Large-Scale In Vitro Multiplication and Phytochemical Analysis of Himantoglossum affine (Boiss.) Schltr.: An Endangered Euro-Mediterranean Terrestrial Orchid
Round 1
Reviewer 1 Report
Revision of the --Manuscript Draft-- Manuscript Number: diversity-2040829
Title: Large-scale in vitro multiplication and phytochemical analysis of Himantoglossum affine (Boiss.) Schltr.: An endangered Euro-Mediterranean terrestrial orchid.
Type: Article
This is a manuscript within the scope of the journal "Diversity (ISSN 1424-2818)" and specifically is suitable for Section (Biodiversity Conservation), and Special Issue (Orchid Conservation and Associated Fungal Diversity). The manuscript is presented in a well-structured manner. The results are relevant for successfully large-scale multiplication of H. affine species and can be a tool for re-introduction of in-vitro raised plants to threatened areas. Therefore, the obtained results have an important implication for orchid germplasm characterization, conservation, and improvement.
The manuscript is scientifically sound and the experimental design is appropriate to test the hypothesis. Their results are very important based on the details given in the methods section. The data are interpreted appropriately and consistently throughout the manuscript. The conclusions are consistent with the evidence and arguments presented.
However, according to the revised manuscript the area weakness is related with:
In general: One problem of the current manuscript is the not appropriate use of Abbreviations for the whole document. Please, consider the suggestions in the attached document e.g., LNs 19-21, 93, 111, 126, 127, 128, 138-141, 144-146, 151, 153, 169, 171, 178, 185, 191, 214, 216, 217, 223, 235-237, 295, 296, 304-306, 330, 393, 424, 507, 564, 583, 628, 635-637, 646-648, 654, 723, 724.
Moreover, I suggest the authors for improving the manuscript to take care with details that need to be accurately corrected for the whole document. See in the attached document the LNs 86, 92, 110, 120, 136-138, 144-146, 150-152, 161, 172, 174, 175, 180, 181, 184, 186, 191, 198, 200-202, 205, 206, 223, 224, 227, 228, 232, 233, 427, 435, 436, 438, 491, 509, 510, 592, 593, 623, 628, 637, 668, 669, 674, 715, 716.
In Materials and Methods: I suggest rewriting the sentence LNs 123-127 for better understanding for readers. It is not clear how you determine the % of ethyl alcohol by alcohol meter. It is not clear how you fix the plant material LNs 222-224.
In Tables and Figures:
Please, the authors in Figure number 1 need to show the scale bar and its size for each photo.
Please, the authors in Figure number 2 need to show the scale bar and its size for each photo. In the case of Figure 2C show the arrows for RZ and LP structures.
Please, the authors in Figure number 3 need to show the scale bar and its size for photo 3B, 3C and 3E.
In Table number 2, LN 435, I suggest modifying the title.
Please, the authors in Figure number 4 need to show the scale bar and size for photo 4J. In the case of possibility for each scale bar try to enhance the actual size of the letters.
Please, the authors in Table number 4 need to modify it according to the suggestions in the attached document, e.g., please, declare in legend LN 585 that FW is fresh weight.
In LN 639, the Reference Takamura and Tanaka (2004) does not appear on the Reference list.
Please, remember that more aspects are mentioned in the revised and attached document.
Comments for author File: 
Comments.pdf
Author Response
Dear reviewer
All, of your suggestions, have been applied throughout of manuscript in red color. We tried to include your valuable advice and suggestions everywhere it was possible. We think your comments have clearly improved the scientific value of the manuscript. With your comments:
Point 1: In general: One problem of the current manuscript is the not appropriate use of Abbreviations for the whole document. Please, consider the suggestions in the attached document e.g., LNs 19-21, 93, 111, 126, 127, 128, 138-141, 144-146, 151, 153, 169, 171, 178, 185, 191, 214, 216, 217, 223, 235-237, 295, 296, 304-306, 330, 393, 424, 507, 564, 583, 628, 635-637, 646-648, 654, 723, 724.
Response 1: The abbreviations were corrected throughout of the manuscript. Moreover, all sugggested corrections were performed for the whole document.
Point 2: Moreover, I suggest the authors for improving the manuscript to take care with details that need to be accurately corrected for the whole document. See in the attached document the LNs 86, 92, 110, 120, 136-138, 144-146, 150-152, 161, 172, 174, 175, 180, 181, 184, 186, 191, 198, 200-202, 205, 206, 223, 224, 227, 228, 232, 233, 427, 435, 436, 438, 491, 509, 510, 592, 593, 623, 628, 637, 668, 669, 674, 715, 716.
Response 2: The manuscript was improved based on your worthwhile suggestions throughout all sections of the manuscript.
Point 3: Materials and Methods: I suggest rewriting the sentence LNs 123-127 for better understanding for readers. It is not clear how you determine the % of ethyl alcohol by alcohol meter. It is not clear how you fix the plant material LNs 222-224.
Response 3: Thanks for your comment. The sentence was long and ready to undrestand therfore we reivsed it by dividing to two sentences. The ethanol % was calculated by adding 96% ethanol in ration to the whole volume of FAA fixation solution. For example to prepare 100 mL of FAA solution we added 65.625 mL 96% ethylic alcohol. To fix the orchid’s plant sample they were simply immersed in FAA solution. We added more information to the corresponding section.
Point 3:
Tables and Figures:
Please, the authors in Figure number 1 need to show the scale bar and its size for each photo.
Please, the authors in Figure number 2 need to show the scale bar and its size for each photo. In the case of Figure 2C show the arrows for RZ and LP structures.
Please, the authors in Figure number 3 need to show the scale bar and its size for photo 3B, 3C and 3E.
In Table number 2, LN 435, I suggest modifying the title.
Please, the authors in Figure number 4 need to show the scale bar and size for photo 4J. In the case of possibility for each scale bar try to enhance the actual size of the letters.
Please, the authors in Table number 4 need to modify it according to the suggestions in the attached document, e.g., please, declare in legend LN 585 that FW is fresh weight.
Response 3: All corrections listed in Point 3 were done all over the manuscript. Regarding the enhancing of actual size of letters, it is not possible as these letters have fixed within the pictures and its default of employed microscopes.
Author Response File: 
Author Response.docx
Reviewer 2 Report
The manuscript ”Large-scale in vitro multiplication and phytochemical analysis of Himantoglossum affine (Boiss.) Schltr.: An endangered Euro-Mediterranean terrestrial orchid” is dealing with in vitro propagation of orchid which tubers are used for the production of nutritional supplement Salep. Authors presented results about in vitro seed germination and propagation by somatic embryogenesis. Also, phytochemical profile of tubers and leaves is presented. In manuscript there were no evaluation of any aspects of plant biodiversity. The manuscript has some merit but it can not be accepted for publication in present form. There are a lot of errors in presentation of the data. Some of them are highlighted in the text. I suggested authors to present numbers in uniform way (for instance 00.00±00.00) in all Tables. Legend for Figure 4 is confusing. What means SB in Figure 4E? What means DL in Figure 4D? How it is possible that tubers has leaf epidermis as it presented in Figure 4D? The usage of each dye may be included in Figure description.
Comments for author File: Comments.pdf
Author Response
Dear reviewer
All, of your suggestions, have been applied throughout of manuscript in blue color. We tried to include your valuable advice and suggestions everywhere it was possible. We think your comments have clearly improved the scientific value of the manuscript. With your comments:
Point 1: Authors presented results about in vitro seed germination and propagation by somatic embryogenesis. Also, phytochemical profile of tubers and leaves is presented. In the manuscript, there was no evaluation of any aspects of plant biodiversity.
Response 1: Thanks for your comment. As the manuscript has been submitted to a special issue “Orchid Conservation and Associated Fungal Diversity” we believe that it fall within the scope of the journal. This fact has been also confirmed by Reviewer 1.
Point 2: There are many errors in the data presentation. Some of them are highlighted in the text. I suggested authors present numbers in a uniform way (for instance 00.00±00.00) in all Tables.
Response 2: Thanks for your comment. All numbers and values were corrected based on your worthwhile advice.
Point 3: Legend for Figure 4 is confusing. What means SB in Figure 4E? What means DL in Figure 4D?
Response 3: Thanks for your comment. The whole of Fig 4 description and legends were revised and corrected based on your advice.
Point 4: How is it possible that the tubers have leaf epidermis as it presented in Figure 4D?
Response 4: Thanks for your comment. That is right. The description for each figure sub-section was confusing. In fact, Fig 4D is for leaves not for tuber. We have already updated and revised the Fig 4 description based on your worthwhile comment.
Point 5: The usage of each dye may be included in Figure description.
Response 5: Thanks for your comment. We have already mentioned the employed dyes in the material and method “2.7. Histochemical investigation” section.
Author Response File: Author Response.docx
Round 2
Reviewer 1 Report
Dear Authors,
I revised with pleasure for the second time the manuscript entitled: Large-scale in vitro multiplication and phytochemical analysis of Himantoglossum affine (Boiss.) Schltr.: An endangered Euro Mediterranean terrestrial orchid.
I believe it has been improved substantially. However, the actual manuscript needs to minor text editing for publication in Diversity (ISSN 1424-2818).
In General, I suggest the authors remember that the Acronyms/Abbreviations should be defined the first time they appear in each of three sections: the abstract; the main text; the first figure or table. Of course, you need to use them in each Section otherwise it is not useful.
- In the Abstract, see the LNs 19, 20.
- In the main text Section: Introduction, see the LNs 89, 90, 98. Materials and Methods, the authors need to correct the LNs 144, 145, 190, 219, 231. Results, see the LN 333.
- In figure number 4, I suggest in scale bars to enhance the size of the letters. It is not readable.
As a guide, please revise the attached document.
With greetings,
Author Response
Response to Reviewer 1 Comments
We have improved the manuscript according to the reviewer’s comments. All corrections have been distinguished with different colors in the text for each reviewer. First reviewer in red color, the second reviewer in blue color. With reviewer comments:
In General, I suggest the authors remember that the Acronyms/Abbreviations should be defined the first time they appear in each of three sections: the abstract; the main text; the first figure or table. Of course, you need to use them in each Section otherwise it is not useful.
Point 1: In the Abstract, see the LNs 19, 20.
Response 1: Based on your secription and you advice, the defnitions in LNs 19-20 are correct as they have bring for first time in abstract and they are in full form.
Point 2: In the main text Section: Introduction, see the LNs 89, 90, 98. Materials and Methods, the authors need to correct the LNs 144, 145, 190, 219, 231. Results, see the LN 333.
Response 2: All coressponding sections were irevised based on your worthwhile suggestions throughout all sections of the manuscript. However, we couldn’t realize which word you mean at LN 231.
Point 3: In figure number 4, I suggest in scale bars to enhance the size of the letters. It is not readable.
Response 3: It was revised.
Reviewer 2 Report
Dear authors,
You significantly improved first version of the manuscript. I still have find some errors in the presentation of the data in Tables. First, what values are presented in Tables? Mean value ± standard error or Mean value ± standard deviation? Please clarify in Material and methods sections. Second, present all values in same format, at two decimals i.e 40.00± 0.15.
Author Response
Response to Reviewer 2 Comments
We have improved the manuscript according to the reviewer’s comments. All corrections have been distinguished with different colors in the text for each reviewer. First reviewer in red color, the second reviewer in blue color. With reviewer comments:
Point 1: You significantly improved first version of the manuscript. I still have find some errors in the presentation of the data in Tables. First, what values are presented in Tables? Mean value ± standard error or Mean value ± standard deviation? Please clarify in Material and methods sections. Second, present all values in same format, at two decimals i.e 40.00± 0.15.
Response 1: Thanks for your comments. The values are average ± standard error. The decriptions were added to the end of each table. Regarding the values’ format, they were revised based on your recommendation in all tables.
