In Vitro Polyploidization of Thymus vulgaris L. and Its Effect on Composition of Essential Oils

Round 1

Reviewer 1 Report

The above manuscript describes a protocol for both in vitro propagation and generation of tetraploid variants of  Thyme (Thymus vulgaris L.) as  historical medicinal  and spice plant.  In addition to the morphological differncies the GC/MS analysis of essential olis revealed  that the tetraploid subclones produced different oil qualities. Toxicity assays testing on C. quinquefasciatus and S. littoralis larvae demonstrate that the altered tetraploid of T. vulgaris essential oil was more toxic for both types of insects than essential oil obtained from diploid (control) plants. Despite of the fact that the poliploidization procedure follows a routine protocol, the provided information about essential oil composition and toxicity this work can be considered as novel contribution and after improvement of this manuscript I recommend the manuscript.

Some questions and comments to be considered:

1. It is not clear what was the basis to select hormones with these concentrations (0.1 mg l-1 IBA and 0.1 mg l-1 BA).
2. In line 248 „All detected polyploids were tetraploids „ this statement indicated that the double genome size was confirmed by both flow cytometry and chromosome counting?
3. As shown by Fig. 3 and Fig. 4. one cannot conclude that the tetraploids possess higher biomass.

In summary, considering the novel information about essential oil composition and insecticidal activities I recommend the manuscript.

Author Response

Dear reviewer,

thank you for your review and comments. Here are our answers to your questions and comments:

1. We selected hormones and their concentrations in culture media on base of previous in vitro experiments with medicinal plants. In a case of thyme, we also tried higher concentration, but this stimulated callus formation on cutting side of shoots and made more difficulties with roots formations.
2. All tetraploids were detected by flow cytometry. We had problems with rooting of shoots, so, chromosomes were counted in those tetraploids, which were vital and rooting.
3. It is true that we did not measure weight of biomass, but tetraploids were and are more burly than diploids, not only in in vitro but on field. Although Figure 3 showed tetraploids smaller, this is on beginning of regeneration after oryzalin treatment. When tetraploids fully regenerated they started grow more robustly.

Reviewer 2 Report

This manuscript presents some very useful data for the food industry and could be applied to the production of botanical insecticides.

The methods are clearly described and the data is clearly presented.  The conclusions are clearly defended by the data in the results and discussion. However, authors could enrich the discussion and involve one recent publication; that also means that in their work they have not proposed for the first time a suitable protocol for the in vitro polyploidization by oryzalin of Thymus vulgaris (l. 403-4).

The statistical evaluation could be proved, using the one-way ANOVA (analysis of variance) test to compare individual groups of tables 1 and 2. These two tables are not mentioned in the 'Results' session, and I suggest they should merge them in one table. I also strongly suggest that authors provide writing/description on the PCA analysis (what about corelations?).

The article adds some more data to that the current literature, but it should be revised to improve the clarity of the presentation. A discussion of the enriched statistical evaluation should be presented as well.  I think that the present manuscript should be accepted after a good revision effort.

Author Response

Responses to reviewer 2

Dear reviewer,

thank you for your review and comments. Here are our answers to your comments and suggestions:

1. We involved new recent publication to discussion, which was focused on the thyme. Yes, we are now not first, however, we have our tetraploid thymes three years in field trials. New reference was added and thank you for your notice. We also added to Discussion other new publication concerning artificial polyploidization via oryzalin treatment.
2. Although the data are not so rich for statistical analysis, we did one-way ANOVA test for individual groups. Significant difference was found in comparison between used procedures. It is now described in manuscript.
3. Thank you for suggestion of merging tables. We did and it is more clear for readers.
4. We also provide more information to PCA analysis.

Reviewer 3 Report

Very interesting research, original and partly pioneering results. Work of high scientific value, clearly presented results and well discussed. Minor errors and necessary additions are listed below.

1) Abbreviations (independently from the list of abbreviations at the end of the manuscript) should be defined in parentheses the first time they appear in the abstract, main text, and in figure or table captions and used consistently therafter.

2) Keywords - better to use different words than in the title, for example thyme instead of Thymus vulgaris

3) Throughout the text, please use italics for Latin plant names. In many places in the manuscript this notation is not correct, starting with lines 28 (Thymus vulgaris L.) and 29 of the Lamiaceae. This is also true for the titles of publications in the literature list.

4) Cultivar names (line 61 and next) should be written in single upper-case apostrophes. The abbreviation "cv." (line 70) is not a correct transcription according to international rules for the writing of cultivar names of cultivated plants. 

5) line 75 - should be [20] instead [19]. In addition, in the case of MS medium (here and in other places in the chapter M&M), additives such as vitamins, sugar, gelling agent (agar?) and pH should be listed. Also with the name/s of producer/s of the chemicals and country of origin.

6) line 75-76 - here and in many places in the text  units given per liter, should be written as dm^-3 according SI system or L^-1, where minus three or minus one are in superscript. 

7) line 87 - "in vitro micropropagation" - I suggest to write just "micropropagation" or "in vitro propagation".

8) line 88 - The Petri dishes... instead The petri dishes

9) line 175-6 please provide the concentration!

10) line 186 - please add after 'Varico' and before 15, the word clone

11) line 193 - cm2 - two should be written in superscript (as cm²)

12) line 185 and 196 - EOs and EO - here and in many places in the text both forms (singular and plural) were used. I suggest to unify this notation throughout the text.

13) Table 3 - The information in the title of the table about the standard deviation (mean ± SD) does not correspond with the heading of columns 3 and 5, where RSD in percent is given.

14) Figures 1-5 - titles should be placed below the figures, not above them.

15) Figure 2 - the sentence "Ploidy levels of both explants were..." is not necessary here. I suggest to remove it. The figure is very bad quality.

16) Figure 4 - Please improve the title of the figure by adding the latin name of plant and numbers of diploid and tetraploid.

17) line 414 (Abbreviations) - similar as remark 12) please decide using EO or EOs; GC/MS is missing.

Author Response

Responses to reviewer 3

Dear reviewer,

thank you for your review and comments. We made changes according all of your comments and suggestions. We remove errors and manuscript is now more correct and clear for readers.

Round 2

Reviewer 2 Report

Dear authors,

congratulation for the manuscript.

There are some more comments based on file: ’agronomy-1120130-peer-review-v2’

1. 25: Please use alphabetical order
2. 41, 42, 44, 59, 62, 255, ….315,….etc : replace ‘in vitro’ by ‘in vitro’ (please check and replace everywhere in the text)
3. 59, 110…436, 439, 448….: replace ‘et al’ by ‘et al’ (please check and replace everywhere in the text)
4. 114, 190….: you have to convert μΜ to mg L^-1 (please check and replace everywhere in the text)
5. 163, …..313, 315,…..: replace ‘ml-1’ by ‘ml^-1 (please check and replace everywhere in the text)
6. 211: replace ‘1^oC’ by ‘1 ^oC’ (please check and replace everywhere in the text)
7. 368: replace ‘morfological’ by ‘morphological’

Author Response

Dear reviewer,

thank you for your comments. We tried to catch all mentioned mistakes and arranged all comments in the manuscript including converting µM to mg L-1.

Kind Regards,

Vladan Ondrej