In Vitro Propagation by Axillary Shoot Culture and Somatic Embryogenesis of Daucus carota L. subsp. sativus, ‘Polignano’ Landrace, for Biodiversity Conservation Purposes
Round 1
Reviewer 1 Report
The manuscript focuses on in vitro propagation and somatic embryogenesis of rare species of carrot in order to conserve this species for the future. I must say I liked this manuscript because it was very well-written, with good English, clear from all points of view and if not for minor comments I would recommend accepting it immediately. Due to the length of the manuscript, I recommend publishing it as Short communication.
Here are my comments:
Figures – to Fig 2 and 3, embryogenesis is misspelled (with "i" instead of "y"). Please correct.
Line 5 – unnecessary comma after name “Blando” (upper index), please remove
Line 285 – Please slightly rewrite the sentence
That is all. Good luck.
Author Response
Dear Reviewer,
Thank you very much for the positive observations you have done to our manuscript.
I have done the changes you have indicated on the Figures 2 and 3.
At Line 5 – the comma after name “Blando” (upper index), is necessary as it is indicated in the Horticulturae template.
At Line 285 –I do not know what you mean ('slighly rewrite the sentence'). I prefer to leave the phrase as it , for not changing the maening.
Author Response File: 
Author Response.docx
Reviewer 2 Report
Dear authors,
I have reviewed your manuscript "In vitro propagation by axillary shoot culture and somatic embryogenesis of Daucus carota L. subsp. sativus, ‘Polignano’ landrace, for biodiversity conservation purposes" submitted for publication in Horticulturae. The manuscript is interesting and presents a valuable collection of information on the in vitro propagation/regeneration of carrot Polignano. However, some points need to be improved before it is published. Statistical analysis: add the complete information about the number of explants and replicates in each stage. Several sentences require the addition of references.
I have highlighted a number of suggestions for your consideration:
Lines 18 and 21: describe the abbreviation (BA and 2,4-D) at the first mention.
Line 18: supplementation on xx medium was enough to obtain
Line 37: [4,5]
Line 38: Add a reference to support this sentence
Line 42: Add a reference to support this sentence
Line 46: Add a reference to support this sentence
Line 50: [3].
Line 56: It would be good to have a sentence about in situ versus ex situ conservation; and then present the ex situ strategies with in vitro culture as an example. Add references to support this.
Line 59: Add a reference to support this sentence
Line 67: in vitro regeneration is advisable for xxx [reference].
Line 70: Add a reference to support this sentence
Line 85: How long it took?
Line 86: Describe NaOCl
Line 88: basal medium (BM, Table 1)
Lines 88-90: supplemented with 6-benzyladenine (BA) at 0.22 and 89 0.44 μM, 2% sucrose, and xx g L agar at pH xx.
Line 90: How about culture conditions? Subculture was performed each xx weeks. did you grow the cultures directly into the light or did you leave them in the dark for a few days? Please add this information
Line 91: Consider adding this table as a supplementary material
Line 93: same BM medium
Line 96: After three subcultures,
Line 98: Percentage of rooted
Line 106: Describe MS and add reference.
Line 107: add the amount of agar and the pH of the media.
Line 108: add the amount of agar and the pH of the media
Line 108: plant growth regulator (PGRs)
Line 108: Percentages of ….. as well
Lines 112-116: You need to add this information earlier.
Line 118: Acclimatization and transfer of xxx to the field
Line 119: xxx-week-old rooted microplants were
Line 128: Statistical analysis_ Add information about the number of explants and replicates in each stage.
Line 139: in BM medium composed of xxxx (Figure 1B).
Figures 1,2, and 3 were not uploaded. It probably was an error during the submission process; therefore, it created confusion during the review process.
Line 141: Describe PC in the figure caption.
Line 150: Describe BA and PC in the table caption.
Line 153: Mean Multiplication Index _ add this information in the material and methods
Line 158: When the mean multiplication index (MMI). Please rewrite this sentence.
Line 166: the average rooting percentage
Line 170: Describe NAA and PC in the table caption.
Line 181: Describe PC in the table caption.
Line 191: Describe PC in the figure caption.
Line 199: Add the concentration
Line 205: Describe PC in the table caption. Describe MS and BM in the table footer
Line 232: Somatic embryogenesis (SE)
Line 232: Describe PC in the table caption
Line 252: were obtained
Line 253: highest BA concentration ( add the concentration here)
Line 254: add the value
Line 260: Add references to support this sentence.
Line 263: rooting percentage
Line 263: Likewise, rooting
Line 268: Consider adding a sentence about the importance of this step within a micropropagation protocol, then add a reference.
Line 271: There is no need to add a new paragraph here. This sentence can be placed next to the one on line 270.
Please carefully revise the format of the references (https://www.mdpi.com/authors/references); lines 348, 350, 352, 354, etc.
Please carefully revise all these points.
Author Response
Dear reviewer,
First of all, we would like to thank you for the time you spent doing a so thorough revision of our manuscript. Your work is highly appreciated and we think it has contributed to improving our manuscript.
Here following, our responses to the suggestions highlighted in your revision. Moreover, the changes are indicated in red in the revised text.
Lines 18 and 21: describe the abbreviation (BA and 2,4-D) at the first mention. Done, in the abstract.
Line 18: supplementation on xx medium was enough to obtain. Done, in the abstract.
Line 37: [4,5] Probably you indicate that a space is needed between 4 and 5, but the manuscript template indicates no space.
Line 38: Add a reference to support this sentence. Yes, a reference has been added: Banga , O. Origin and domestication of the western cultivated carrot. Genetica Agraria 1963, 17, 357-370.
Line 42: Add a reference to support this sentence. Yes, a reference has been added: Banga, O. The development of the original European carrot material. Euphytica 1957, 6, 64-76.
Line 46: Add a reference to support this sentence. Yes, a reference has been added: Renna, M.; Serio, F.; Signore, A.; Santamaria, P. The yellow–purple Polignano carrot (Daucus carota L.): a multicoloured landrace from the Puglia region (Southern Italy) at risk of genetic erosion. Gen. Res. Crop Evol. 2014, 61, 1611–1619. DOI: 10.1007/s10722-014-0155-9
Line 50: [3]. OK!
Line 56: It would be good to have a sentence about in situ versus ex situ conservation; and then present the ex situ strategies with in vitro culture as an example. Add references to support this.
The sentence has been improved adding others details and references, including a work from the author:
- Ruta, C.; Lambardi, M.; Ozudogru, E.A. Biobanking of vegetable genetic resources by in vitro conservation and cryopreservation. Biodivers. Conserv. 2020, 29, 3495–3532. https://doi.org/10.1007/s10531-020-02051-0
- Panis, B.; Lambardi, M. Status of cryopreservation technologies in plants (crops and forest trees). In The role of biotechnology in exploring and protecting agricultural genetic resources; Ruane, J., Sonnino, A., Eds; FAO, Rome, 2006; pp. 61–78.
- Engelmann, F. Use of biotechnologies for the conservation of plant biodiversity. In Vitro Cell. Dev. Biol. - Plant 2011, 47(1), 5–16. http://www.jstor.org/stable/23016584
Line 59: Add a reference to support this sentence. Yes, a reference has been added: Rao, R.V; Hodgkin, T. Genetic diversity and conservation and utilization of plant genetic resources. Plant, Cell Tissue Organ Cult. 2002, 68, 1-19. https://doi.org/10.1023/A:1013359015812
Line 67: in vitro regeneration is advisable for xxx [reference]: . … is advisable for propagation (18. Bidabadi, S.S.; Jain, S.M. Cellular, molecular, and physiological aspects of in vitro plant regeneration. Plants 2020, 9, 702. DOI: 10.3390/plants9060702
Line 70: Add a reference to support this sentence. Yes, a reference has been added (19) George, E.F.; Hall, M.A.; De Klerk, G.-J. Plant Propagation by Tissue Culture, Volume 1. The Background; Springer Dordrecht, Netherlands, 2008. https://doi.org/10.1007/978-1-4020-5005-3
Line 85: How long it took? It has been added in the text (15 days).
Line 86: Describe NaOCl. Done
Line 88: basal medium (BM, Table 1). OK
Lines 88-90: supplemented with 6-benzyladenine (BA) at 0.22 and 89 0.44 μM, 2% sucrose, and xx g L agar at pH xx. This information have been added later on as a new sub-section called ‘Culture conditions’.
Line 90: How about culture conditions? Subculture was performed each xx weeks. did you grow the cultures directly into the light or did you leave them in the dark for a few days? Please add this information. The information have been added in the ‘Culture conditions’ sub-section.
Line 91: Consider adding this table as a supplementary material Yes, Table 1 has been moved to Supplementary Material.
Line 93: same BM medium. The sentence has been better formulated, see line 146-147.
Line 96: After three subcultures, Yes, it has been changed, removing ‘multiplication’
Line 98: Percentage of rooted. Yes, it has been changed, changing % into ‘percentage’.
Line 106: Describe MS and add reference. Yes, it has been changed and reference on MS medium has been added.
Line 107: add the amount of agar and the pH of the media. The information have been added in the ‘Culture conditions’ sub-section.
Line 108: add the amount of agar and the pH of the media The information have been added in the ‘Culture conditions’ sub-section.
Line 108: plant growth regulator (PGRs). Yes, it has been changed.
Line 108: Percentages of ….. as well. The sentence has been changed as suggested.
Lines 112-116: You need to add this information earlier. The information have been added in the ‘Culture conditions’ sub-section.
Line 118: Acclimatization and transfer of xxx to the field. The sentence has been changes as ‘Acclimatization and transfer of micropropagated and regenerated plants to the field’.
Line 119: xxx-week-old rooted microplants were. The age of the microplants (four weeks-old) has been added.
Line 128: Statistical analysis_ Add information about the number of explants and replicates in each stage. The information has been added in a details manner in the ‘Statistical analysis' section.
Line 139: in BM medium composed of xxxx (Figure 1B). The sentence has been changed.
Figures 1,2, and 3 were not uploaded. It probably was an error during the submission process; therefore, it created confusion during the review process. Figures have been correctly uploaded.
Line 141: Describe PC in the figure caption. OK, done.
Line 150: Describe BA and PC in the table caption. OK, done.
Line 153: Mean Multiplication Index _ add this information in the material and methods. The significance of the MMI has been expressed as a footnote of Table 1.
Line 158: When the mean multiplication index (MMI). Please rewrite this sentence. The paragraph has been removed, as it can be misunderstood, and it is not so important, finally.
Line 166: the average rooting percentage. Yes, it has been changed, changing % into ‘percentage’.
Line 170: Describe NAA and PC in the table caption. Ok, done.
Line 181: Describe PC in the table caption. Ok, done
Line 191: Describe PC in the figure caption. Ok, done
Line 199: Add the concentration. Ok, done
Line 205: Describe PC in the table caption. Describe MS and BM in the table footer. Ok, done
Line 232: Somatic embryogenesis (SE). It has been introduced SEs indicating ‘Somatic Embyos’
Line 232: Describe PC in the table caption. Ok, done
Line 252: were obtained. OK, thanks!
Line 253: highest BA concentration ( add the concentration here). The concentration has been added.
Line 254: add the value. The value has been added (‘from 50 to 60%')
Line 260: Add references to support this sentence. It is a hypothesis formulated by the authors.
Line 263: rooting percentage. Yes, it has been changed, changing % into ‘percentage’.
Line 263: Likewise, rooting. OK, thanks!
Line 268: Consider adding a sentence about the importance of this step within a micropropagation protocol, then add a reference. The sentence has been added: ‘Clonal stability of micropropagated plants, in fact, is a prerequisite to maintain the advantages of desired elite genotypes [28].’ Reference: Lata, H.; Chandra, S.; Techen, N.; Khan, I.A.; ElSohly, M.A. Molecular analysis of genetic fidelity in Cannabis sativa L. plants grown from synthetic (encapsulated) seeds following in vitro storage. Biotechnol. Lett. 2011, 33, 2503–2508.
Line 271: There is no need to add a new paragraph here. This sentence can be placed next to the one on line 270. OK
Please carefully revise the format of the references (https://www.mdpi.com/authors/references); lines 348, 350, 352, 354, etc. OK, done.
Author Response File: Author Response.docx
