Study of the Component Composition of Essential Oil, Morphology, Anatomy and Ploidy Level of Hyssopus officinalis f. cyaneus Alef
, Iliya Vladimirovich Bulavin 1, Natalia Nikolaevna Ivanova 1, Nataliya Nikolaevna Miroshnichenko 1, Nikita Maximovich Saplev 1, Oksana Michailovna Shevchuk 1,2, Sergey Alexandrovich Feskov 1 and Tatiana Sergeevna Naumenko 1,2
Round 1
Reviewer 1 Report
The study reports on the isolation of a promising sample of Hyssopus officinalis f. cyaneus with a high content of isopinocamphone in the essential oil, which was created using breeding methods. Overall, the study suggests that breeding methods and biotechnological approaches can be used to generate new hyssop cultivars with desirable traits for pharmaceutical use. Although the M&M and results were correctly presented discussion is lacking with multiple issues. The author needs to revise the discussion correlating the following comments with the results (3-8). If the author could provide the rationale for their findings, it will help to improve the presentation of results and discussion section.
The title lacks clarity and specificity. Additionally, the title does not follow the standard scientific title format, which usually includes a clear and concise statement of the research question or objective. A more appropriate and informative title could be: "Essential Oil composition, Morphology, Anatomy, and Ploidy Analysis of Hyssopus officinalis L. f. cyaneus ".
The author needs to work on the following comments;
1. Abstract is too long. The abstract could benefit from being more concise and focused. The author should consider including only essential information and clearly state the aim of the study at the end. This would help the reader to quickly understand the purpose and key findings of the research.
2. Has the promising sample of Hyssopus officinalis f. cyaneus with increased content of isopinocamphone been tested for its effectiveness and safety in human clinical trials? How does the author want to address this issue?
3. I was unable to find the information regarding; how the genetic stability of the promising sample of Hyssopus officinalis f. cyaneus assessed and were any concerns about genetic drift or instability addressed.
4. Was the impact of cultivation and mass production of the new hyssop sample on local communities or economic factors considered in the study?
5. Were any comparisons made between the promising sample of Hyssopus officinalis f. cyaneus and other commonly used medicinal plants to determine its potential advantages or disadvantages in terms of efficacy, safety, and production costs?
6. Is the observed decrease in the cuticle layer and the degree of development of collenchyma near vascular bundles in Hyssopus officinalis f. cyaneus grown in vitro on MS medium with 0.4 and 0.5 mg/l of BAP reversible upon culturing the plant ex situ?
7. How author will explain the mechanism responsible for the consistently high content of isopinocamphone in the essential oil of the selected sample of Hyssopus officinalis f. cyaneus over five months of analysis?
8. Author needs to add recent references as well.
9. Figure 3 (All) should be replaced with a high-resolution figures and it should be clear that the figures are from during in vitro culture. For example; I am unable to differentiate whether figure 3a is in vitro culture or not. The author needs to replace all the figures and provide the stabilized culture with a nutrient medium.
10. Figure for Histograms should be Figure 5. However, I suggest it be put in a supplementary file.
11. It would be better if the author can add some limitations and future prospects of the study in the conclusion section.
The following suggestions could be helpful for author's future research objectives;
1. Consider conducting molecular and genetic analyses of Hyssopus officinalis f. cyaneus to understand better its genetic diversity, population structure, and potential for further breeding and improvement. This could include DNA sequencing, SNP genotyping, and marker-assisted selection.
2. Collaborate with local farmers and herbalists to promote the cultivation and use of Hyssopus officinalis f. cyaneus as a valuable medicinal and aromatic plant, and to explore potential commercial applications in the food, cosmetics, and fragrance industries.
Author Response
Response to Reviewer 1 Comments
Point 1:
Abstract is too long. The abstract could benefit from being more concise and focused. The author should consider including only essential information and clearly state the aim of the study at the end. This would help the reader to quickly understand the purpose and key findings of the research.
Response 1: Was corrected.
Point 2:
Has the promising sample of Hyssopus officinalis f. cyaneus with increased content of isopinocamphone been tested for its effectiveness and safety in human clinical trials? How does the author want to address this issue?
Response 2: This is our future task in colobaration with others medical institutions.
Point 3:
I was unable to find the information regarding; how the genetic stability of the promising sample of Hyssopus officinalis f. cyaneus assessed and were any concerns about genetic drift or instability addressed.
Response 3: In our investigation, ploidy level analysis was made and according to the literature, plant in vitro genetic stability is also determined by this method. Genetic drift has not been given attention, since this phenomenon is related to seed-renewed populations. We used the material in vitro, propagated vegetatively by microcuttings.
Point 4:
Was the impact of cultivation and mass production of the new hyssop sample on local communities or economic factors considered in the study?
Response 4: Hyssopus officinalis is not invasive species in the conditions of dry subtropic.
Economic factors were not investigated.
Point 5:
Were any comparisons made between the promising sample of Hyssopus officinalis f. cyaneus and other commonly used medicinal plants to determine its potential advantages or disadvantages in terms of efficacy, safety, and production costs?
Response 5: Hyssopus officinalis f. cyaneus is a singe form that contains isopinocamphone in such amount (70%). Other plants such as Rosmarinus sp, Mentha sp., and Artemisia sp. have a trace amount of this component. So, maybe, we will do this in our future experiment to determine biological activity as a comparative analysis.
Point 6:
Is the observed decrease in the cuticle layer and the degree of development of collenchyma near vascular bundles in Hyssopus officinalis f. cyaneus grown in vitro on MS medium with 0.4 and 0.5 mg/l of BAP reversible upon culturing the plant ex situ?
Response 6: The reduction of the cuticular layer and collenchyma developmental degree is reversible and is directly related to in vitro conditions. After the plant's adaptation and further growth, tissue formation and development occur appropriately.
Point 7:
How author will explain the mechanism responsible for the consistently high content of isopinocamphone in the essential oil of the selected sample of Hyssopus officinalis f. cyaneus over five months of analysis?
Response 7: In author's opinion, high content of isopinocamphone in the essential oil of the selected sample of Hyssopus officinalis f. cyaneus is explained genetically.
Point 8:
Author needs to add recent references as well.
Response 8: The new references were added.
Point 9:
Figure 3 (All) should be replaced with a high-resolution figures and it should be clear that the figures are from during in vitro culture. For example; I am unable to differentiate whether figure 3a is in vitro culture or not. The author needs to replace all the figures and provide the stabilized culture with a nutrient medium.
Response 9:
The parts of Figure 3 have been replaced. At the same time, the authors consider leaving the figure order and its amount as advisable since the manuscript reports on the effect of various BAP concentrations on in vitro morphogenesis.
Point 10:
Figure for Histograms should be Figure 5. However, I suggest it be put in a supplementary file.
Response 10: Was corrected.
Point 11:
It would be better if the author can add some limitations and future prospects of the study in the conclusion section.
Response 11: Our investigation is a first step in the task-oriented selection of Hyssopus sp. We planned to study of essential oil and water extract′s biological activity for its possible usage.
The following suggestions could be helpful for author's future research objectives;
1. Consider conducting molecular and genetic analyses of Hyssopus officinalis f. cyaneus to understand better its genetic diversity, population structure, and potential for further breeding and improvement. This could include DNA sequencing, SNP genotyping, and marker-assisted selection.
Response 2: Conducting of molecular and genetic analyses of Hyssopus officinalis f. cyaneus will be performed in our subsequent investigation.
2. Collaborate with local farmers and herbalists to promote the cultivation and use of Hyssopus officinalis f. cyaneus as a valuable medicinal and aromatic plant, and to explore potential commercial applications in the food, cosmetics, and fragrance industries.
Response 2: Will be done in future investigation.
Reviewer 2 Report
The manuscript "Study of the component composition of essential oil, morphology, anatomy and ploidity of Hyssopus officinalis L. f. cyaneus" is
Abstract,
line 18: Hyssopus officinalis f. cyaneus correct to H. officinalis f. cyaneus
line 21: A total of 68 components were identified in the...
Results and discussion
The results are clearly presented, however the discussion is very short.
There are one paper dealing with influence of year conditions on hyssop essential oil quality: https://doi.org/10.3390/pr9071152
Include this paper in the discussion!
It is necessary to explain the variation in the chemical composition of the oil depending on the year, only table is not enough!
Explain why exactly f. cyaneus is used for investigations!
Include more papers in the discussion!
Author Response
Response to Reviewer 2 Comments
Point 1:
line 18: Hyssopus officinalis f. cyaneus correct to H. officinalis f. cyaneus
Response 1: Was corrected.
Point 2:
line 21: A total of 68 components were identified in the...
Response 2: Was corrected.
Point 3:
Results and discussion
The results are clearly presented, however the discussion is very short.
There are one paper dealing with influence of year conditions on hyssop essential oil quality: https://doi.org/10.3390/pr9071152
Include this paper in the discussion!
Response 3: Recommended article as well others references were included in the manuscript.
Point 4:
It is necessary to explain the variation in the chemical composition of the oil depending on the year, only table is not enough!
Response 4: In our opinion, one of the most important factors affecting the chemical composition of the essential oil of Hyssopus officinalis is intraspecific diversity as a result of adaptation to habitat conditions and cultivation.
Point 5:
Explain why exactly f. cyaneus is used for investigations!
Include more papers in the discussion!
Response 5: In our collection, 3 forms of Hyssopus officinalis with white, blue, and pink flowers are presented. The forms with white flowers (f. albus) was patented as cv. Nikitskiy Beliy. The next step was the investigation of f. cyaneus as a prospective pharmacological culture. So, we have an idea to study the component composition of the essential oil of this form, morphology, anatomy, and ploidy level of microshoots in vitro on a nutrient medium with BAP in comparison with ex situ samples to develop a cultivation technique with preservation of a valuable trait and use in task-oriented selection.
Round 2
Reviewer 1 Report
The author has revised the MS successfully.
Author Response
Thanks for the work done in reviewing our article.
Reviewer 2 Report
The discussion part is still poor. Just citing a reference does not mean a discussion!
What about weather conditions during investigated years?
Please find correlation and discuss!
What about chemical composition stability?
It is necessary to delve into the topic (study is conducted during 5 years!)!
Author Response
Point 1: The discussion part is still poor. Just citing a reference does not mean a discussion!
Response 1: We have improved the discussion by including data on the influence of various factors on essential oil main components.
Point 2: What about weather conditions during investigated years?
Please find correlation and discuss!
Response 2: We would like to note that the study of the influence of weather conditions was not related to the topic of our investigation. We took accessions from the seed population and determined a promising variant based on the results of subsequent isolation of essential oil and studying its component composition for five years. However, we have included information about air temperature and precipitates for three months (May, June, and July). A graph describes the influence of these factors on the main component content of the essential oil in July, since its extraction was performed during this period.
Point 3: What about chemical composition stability?
Response 3: On the base of literature data and comparison of weather condition, we assumed that the content of the essential oil main components in different samples was determined genetically.
Point 4: It is necessary to delve into the topic (study is conducted during 5 years!)!
Response 4: Thank you for your comments, which are aimed at clarifying the content of the manuscript.
Round 3
Reviewer 2 Report
The manuscript "Study of the component composition of essential oil, morphology, anatomy and ploidy level of Hyssopus officinalis f. cyaneus Alef." is very complex dealing with essential oil composition, morphology, anatomy and ploidy level of microshoots grown in vitro on a nutrient medium, and compare with ex situ samples.
The manuscript is quite good, but the importance of research and application in practice should be further emphasized.
