Production of Black Cumin via Somatic Embryogenesis, Chemical Profile of Active Compounds in Callus Cultures and Somatic Embryos at Different Auxin Supplementations
, Neama Abdalla 5,6,*,†and Judit Dobránszki 6,†Round 1
Reviewer 1 Report
In this study, the induction of callus and somatic embryo of this important medicinal plant Nigella sativa was studied, and the metabolites of different types of tissues were determined by GS. This study will help to further develop and utilize the medicinal value of this important medicinal plant. However, there are major deficiencies in the introduction, result analysis and discussion of this study. The introduction lacks the research progress on the effects of auxin types and concentrations on somatic embryogenesis, the histological observation of the results is not clear enough, the samples selected for the comparison of metabolite determination do not have phenotypic differences, and the discussion lacks logic and concision of the highlights of the study, so it is recommended to reject the paper and resubmit it.
Most of the research content is about the effects of different types and concentrations of auxin on callus induction and somatic embryo development, but there is no corresponding progress summary in the preface, so the preface needs to be adjusted.
How is the callus differentiation ratio and embryo differentiation ratio calculated? Especially the embryo, the embryo is the tissue that has entered the differentiation stage, what is the differentiation ratio of the embryo? In other words, the author should give a clear explanation of the indicators in the paper, and accompany it with a detailed calculation formula for others to repeat.
Line183-185, Fig.1.A doesn’t reflect that cotyledon was the best induced explants.
Line 195-196, Fig.1.D represents the state of embryo formation in the optimal medium, while in line207-209, it also refers to complete plants developed and formed under light culture environment, which is inconsistent. In addition, Fig.1.D and Fig.1.F are both quoted here, and the organization of these two pictures is obviously different. Fig.2 lacks of scale and rough physiological observation of somatic embryo development process, lacks of description of morphology and physiological characteristics of spherical embryo, cardioid embryo and other stages of embryo, typical histological characteristics of each stage have not been observed, it is recommended to select new materials for histological observation.
Conclusion 3.4: â‘ There is a lack of duplication in the determination of metabolites in each sample, and it is recommended that there should be at least 3-5 biological replicates in the determination of metabolites in each sample. â‘¡ From callus samples, samples treated with two different concentrations of NAA were selected based on what? The above does not indicate that the samples treated with these two concentrations have obvious morphological differences or are significantly different from other samples. Similarly, the reasons for the selection of embryo samples are also to be investigated. The author points out that (line265-267) two optimal treatments for embryo formation are selected, but only for cotyledon tissue. Why only cotyledon tissue is considered? At the same time, the effects of the two treatments are similar, why do we compare the differences in metabolites?
Discussion is illogical, the author did not discuss the main conclusions of this study with predecessors’ works. Most of the discussion is on the potential medicinal value of callus, which is not related to the main results of this study. It is suggested that the author make major revisions to the discussion to clarify the highlights of the study.
Author Response
Reviewer 1#_
Comments and Suggestions for Authors
In this study, the induction of callus and somatic embryo of this important medicinal plant Nigella sativa was studied, and the metabolites of different types of tissues were determined by GS. This study will help to further develop and utilize the medicinal value of this important medicinal plant. However, there are major deficiencies in the introduction, result analysis and discussion of this study. The introduction lacks the research progress on the effects of auxin types and concentrations on somatic embryogenesis, the histological observation of the results is not clear enough, the samples selected for the comparison of metabolite determination do not have phenotypic differences, and the discussion lacks logic and concision of the highlights of the study, so it is recommended to reject the paper and resubmit it.
Response: Many thanks for your comments which aimed to improve the MS. All changes or corrections you asked us to do are highlighted in yellow color.
Most of the research content is about the effects of different types and concentrations of auxin on callus induction and somatic embryo development, but there is no corresponding progress summary in the preface, so the preface needs to be adjusted.
Response: Many thanks for your comments. One new paragraph has been added to the introduction. Also, we referred to some previous reports regarding to tissue culture of this important plant species; mainly on callus, somatic embryos and secondary metabolites production.
How is the callus differentiation ratio and embryo differentiation ratio calculated? Especially the embryo, the embryo is the tissue that has entered the differentiation stage, what is the differentiation ratio of the embryo? In other words, the author should give a clear explanation of the indicators in the paper, and accompany it with a detailed calculation formula for others to repeat.
Response: The equations used in the study have been added to the revised MS
The title No. 2.2 in M and M section has changed into "Callus induction and differentiation, and somatic embryo formation and conversion to plantlets"
Some sentences in the text have changed
The title No. 3.2 in Results section also changed to " Differentiation of calli into shoots/plants and embryos conversion to plantlets"
Some sentences in the text have changed and in the table 2 as well.
Line183-185, Fig.1.A doesn’t reflect that cotyledon was the best induced explants.
Response: We can't notice from the photo 1A that cotyledon was the best induced explants, because the explant changed totally into callus tissue. However, all the measurements of callus induction indicated statistically that cotyledon is the best, this is the truth and the photo is correct it is for cotyledon explant. We modified some words in the title of figure (1).
Line 195-196, Fig.1.D represents the state of embryo formation in the optimal medium, while in line207-209, it also refers to complete plants developed and formed under light culture environment, which is inconsistent. In addition, Fig.1.D and Fig.1.F are both quoted here, and the organization of these two pictures is obviously different. Fig.2 lacks of scale and rough physiological observation of somatic embryo development process, lacks of description of morphology and physiological characteristics of spherical embryo, cardioid embryo and other stages of embryo, typical histological characteristics of each stage have not been observed, it is recommended to select new materials for histological observation.
Response: Ok. Photo 1E replaced by other one which is clearer and more suitable. We interested to determine/detect the different developmental stages of embryos to ensure that we have somatic embryos not other structures. So, there is no need to describe the morphology and physiology of the getting embryos accurately. We can't repeat the histology because the work is already finished and we did not have more samples for this. Anyway thanks for your valuable comment and we will consider it in our upcoming studies.
Conclusion 3.4:
â‘ There is a lack of duplication in the determination of metabolites in each sample, and it is recommended that there should be at least 3-5 biological replicates in the determination of metabolites in each sample.
Response: There were three replicates in each sample (callus, embryos and seed extract) when we determined the metabolic compounds. We refereed to this information in M and M section under the title " 2.4. Gas Chromatography analysis of extracts from seeds, calli and embryos
â‘¡ From callus samples, samples treated with two different concentrations of NAA were selected based on what? The above does not indicate that the samples treated with these two concentrations have obvious morphological differences or are significantly different from other samples. Similarly, the reasons for the selection of embryo samples are also to be investigated. The author points out that (line265-267) two optimal treatments for embryo formation are selected, but only for cotyledon tissue. Why only cotyledon tissue is considered? At the same time, the effects of the two treatments are similar, why do we compare the differences in metabolites?
Response: Selection of callus samples produced on 1 and 3 mg L-1 NAA and, embryos on 2 and 3 mg L-1 IBA for metabolic analysis was based on the statistical analysis, which indicated that the mentioned concentrations were the best two treatments for callus induction and embryos formation; respectively. The criteria for selection those treatments was the statistical differences, I mean
Moreover, cotyledon was the superior explant from the two examined explants under study. It was statistically better than hypocotyl explant
If the effects of the two treatments are similar, why do we compare the differences in metabolites? Because we did not know how the two different best treatments will affect the metabolite content in the measured samples.
Finally, we have to select the best explant, and the best treatment for callus induction and embryos formation, it is logic in order to have enough plant material for large scale production of the valuable metabolites for industry or medicinal purposes.
Discussion is illogical, the author did not discuss the main conclusions of this study with predecessors’ works. Most of the discussion is on the potential medicinal value of callus, which is not related to the main results of this study. It is suggested that the author make major revisions to the discussion to clarify the highlights of the study.
Response: We have already refereed to the main results of the previous studies on callus induction, regeneration, somatic embryos formation and differentiation in Nigella sativa. Also, the most important metabolites in callus, seeds in the previous reports and in the current study.
Reviewer 2 Report
The manuscript entitled (Production of Black Cumin by Somatic Embryogenesis and Chemical Profile of Active Compounds in Callus Cultures and Somatic Embryos at different Auxin Supplementations) by Higazy et al., reported, the effects of different auxins on callus induction and subsequent somatic embryo formation. In addition, the phytoconstituents were determined in seeds, calli and embryos extracts by GC-MS. This this good idea and can be used to increase the production of secondary metabolites in medicinal plants. This manuscript could be accepted after minor corrections.
1- Some minor English and grammatic mistakes should be corrected throughout the manuscript.
2- Plant family name should be added in abstract and keywords.
3- The abbreviations should be written in full names when mentioned for the first time in manuscript as in Abstract.
4- Plant taxonomist and voucher specimen should be added to plant material, because there are many varieties of Black seed.
5- Under each Tables (1, 2, and 3), please draw chemical structures for each compound isolated from extract and put a star on structures for major metabolites.
6- Resolution of Figure 2 for A, B, C, and D not good. Please, increase resolution.
7- GC chromatogram should be provided as supplementary materials.
Some minor English and grammatic mistakes should be corrected throughout the manuscript.
Author Response
Reviewer 2
Comments and Suggestions for Authors
The manuscript entitled (Production of Black Cumin by Somatic Embryogenesis and Chemical Profile of Active Compounds in Callus Cultures and Somatic Embryos at different Auxin Supplementations) by Higazy et al., reported, the effects of different auxins on callus induction and subsequent somatic embryo formation. In addition, the phytoconstituents were determined in seeds, calli and embryos extracts by GC-MS. This this good idea and can be used to increase the production of secondary metabolites in medicinal plants. This manuscript could be accepted after minor corrections.
Response: All corrections and changes throughout the MS are highlighted in yellow
1- Some minor English and grammatical mistakes should be corrected throughout the manuscript.
Response: We checked the English language and tried to improve it
2- Plant family name should be added in abstract and keywords.
Response: Ok done
3- The abbreviations should be written in full names when mentioned for the first time in manuscript as in Abstract.
Response: Ok done
4- Plant taxonomist and voucher specimen should be added to plant material, because there are many varieties of Black seed.
Response: Ok, done. The needed information has been added to M and M section under title "2.1. Plant material and seed germination"
The source of the cultivar used in the study is belonging to one of authors. So, there is no voucher specimen
5- Under each Tables (1, 2, and 3), please draw chemical structures for each compound isolated from extract and put a star on structures for major metabolites.
Response: You mean Tables 3, 4 and 5 which includes the chemical compounds isolated from calli, embryos and seeds extracts
Ok, done. The special programs, which could be used for drawing the chemical structure of compound, are not available for us. We have used the free websites; we could find most of chemical compounds mentioned in the MS not all of them. We hope it is acceptable for you and the journal as well.
6- Resolution of Figure 2; for A, B, C, and D photos are not good. Please, increase resolution.
Response: Ok, we have provided good photos. We modified them as we can
7- GC chromatogram should be provided as supplementary materials.
Response: Ok, done. We submitted them as supplementary materials.
Comments on the Quality of English Language
Some minor English and grammatical mistakes should be corrected throughout the manuscript.
Response: Ok, we revised it
Many thanks for your encouragement and the valuable comments
Reviewer 3 Report
Dear Authors,
The presented topic is very relevant for medicinal plant growers and the medical/pharmaceutical industry.
There are a few points that should be addressed by the authors:
- Is it possible to quantify the phytochemical compounds? Also to make a statistical comparison between calli derived from cotyledon explant cultured, embryos produced on calli derived from cotyledon explant, and seed extract? It could be used in the same statistical analysis as in Tables 1 and 2.
- This method of culture and extraction of valuable phytochemical has an economic background?
In my opinion, the Introduction, Materials and Methods, Results and Discussion chapters are well structured.
Thank you!
Author Response
Reviewer_3
Comments and Suggestions for Authors
Dear Authors,
The presented topic is very relevant for medicinal plant growers and the medical/pharmaceutical industry.
There are a few points that should be addressed by the authors:
- Is it possible to quantify the phytochemical compounds? Also to make a statistical comparison between calli derived from cotyledon explant cultured, embryos produced on calli derived from cotyledon explant, and seed extract? It could be used in the same statistical analysis as in Tables 1 and 2.
Response: for the first part of the question; yes, it is possible to estimate the exact quantity of the phytochemical compounds in calli, embryos and seeds extracts. But we need for quantification analysis standard compounds which are little bit expensive. We will take in consideration in the future in further studies.
About the second part; there is no possibility for statistical comparison of phytochemical compound content between callus, embryo and seed extracts. Because the chemical analysis by GC-MS measured the peak areas of each compound relative to the other compounds in the same extract. It is mainly detects the presence or absence of the compound in the extract. The comparison here has no meaning in this situation or not fair, I mean. However, if we can measure the exact quantity of the compound, in this case we can make statistical comparison between callus, embryo and seeds extracts regarding to their actual content of such chemical metabolites. In Tables 1, and 2 the calli and embryo production were comparised to see the differences in response to auxin treatments
- This method of culture and extraction of valuable phytochemical has an economic background?
Response: Yes, callus and embryos cultures could be used for enhancing the accumulation of secondary metabolites. Optimizing the culture conditions (i.e., medium composition, PGR type and concentration, incubation conditions, etc), or using elicitors or nanoparticles could be applied for large scale production of the most valuable secondary products in calli or embryo cultures for industrial or pharmaceutical/ medicinal purposes with economic benefits
In my opinion, the Introduction, Materials and Methods, Results and Discussion chapters are well structured.
Response: many thanks for your encouraging comments
we added supplementary please check
