried various polyploidy analysis technique for the double confirmation of the produced polyploids for horticulture evaluation and future field application to test there suitability for desirable characters for commercial application.
In our study the main objective of this experiment is to find out most efficient and safe method for induction of tetraploidy for handling large number of seeds and to reduce safety risks associated with the colchicine handling. So in this experiment we have demonstrated most safe, economically viable and technologically feasible method for induction of tetraploidy
Even though chromosome counting by cytological technique is accurate it is cumbersome and time-consuming, hence use of FCM is more common because of rapid delivery of results and less time consuming, only the limitation is higher cost of the equipment. Which was also mentioned in the previous report [37] (this para incorporated in the main document as an additional support).
Q16. Conclusions. The authors are making a general summary of the study and in the conclusions it should be mentioned what was concluded after all the study and discussion of the results.
Modified version of the conclusion with correction is incorporated with red lettering. Kindly see.
Author Response File: Author Response.docx
Reviewer 3 Report
Here is my comments for manuscript ID agronomy-2340037:
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Authors should address some points:
Line 19: Authors should give a blank space of "for24h".
Lines 44-46 and so on: Authors should check the scientific name of plant samples (Italic font). Comment: Please check carefully!
Lines 65-71: Authors should add the references for each sentence.
Line 80: Authors should give a blank space for "[18].Presently"
Lines 91-93: When did Authors collect samples? Did Authors do the pre-tests of scarification or disinfection methods for previously harvested and store seeds?
Lines 95-97: Authors should give the information of chamber (temperature, light intensity for cotyledons...)
Lines 112-113: Authors should add the references of flow cytometry method.
Lines 114-125: Authors should add the references of describing methods for ploidy analysis.
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Author Response
Reviewer 3
Authors should address some points:
Line 19: Authors should give a blank space of "for24h".
Correction incorporated in the document
Lines 44-46 and so on: Authors should check the scientific name of plant samples (Italic font). Comment: Please check carefully!
Correction incorporated in the document with red lettering.
Lines 65-71: Authors should add the references for each sentence.
Correction incorporated in the document
Line 80: Authors should give a blank space for "[18].Presently"
Correction incorporated in the document
Lines 91-93: When did Authors collect samples? Did Authors do the pre-tests of scarification or disinfection methods for previously harvested and store seeds?
Freshly harvested fruits were from ICAR-CCRI field grown rootstocks.
No pre test of scarification conducted as seeds were extracted from freshly harvested fruits.
Lines 95-97: Authors should give the information of chamber (temperature, light intensity for cotyledons...)
Inoculated seeds were maintained at 26 ± 1°C with 5.000 lux white-light intensity
Lines 112-113: Authors should add the references of flow cytometry method.
Correction incorporated in the document under 2.3 in materials and methods
Lines 114-125: Authors should add the references of describing methods for ploidy analysis.
Correction incorporated in the document under 2.3 and 2.5 of material and methods with red lettering.
Author Response File: Author Response.docx
Round 2
Reviewer 2 Report
The main changes were made, with a significant improvement in the text and in the presentation of results. So my advice is to accept the article in its current format. However, before publication, it is necessary to reformulate the tables, as they are misconfigured, difficult to understand.
Author Response
Thank you for the comment. We revisited all the tables and improved and simplified the table contents and titles for better understanding. Kindly see the tables in pdf form, in order to avoid the problem of misconfiguration and also for more clarity and better understanding. In MS word document when it is transferred from computer to computer, maybe there is misconfiguration or dislocation of titles and footnotes of the tables. So, kindly note this point and bring the necessary modification from your side.
Author Response File: Author Response.docx
Reviewer 3 Report
Lines 95-96: Authors responded to the Reviewer that they used 5.000 lux white-light intensity, however, the manuscript was shown 3.000 lux white-light intensity.
Author Response
Reviewer 3 comments: Lines 95-96: Authors responded to the Reviewer that they used 5.000 lux white-light intensity, however, the manuscript was shown 3.000 lux white-light intensity.
Thank you for the comment. It was a typographical error in reviewer's reply. We have used 3000 lux white light intensity which was indicated as 44.46 µ mol m -2 s-1 light intensity (in page number 2, line no. 96).
The changes were incorporated with blue ink.
