Histochemical Localization of Alkaloids in the Bulbs of In Vitro-Regenerated Snake’s Head Fritillary (Fritillaria meleagris L.): The Effect of a Temperature Regime

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

Overall, the work has potential when it comes to Communication; but some points of the methodology need to be better described.

The abstract needs to be improved. Please describe in this section what the work treatments are.

The introduction of the manuscript is consistent with what is proposed in the work.

 

In methodology:

Authors must make it clear how much NaClO was used; for example, was 20% of 4% NaClO used? Or was 4% used? This became confusing. Do the calculations and describe the exact concentration of the aseptic solution, that's all. Another point, how many times were the bulbs rinsed after soaking in NaClO?

 

Regarding the cultivation medium; describe it more fluidly to the reader. For example:

Five explants were placed in each Petri dish filled with 15 mL of Murashige and Skoog (MS) medium [26] supplemented with 30 g L-1 sucrose, 7 g L-1 agar, 250 mg L-1 casein hydrolysate, 250 mg L-1 L-proline, 80 mg L-1 adenine sulfate; and 1 mg L-1 thidiazuron (TDZ).

After 4 weeks of cultivation, bulblet formation was observed [25]. Obtained bulblets were cut longitudinally into four scale sections and cultured on MS – as previously described, except this time 0.1 mg L-1 TDZ was used – for an additional 4 weeks, where new bulblets were formed [25] and used to establish stock cultures.

 

Line 120: (unless stated otherwise).???

Line 121 to 127: Describe the treatments better, this was very confusing. State clearly and objectively what the treatments were. Were there only 2 treatments? Or were there 3? Hard to follow. Furthermore, describe the n sample of each treatment. How many explants were used per Erlenmayer flask and how many Erlenmayer flasks were used in each treatment? Only the number of explants was determined.

 

Author Response

Responses to the Reviewer 1 Comments

 

Thank you for reading the manuscript horticulturae-2755084. We appreciate your comments and suggestions as they have helped us to improve the text. Manuscript has been modified according to the reviewer’s suggestions, and the changes are clearly visible in the Track Changes version of the revised manuscript. Responses to the reviewer’s comments are provided below.

 

Overall, the work has potential when it comes to Communication; but some points of the methodology need to be better described.

We have described certain points of the methodology in more detail.

 

The abstract needs to be improved. Please describe in this section what the work treatments are.

We have described the work treatments in the section Abstract as follows: “Histochemical localization of alkaloids was performed using Wagner and Dragendorff reagents in fresh sections of bulbs cultured at 24 °C or 7 °C for 4 weeks, as well as those cultured at 24 °C following the 4-week chilling treatment, which were sampled at the beginning of sprouting.”

The introduction of the manuscript is consistent with what is proposed in the work.

 

In methodology:

Authors must make it clear how much NaClO was used; for example, was 20% of 4% NaClO used? Or was 4% used? This became confusing. Do the calculations and describe the exact concentration of the aseptic solution, that's all. Another point, how many times were the bulbs rinsed after soaking in NaClO?

In our case, the aseptic cultures were obtained by surface sterilization of seeds with 20% commercial bleach solution; non-diluted commercial bleach contained 4% active chlorine. This means that the final concentration of NaClO in the aseptic solution was 0.8% (w/v), which is now specified in the text. 

We have also added that the seeds were rinsed three times with sterile water after soaking in NaClO.

 

Regarding the cultivation medium; describe it more fluidly to the reader. For example:

Five explants were placed in each Petri dish filled with 15 mL of Murashige and Skoog (MS) medium [26] supplemented with 30 g L-1 sucrose, 7 g L-1 agar, 250 mg L-1 casein hydrolysate, 250 mg L-1 L-proline, 80 mg L-1 adenine sulfate; and 1 mg L-1 thidiazuron (TDZ). After 4 weeks of cultivation, bulblet formation was observed [25]. Obtained bulblets were cut longitudinally into four scale sections and cultured on MS – as previously described, except this time 0.1 mg L-1 TDZ was used – for an additional 4 weeks, where new bulblets were formed [25] and used to establish stock cultures.

We have changed the description of the cultivation medium according to the reviewer’s suggestion.

 

Line 120: (unless stated otherwise).???

This phrase remained, by mistake, from the initial draft of the manuscript (where the description of culture conditions was supposed to refer to all cultures, including those that underwent chilling treatment). We have deleted it from this sentence, because the cultivation temperature of 24 °C matches exactly the conditions of culture initiation, bulb formation and multiplication experiments described in the preceding paragraphs.

 

Line 121 to 127: Describe the treatments better, this was very confusing. State clearly and objectively what the treatments were. Were there only 2 treatments? Or were there 3? Hard to follow. Furthermore, describe the n sample of each treatment. How many explants were used per Erlenmayer flask and how many Erlenmayer flasks were used in each treatment? Only the number of explants was determined.

There were two chilling treatments: 1) chilling treatment (at 7 °C) for 4 weeks, after which the bulbs were sampled immediately; sprouting rarely occurred at this temperature, but if it did, those bulbs were sampled at that time point (e.g. 7 or 14 days after the beginning of the chilling treatment), and 2) chilling treatment (at 7 °C) for 4 weeks followed by cultivation at 24 °C; after transfer to 24 °C, bulbs started to sprout and were sampled as soon as sprouting began. Bulbs cultured at 24 °C for 4 weeks were used as control. Both treatments are now described better in the text, as so is their sampling time.

For each treatment, the total number of bulbs was 20. Five explants were used per Erlenmayer flasks, and 4 Erlenmayer flasks were used in each treatment. Therefore, we added the sentence “Four replicates, each consisting of five explants, were used per treatment (n = 20).”

 

Reviewer 2 Report

Comments and Suggestions for Authors

The work called “Histochemical localization of alkaloids in the bulbs of in vitro regenerated snake's head fritillary (Fritillaria meleagris L.): effect of temperature regime”, is very interesting and raises the importance of obtaining in vitro culture and alkaloids , based on the medicinal properties of the plant

 

From my point of view, it is preliminary work

 

It is necessary to integrate the figures to observe in comparison with the control

mark on the images the information that is mentioned in the text.

Integrate a size reference bar in images.

The discussion is very simple, it would be desirable to discuss based on the factors that affect the accumulation of the compounds of interest.

In vitro culture represents an important advance in this study.

Go deeper into the histochemical analysis and make the analysis of the results of in vitro culture interesting.

The importance of the results is not appreciated.

Improve writing.

Why were those temperature conditions taken for the test?

The work is not justified.

What about the use of plant growth regulators and plant regeneration?

Are the objective expectations met?

 

See notes in the attached document. ​

Comments for author File: Comments.pdf

Author Response

Responses to the Reviewer 2 Comments

 

 

The work called “Histochemical localization of alkaloids in the bulbs of in vitro regenerated snake's head fritillary (Fritillaria meleagris L.): effect of temperature regime”, is very interesting and raises the importance of obtaining in vitro culture and alkaloids, based on the medicinal properties of the plant

Thank you for reading the manuscript horticulturae-2755084. We appreciate your comments and suggestions as they have helped us to improve the text. Manuscript has been modified according to the reviewer’s suggestions, and the changes are clearly visible in the Track Changes version of the revised manuscript. Responses to the reviewer’s comments, including those made in the pdf version of the manuscript, are provided below.

 

From my point of view, it is preliminary work

We agree with this comment. We already stated that this work was a preliminary study for a larger comprehensive investigation of the effects of different physical, nutritional, and hormonal factors, plant age, and bulb size on alkaloid production in F. meleagris.

 

It is necessary to integrate the figures to observe in comparison with the control

We have integrated figures 2, 3 and 4 into one figure (now designated as Figure 2), so that it would be easier to make comparisons with control.

 

mark on the images the information that is mentioned in the text.

The information mentioned in the text is now marked in the figures, and explained in the figure legend.

 

Integrate a size reference bar in images.

We have inserted scale bars according to the reviewer’s suggestion. Their size is given in the figure legend.

 

The discussion is very simple, it would be desirable to discuss based on the factors that affect the accumulation of the compounds of interest.

The discussion based on the factors that affect the accumulation of the compounds of interest is added to the section Discussion.

 

In vitro culture represents an important advance in this study.

Yes, we agree. We already stated that “Since F. meleagris is very rare and endangered [...], in vitro propagation techniques provide a very effective method for its large-scale propagule generation, thus expanding the source of the sought alkaloids.” There are different techniques for propagation of F. meleagris, as shown in our previous studies, but it is important to explore the possibilities of increasing the production of pharmacologically important metabolites in bulbs of in vitro cultured plants. One way to approach this is to perform a histolocalization screening of the bulbs grown under specific in vitro culture conditions, in order to reveal bioactive metabolites accumulating under such conditions and potentially improve their production.

 

Go deeper into the histochemical analysis and make the analysis of the results of in vitro culture interesting.

Bulbs (non-sprouted and sprouted) used for histochemical analysis were cut in halves, longitudinally or transversely, and examined after staining. As these slices were quite thick, we did not venture to deepen the histochemical analysis in terms of identifying individual cells containing alkaloids. We suspect that the positive reactions to the two reagents used were localized in the idioblasts (parenchyma cells known to store various metabolites, including alkaloids) of the bulb scales and sprouts. However, due to the thickness of the sections, we were unable to identify these particular cells, so we relied largely on our previous histological observations of the bulb scales subjected to the respective temperature treatments. For the same reason, we did not mark the amyloplasts in these sections, because individual amyloplasts cannot be discerned (see below, our response to the reviewer’s comment made in the pdf document).

We have added some text to the section Discussion, and we hope that they improved the analysis of the results of in vitro culture.

 

The importance of the results is not appreciated. The work is not justified.

This preliminary investigation revealed that pre-chilled sprouted bulbs of in vitro cultured F. meleagris are potentially preferable in vitro plant material for the isolation of alkaloids. The results presented in this work indicate that low temperature probably increased alkaloid synthesis in F. meleagris. We are not dismissing the possibility of the developmental stage, triggered by temperature regime, being an important cue for the metabolite production. That is why we intend to conduct further research to establish the hypothesized relationship between the developmental stage and alkaloid production, by monitoring the alkaloid shift with the progression of ontogenetic changes.

 

Why were those temperature conditions taken for the test?

Temperature plays an important role as a dormancy-inducing/releasing signal in various geophytes. As in many bulbous plant species, flower development in F. meleagris is induced at higher temperatures (20-25 °C), but subsequent elongation of the flower stalk and proper flowering, as well as growth, relies on an extended period of low temperature (usually < 10 °C). Our previous experiments at this chilling temperature (7 °C) proved satisfactory for bulb dormancy breaking and sprouting (see doi: 10.3390/plants9111449, doi: 10.3390/plants9111573), when compared with earlier experiments conducted at 4 °C. Chilling temperatures that are common during early spring season in temperate regions (0-15 °C) often substantially compromise plant productivity and plants in such areas developed specialized mechanisms to survive exposure to low temperatures. At the same time, such period of low temperature in bulbous plants is required for the dormancy breaking and subsequent sprouting.

 

What about the use of plant growth regulators and plant regeneration?

In this experimental work, we used a common model for the multiplication of F. meleagris bulbs, one that we commonly use in our laboratory for a number of years now, and which proved to be quite efficient. Our intention was not to investigate plant regeneration that was already described in the work cited as []. Plant growth regulator TDZ was used only for the stock cultures and bulb multiplication, but in this brief communication we did not investigate the effect of plant growth regulators on alkaloid production. Rather, we were interested in the effect of chilling and bulb sprouting on alkaloid accumulation in bulbs. We figured that, once we establish the right temperature treatment and developmental stage, we will include plant growth regulators in the experimental setup, in order to investigate other factors that could enhance alkaloid production in the bulbs of F. meleagris.

 

Are the objective expectations met?

The objective expectations – to localize alkaloids in the bulbs cultured at different growth regimes – are met.

 

 

Corrections made in the attached pdf document:​

 

Italics letter - in vitro

According to MDPI Style Guide, the term “in vitro” need not be italicized: “Foreign words do not need to be highlighted or italicized, including Greek/Latin terms, such as i.e., e.g., etc., et al., vs., ca., cf., in vivo, ex vivo, in situ, ex situ, in vitro, in utero, ad hoc, in silico, ab initio, vice versa, and via.” (https://www.mdpi.com/authors/layout#_bookmark15)

This term is also not italicized in already published papers of this special issue ("Innovative Micropropagation of Horticultural and Medicinal Plants") of Horticulturae.

 

 

Murashige and Skoog  medium ADDED WITH 1 mg L-1 thidiazuron (TDZ) ???

Please define BM at the first mention

We have changed the description of the cultivation medium according to the reviewers’ suggestion. Instead of using the term ‘basal medium (BM)’, we simplified it as follows: “Murashige and Skoog (MS) medium [26] supplemented with 30 g L^-1 sucrose, 7 g L^-1 agar, 250 mg L^-1 casein hydrolysate, 250 mg L^-1 L-proline, 80 mg L^-1 adenine sulfate and 1 mg L^-1 thidiazuron (TDZ).”

 

 

This title is not clear - [Figure 1. In vitro cultured bulbs of F. meleagris, used for localization of alkaloids.]

The title of the Figure 1 has now been changed into „In vitro cultured bulbs of F. meleagris collected at the beginning of sprouting, at different time points of different treatments, which were used for localization of alkaloids.”

 

Point out in the figures the structures which are mentioned in the text; For example, amyloplasts, and the abundance of alkaloids

We have marked in the figures the information mentioned in the text.

Individual amyloplasts cannot be discerned hence pointed out in the sections of this thickness and at this magnification, but it is clear from histological analysis that they are particularly abundant and densely packed in the cells of the chilled bulbs, and they are known to contain starch that stains specifically dark-blue with Wagner’s reagent. Therefore, the dark-blue dots in the bulb sections shown in Figure 2 represent an accumulation of densely packed amyloplasts in the multitude of cells, of superimposed cell layers, visible in these sections.

 

It is necessary to integrate a reference bar for all the figures

Scale bars are integrated.

Round 2

Reviewer 2 Report

Comments and Suggestions for Authors

This document was satisfactorily corrected