Long-Term Field Evaluation of Conventional vs. Micropropagated Plants of Chrysanthemum cinerariifolium

Round 1

Reviewer 1 Report

Long-term field evaluation of conventional vs. micropropagated plants of Chrysanthemum cinerariifolium

 

In line 20-21: The authors mentioned, Since plantlets derived from in vitro regeneration may cause ploidy changes. What factors cause polyploidy in in vitro culture?

Is it possible in others subculture we observe polyploidy?

Do metabolites is influenced by medium culture and hormone in in vitro culture?

In line 71: (plantlets derived from in vitro culture may exhibit ploidy variations). Is ploidy positive or negative in production of secondary metabolite?

Materials and Methods section, The authors mentioned: plantlets were transferred in sterile Magenta boxes containing 60 mL MS medium. Was the medium culture free hormone?

I also recommend to enrich introduction and discussion part with the relevant literature. For example I recommend to address the following papers:

Hassankhah A, Vahdati K, Lotfi M, Mirmasoumi M, Preece J, Assareh MH (2014) Effects of ventilation and sucrose concentrations on the growth and plantlet anatomy of micropropagated Persian walnut plants. International Journal of Horticultural Science and Technology. 1(2) 111-120.

Sadat-Hosseini M, Vahdati K, Leslie CA. (2019) Germination of Persian walnut somatic embryos and evaluation of their genetic stability by ISSR fingerprinting and flow cytometry. HortScience 54(9): 1576-1580.

Author Response

1. In line 20-21: The authors mentioned, Since plantlets derived from in vitro regeneration may cause ploidy changes. What factors cause polyploidy in in vitro culture?
2. Is it possible in others subculture we observe polyploidy?
3. In line 71: (plantlets derived from in vitro culture may exhibit ploidy variations). Is ploidy positive or negative in production of secondary metabolite?

Authors:

We thank the reviewer for advice. As we can see, these three observations are all related to polyploidy, hence our decision to merge them in order to give a comprehensive response. Actually, after reading your comments we realized that polyploidy, and its role in our work, required more detailed explanations. Hence, some new parts were added. In detail, the sentence “In vitro cultures are prone to mutations induced by several factors. Explant source and type, age of culture and number of subculture cycles, plant growth regulators used in propagation and regeneration trials influence the onset of somaclonal variability” has been added with the references (SAHIJRAM, L., SONEJI, J. R., & BOLLAMMA, K. (2003). INVITED REVIEW: ANALYZING SOMACLONAL VARIATION IN MICROPROPAGATED BANANAS (Musa SPP.) Plant. https://doi. org/10.1079; Sadat-Hosseini M, Vahdati K, Leslie CA. (2019) Germination of Persian walnut somatic embryos and evaluation of their genetic stability by ISSR fingerprinting and flow cytometry. HortScience 54(9): 1576-1580). We hope that, in this form, our meanings are better convoyed. The basic idea is that various factors, such as explant source, ploidy level, genotype, regeneration system, plant growth regulators, and culture conditions, can contribute to somaclonal variation (Brar and Jain, 1998). Therefore, homogeneity and trueness-to-type of regenerated plantlets must be checked. As concerns your other questions, the answer to the first is yes, polyploidy can be observed in other subcultures. As reported in the paper “INVITED REVIEW: ANALYZING SOMACLONAL VARIATION IN MICROPROPAGATED BANANAS (Musa SPP.) (https://doi. org/10.1079), where the authors analyse factors influencing somaclonal variations, number of subcultures may affect genetic stability and lead to somaclonal variation. Your last related question opens many possibilities: ploidy per se is neither positive nor negative in the production of secondary metabolites, since its effect is widely variable according to the plant species and the involved metabolites. Within the 2010 “Handbook of Essential oils” (Eds. K. Hüsnü Can Baser and Gerhard Buchbauer), many examples are given of medicinal and aromatic plants (e.g., Achillea spp.) whose metabolism is affected by the ploidy level, and the strong commercial success of tetraploid chamomiles is mostly due to their enhanced content in proazulene and flavonoids. In pyrethrum, the relationship between ploidy level and secondary metabolism is still uncertain, although some rather old paper (Tominaga and Yokohata, 1958) stated that triploid individuals had a pyrethrins content higher than the diploid and tetraploid parentals. However, although deeply interesting, this topic falls a bit beyond the scope of our paper.

Reviewer:

Do metabolites is influenced by medium culture and hormone in vitro culture?

Authors:

We thank the reviewer for advice. Plants produced in vitro can produce secondary metabolites with an efficiency that can exceed that of greenhouse-grown plants or wild plants. Several papers report data about the influence of culture medium on secondary metabolites production. Usually the influence is positive with a significative increase in production [Smetanska, I. (2008). Production of Secondary Metabolites Using Plant Cell Cultures. In: Stahl, U., Donalies, U.E., Nevoigt, E. (eds) Food Biotechnology. Advances in Biochemical Engineering/Biotechnology, vol 111. Springer, Berlin, Heidelberg. https://doi.org/10.1007/10_2008_103]

Reviewer:

In Materials and Methods section, the authors mentioned: plantlets were transferred in sterile Magenta boxes containing 60 mL MS medium. Was the medium culture free hormone?

 

Authors:

The medium used for culture was hormone free.

We changed the sentences as reported: Subsequently, in order to obtain enough plant material for rooting step, plantlets were transferred in sterile Magenta boxes containing 60 mL MS medium with no plant growth regulators and subcultured at 30-days intervals.

Reviewer:

I also recommend to enrich introduction and discussion part with the relevant literature. For example I recommend to address the following papers:

Hassankhah A, Vahdati K, Lotfi M, Mirmasoumi M, Preece J, Assareh MH (2014) Effects of ventilation and sucrose concentrations on the growth and plantlet anatomy of micropropagated Persian walnut plants. International Journal of Horticultural Science and Technology. 1(2) 111-120.

Sadat-Hosseini M, Vahdati K, Leslie CA. (2019) Germination of Persian walnut somatic embryos and evaluation of their genetic stability by ISSR fingerprinting and flow cytometry. HortScience 54(9): 1576-1580.

 Authors:

Thank you for your suggestion; we added the given references in introduction and discussion sections.

 

 

Author Response File: Author Response.pdf

Reviewer 2 Report

The paper with the title “Long-term field evaluation of conventional vs. micropropagated plants of Chrysanthemum cinerariifolium” investigated the ploidy variation occurring in micropropagated plants of pyrethrum, a plant used for the extraction of insecticides. The results are remarkably valuable.

 

Line 75 change microprogation to micropropagation

 

At the end of the introduction section, the aim and objectives are not clearly expressed. Only the the general "goal" of the paper. I suggest authors to consider to outline aim and objectives, because these will further guide the reader throughout the paper. Ideally, the objectives are defined as the steps considered for reaching the aim, and the conclusions should mirror the objectives. Possible applications and practical importance can be also specified, as mentioned in the goal of the paper, prior or after the objectives are defined.

The research is interesting and valuable considering the number of years of observation.

 

Line 171 says that climatic data is presented in figure 2, but no figure 2 is found in the manuscript

Authors present in the table 1 the most important data obtained. I suggest to the authors to present some part of their results (the most significant data they consider highly relevant) as graphs, for an easy visual comparison, that would be more attractive for readers.

 

Best regards.

Author Response

General comments

The paper with the title “Long-term field evaluation of conventional vs. micropropagated plants of Chrysanthemum cinerariifolium” investigated the ploidy variation occurring in micropropagated plants of pyrethrum, a plant used for the extraction of insecticides. The results are remarkably valuable.

Thank you for your appreciation and your kind words.

 Reviewer:

Line 75 change microprogation to micropropagation

Authors:

Many thanks for noting it; we substituted microprogation with micropropagation

 Reviewer:

At the end of the introduction section, the aim and objectives are not clearly expressed. Only the general "goal" of the paper. I suggest authors to consider to outline aim and objectives, because these will further guide the reader throughout the paper. Ideally, the objectives are defined as the steps considered for reaching the aim, and the conclusions should mirror the objectives. Possible applications and practical importance can be also specified, as mentioned in the goal of the paper, prior or after the objectives are defined.

The research is interesting and valuable considering the number of years of observation.

Authors:

Thank you for this observation, which allowed us to improve a crucial part of our work. To obtain a clearer statement of aims and objectives of the experiment we modified the “introduction” section according to your recommendation, also moving to the conclusions the general “goal”. We hope that our efforts were satisfactory. 

 Reviewer:

Line 171 says that climatic data is presented in figure 2, but no figure 2 is found in the manuscript.

 

Authors:

We apologize, but something must have happened in uploading the final version of our manuscript, and figure 2 disappeared from both the PDF and DOC files. The figure was properly inserted again.

 

 

Reviewer:

Authors present in the table 1 the most important data obtained. I suggest to the authors to present some part of their results (the most significant data they consider highly relevant) as graphs, for an easy visual comparison, that would be more attractive for readers.

 

Authors:

Your suggestion is correct, and graphs are surely more readable and attractive than tables. Unfortunately, in our case, turning a part of the reported data to a graphical form would force us to hide the ANOVA procedures (as expressed by the DF) and their results (the F values), only allowing to save the letters from the Tukey’s test. Furthermore, in our opinion, the tabular form allows to catch immediately the simultaneous variation of the different variables, whereas graphs, reported in different scales, are more suitable to compare data of the same category. Hence, we do prefer to leave table 1 in its present form.

 

Author Response File: Author Response.pdf

Reviewer 3 Report

Article Long-term field evaluation of conventional vs. micropropagated plants of Chrysanthemum cinerariifolium Catalano C., Carra A., Carimi F., Motisi A., Abbate L., Sarno M., Carrubba A. is extremely sloppy.

Abstract: It is not clear, what is the relationship between the content of pyrethrins and the level of ploidy in Chrysanthemum cinerariifolium plants? Although the title of the article speaks of a long-term field assessment, the results of this assessment are reflected in the text of the Abstract in only one single sentence!

Introduction: The need to study the level of ploidy in Chrysanthemum cinerariifolium plants is not substantiated at all. Indeed, in vitro culture, the ploidy of plant tissues can change, but so what! The main valuable product of this plant is secondary metabolites - pyrethrins. The authors do not give in the Introduction a single mention of works where a change in the content of pyrethrins would be somehow related to a change in ploidy. Also not discussed is how many chromosomes are in the genome of Chrysanthemum cinerariifolium? Are they large or small? Is it convenient to observe (count) them? And why did the authors choose the expensive FCM method instead of the cheap and reliable root tip staining Felgen method. In addition, it is unlikely that FCM is able to unambiguously detect aneuploids, which can greatly affect the content of pyrethrins. The Chrysanthemum cinerariifolium plant has different organs and tissues, but the authors do not argue why they used leaves for FCM rather than flowers, where the accumulation of pyrethrins is greatest. Why then are roots or stems not considered? The last sentence of the Introduction section, where the aim of the study is stated, does not correspond in any way with the title of the article.

Materials and methods: It is unclear when samples were taken for Flow Cytometric Analysis. Was it only once? What year? Or was it done every year?

Results: Where is Figure 2? Table 1 shows the results of morphometry from 2012 to 2019. It is not clear if these results are for cuttings or for in vitro micropropagated plants? It would be necessary to provide similar indicators for each category. Below are Cuttings and In vitro micropropagated. Are these averages for all years or for a specific year?

Analysis of ploidy level: From figure 3 it is not clear, what was the control and what was compared? Maybe flowers, roots or stems should be taken as controls? In figure 3, only one peak is visible, and it is not clear what it has to do with? To mother plants or to plants micropropagated in vitro?

Discussion: Applied aspects (production of pyrethrins) and connection with morphometry of cuttings and in vitro micropropagated plants are not discussed. Where is the connection with the change in ploidy?

Similar results obtained on other crops are not discussed: the change in parameters (eg, biochemical) in plants in vitro micropropagated and planted in an open field, and in plants propagated by cuttings. Such studies have been carried out in some aromatic plants.

Lines 307-311. The sentence is not clear at all.

Conclusion - missing.

References - not filled out according to MDPI rules.

Author Response

We thank the reviewer for suggestions.

Reviewer:

It is not clear, what is the relationship between the content of pyrethrins and the level of ploidy in Chrysanthemum cinerariifolium plants?

Authors:

We have partially replied to this observation in our first response to rev. 1. The relationship between the content of pyrethrins and the level of ploidy in pyrethrum is surely an interesting issue, but it is not included in the scope of the present work.

Reviewer:

Although the title of the article speaks of a long-term field assessment, the results of this assessment are reflected in the text of the Abstract in only one single sentence!

Authors:

We thank the reviewer for advice. In writing the abstract, the need for brevity caused us to rule some important concept out.

 Reviewer:

The need to study the level of ploidy in Chrysanthemum cinerariifolium plants is not substantiated at all.

Authors:

We thank the reviewer for advice. This part was deeply amended, and the importance of studying the ploidy levels was better detailed. See also the response to Rev. 1.

Reviewer:

The main valuable product of this plant is secondary metabolites - pyrethrins.

Authors:

Of course, we agree. However, being pyrethrins contained in flowers, the marketable part of the plant is represented by the flowers themselves.  

Reviewer:

The authors do not give in the Introduction a single mention of works where a change in the content of pyrethrins would be somehow related to a change in ploidy.

Authors:

See our previous reply. Although this is a topic of great interest, our work was not addressed to evaluate pyrethrin content.

 Reviewer:

Also not discussed is how many chromosomes are in the genome of Chrysanthemum cinerariifolium? Are they large or small? Is it convenient to observe (count) them? And why did the authors choose the expensive FCM method instead of the cheap and reliable root tip staining Felgen method.

Authors:

Thank you. In the present work, flow cytometric analysis was used to determine the genetic stability of micropropagated plants in vitro.

We have not done cytogenetic studies of the species Chrysanthemum cinerariifolium

 Reviewer:

The Chrysanthemum cinerariifolium plant has different organs and tissues, but the authors do not argue why they used leaves for FCM rather than flowers, where the accumulation of pyrethrins is greatest. Why then are roots or stems not considered?

Authors:

Our work was not addressed to evaluate pyrethrin content.

Reviewer:

The last sentence of the Introduction section, where the aim of the study is stated, does not correspond in any way with the title of the article.

Authors:

We thank the reviewer for comment. We agree with this remark. The final part of introduction has been changed as here reported: “In this study we compared the field performance of pyrethrum plants regenerated in vitro with plants obtained by conventional cuttings after verifying ploidy stability by FCM analysis.”

This new version, in our opinion, correspond with the title and is consistent with the structure of the paper.

Reviewer:

It is unclear when samples were taken for Flow Cytometric Analysis. Was it only once? What year? Or was it done every year?

Authors:

For each samples of micropropated plants and internal diploid standard, 3 replicates were performed. This sentence was inserted into the text.

 Reviewer:

Where is Figure 2?.

Authors:

Due to a layout problem, Fig. 2 had disappeared. In the new version of the manuscript we have added Figure 2.

Reviewer:

Table 1 shows the results of morphometry from 2012 to 2019. It is not clear if these results are for cuttings or for in vitro micropropagated plants? It would be necessary to provide similar indicators for each category. Below are Cuttings and In vitro micropropagated. Are these averages for all years or for a specific year?

Authors:

As explained in the caption to table 1, the values reported in the body of the table are the mean values of the main factors (“Year” and “Propagation Method”). Of course, the factor Y is averaged across propagation methods, and the factor PM is averaged across years. As shown by the corresponding F values, for all variables, the interaction between factors (YxPM) resulted not significant at the ANOVA, hence the interaction values (averaged across repetitions) are not individually reported.

 Reviewer:

Analysis of ploidy level: From figure 3 it is not clear, what was the control and what was compared? In figure 3, only one peak is visible, and it is not clear what it has to do with? To mother plants or to plants micropropagated in vitro? Maybe flowers, roots or stems should be taken as controls?

Authors:

The analysis of ploidy level was conducted by simultaneously analyzing the nuclei of the micropropagated plants with the nuclei of the mother plants. A single peak shows that there have been no changes in ploidy. The analysis was done by choppetting together leaves of micropropagated plants and mother plants (control plants).

 Reviewer:

Applied aspects (production of pyrethrins) and connection with morphometry of cuttings and in vitro micropropagated plants are not discussed. Where is the connection with the change in ploidy?

Authors:

The flow cytometric analysis in the present work was carried out to verify the genetic stability of the micropropagated plants compared to the plants from which the cuttings were taken. We did not evaluate the pyrethrins content assuming that no change in yield is evident when genetic stability is highlighted.

Reviewer:

Similar results obtained on other crops are not discussed: the change in parameters (eg, biochemical) in plants in vitro micropropagated and planted in an open field, and in plants propagated by cuttings. Such studies have been carried out in some aromatic plants.

Authors:

That is surely true, and probably similar experiments were carried out on other species. However, we just do not believe that making references to other species would add anything substantial to our work, rather this would make the introduction section excessively long.

Reviewer:

Lines 307-311. The sentence is not clear at all.

Authors:

We thank the reviewer for the advice. The sentence was rearranged.

 Reviewer:

Conclusion - missing.

Authors:

We thank the reviewer for the suggestion. Conclusions were added as a separate section.

Reviewer:

References - not filled out according to MDPI rules.

Authors:

We thank the reviewer for noticing this point. All references were thoroughly checked.

 

Author Response File: Author Response.pdf

Round 2

Reviewer 3 Report

Abstract. It is not necessary in this section to describe the plant you have chosen for research. This is best placed in the Materials and Methods section.

A very strange Introduction. More than 70 lines are devoted to the topic of pyrethrins and toxicity (lines 77-150). And about 24 lines are devoted to other aspects (lines 152-174). Why such a ratio? In the text, the authors no longer refer to pyrethrins. Neither in Materials and Methods, nor in Results. The work of the authors did not include the determination of the number of secondary metabolites at all, so why devote so much space in the Introduction to pyrethrins? 3-4 sentences are enough.

Discussion. What is the point of presenting Figure 2 in the manuscript if these results are not discussed?

Lines 704-706 “Genetic changes in micropropagated plants can be detected by flow cytometry…. by calculating nuclear DNA content” - An absolutely erroneous statement. Due to flow cytometry, you can only roughly determine the amount of DNA roughly linked to the ploidy level (2C, 4C, 8C, and so on). This method is completely impossible to determine genetic changes. You can't even tell a dihaploid from an aneuploid, much less different genetic changes: rearrangements of chromosomes, exchange of chromosome fragments, as well as such fine changes as mutations at the level of the promoter or coding gene. Namely, such subtle changes can dramatically affect the production of secondary metabolites. And flow cytometry cannot detect this at all. Therefore, this assertion is false. Correct this suggestion.

Author Response

Reviewer:

Abstract. It is not necessary in this section to describe the plant you have chosen for research. This is best placed in the Materials and Methods section.

Authors:

The abstract was shortened, and the description of plant was reduced as much as possible.

 

Reviewer:

A very strange Introduction. More than 70 lines are devoted to the topic of pyrethrins and toxicity (lines 77-150). And about 24 lines are devoted to other aspects (lines 152-174). Why such a ratio? In the text, the authors no longer refer to pyrethrins. Neither in Materials and Methods, nor in Results. The work of the authors did not include the determination of the number of secondary metabolites at all, so why devote so much space in the Introduction to pyrethrins? 3-4 sentences are enough.

Authors:

This section was shortened, and chemical description of pyrethrins was eliminated.

Reviewer:

Discussion. What is the point of presenting Figure 2 in the manuscript if these results are not discussed?

Authors:

We believe that in a long-term field trial, the trend of climatic parameters cannot be ruled out. Actually, the climatic pattern is discussed in the “Results” section, where the effects of thermal sums and rainfall on plants’ performance and flower yields are taken into account. A reference to figure 2 was however added at line 267.

Reviewer:

Lines 704-706 “Genetic changes in micropropagated plants can be detected by flow cytometry…. by calculating nuclear DNA content” - An absolutely erroneous statement. Due to flow cytometry, you can only roughly determine the amount of DNA roughly linked to the ploidy level (2C, 4C, 8C, and so on). This method is completely impossible to determine genetic changes. You can't even tell a dihaploid from an aneuploid, much less different genetic changes: rearrangements of chromosomes, exchange of chromosome fragments, as well as such fine changes as mutations at the level of the promoter or coding gene. Namely, such subtle changes can dramatically affect the production of secondary metabolites. And flow cytometry cannot detect this at all. Therefore, this assertion is false. Correct this suggestion.

Authors:

Done. We changed the sentence according to the suggestions