The Breeding of High-Quality Dandelions by NaCl Induced Callus Variation Combined with a Drosophila Tumor Cell Migration Test
Round 1
Reviewer 1 Report
Manuscript entitled “Breeding of high-quality dandelion by NaCl induced callus variation combined with Drosophila tumor cell migration test” by Wu et al. address the study of new dandelion lines and the effects of their leaf extracts on cell migration in Drosophila melanogaster.
The authors obtain different mutant lines from a mother plant by cultivation in the presence of NaCl and then regenerate plants. This study focuses mainly on one of these lines (Binpu 2).
The work is interesting but, in my opinion, the manuscript presents some issues mainly in the Introduction, and other things, that need to be checked to make this work suitable for publication.
1. Main issues:
1.1. Regarding section 1. Introduction
The introduction should reflect the importance of Drosophila in cancer research, and it only has two lines about this topic.
2. Additional comments
Please standardize in the text “cichoric” or “chicory”
Lines 30 to73. Please justify the text
Line 35. Please correct “Mazz., a” with “Mazz., (dandelion), a”.
Lines 56. Please, “Drosophila melanogaster” should be in italics
Line 64. Please, “Taraxacum mongolicum” should be in italics
Line 76. The protocol of “Fresh sterilized leaves” should be detailed or referenced
Line 76, 81. cm2. The “2” should be in superscript.
Line 77. MS medium should be detailed or referenced.
Line 78. The conditions of the growth chamber (culture room) should be specified
Line 93. Please correct “Table A.1” with “Table 1”.
Line 132. Please the “standard protocols” should be referenced.
Table 1. The units of “Leaf length” and “Leaf width” are missing. The values of “Leaf length” and “Leaf width” have no deviation.
Line 191. Please add the “n” for these results
Line 206. Please correct “Y2was” with “Y2 was”
Line 220. Please correct “0.05)..” with “0.05).”
Line 22 and 235. Please correct “0.2g/mL” with “0.2 g/mL”
Line 242. Please check “Figure 4g-I”
Figure 2. The scale bars of ”e” and “e´” are missing
Line 262 and 271. Please the units should appear separated from the numbers
Line 272. Please correct “Ck” with “CK”.
Figure 3. In panel a-f” what is the meaning of +
Lines 305, 309-310, 313, 317, 319, 321, 323, 325, 331, 333, 340, 345 and 347. Please, species should be in italics
Figure S1. The figure legend is somewhat confusing in its present version. My suggestion: Progenies of dandelion tissue culture plantlets grown on saline-alkali land. Panels A to F show phenotypes of lines obtained from treatments Y1 to Y6, respectively. Panel G show a parental line (“Daye”). Panel H show the observation nursery.
Author Response
Thank you very much for the revision, we have checked the whole manuscript and replies are follows:
- Main issues:
1.1. Regarding section 1. Introduction
The introduction should reflect the importance of Drosophila in cancer research, and it only has two lines about this topic.
Reply: we have added the reason for selecting D. melanogaster and cited the related references.
- Additional comments
Please standardize in the text “cichoric” or “chicory”
Reply: we confirmed that is cichoric acid.
Lines 30 to73. Please justify the text
Reply: we have adjusted the format.
Line 35. Please correct “Mazz., a” with “Mazz., (dandelion), a”.
Reply: we have corrected it.
Lines 56. Please, “Drosophila melanogaster” should be in italics
Reply: we have corrected it.
Line 64. Please, “Taraxacum mongolicum” should be in italics
Reply: we have corrected it.
Line 76. The protocol of “Fresh sterilized leaves” should be detailed or referenced
Reply: we have added references. Here the leaf sterilization and later mentioned MS medium were regular laboratory method or formula, so we did not give details, but gave the references.
Line 76, 81. cm2. The “2” should be in superscript.
Reply: we have corrected it.
Line 77. MS medium should be detailed or referenced.
Reply: we have added references. See the above answer.
Line 78. The conditions of the growth chamber (culture room) should be specified
Reply: we have supplemented the details. See the revised manuscript.
Line 93. Please correct “Table A.1” with “Table 1”.
Reply: we have confirmed here citation was Table S1.
Line 132. Please the “standard protocols” should be referenced.
Reply: we have added the reference.
Table 1. The units of “Leaf length” and “Leaf width” are missing. The values of “Leaf length” and “Leaf width” have no deviation.
Reply: we have corrected and added them
Line 191. Please add the “n” for these results
Reply: we have added it.
Line 206. Please correct “Y2was” with “Y2 was”
Reply: we have corrected it.
Line 220. Please correct “0.05)..” with “0.05).”
Reply: we have corrected it.
Line 22 and 235. Please correct “0.2g/mL” with “0.2 g/mL”
Reply: we have corrected them.
Line 242. Please check “Figure 4g-I”
Reply: we have corrected it as Figure 3g-i.
Figure 2. The scale bars of ”e” and “e´” are missing
Reply: we have added it.
Line 262 and 271. Please the units should appear separated from the numbers
Reply: we have corrected them.
Line 272. Please correct “Ck” with “CK”.
Reply: we have corrected it.
Figure 3. In panel a-f” what is the meaning of +
Reply: + means positive control or Drosophila tumor cell migration model, an necessary control treatment for comparing the variations after giving medical treatment.
Lines 305, 309-310, 313, 317, 319, 321, 323, 325, 331, 333, 340, 345 and 347. Please, species should be in italics
Reply: we have corrected them.
Figure S1. The figure legend is somewhat confusing in its present version. My suggestion: Progenies of dandelion tissue culture plantlets grown on saline-alkali land. Panels A to F show phenotypes of lines obtained from treatments Y1 to Y6, respectively. Panel G show a parental line (“Daye”). Panel H show the observation nursery.
Reply: thanks for your suggestion, we have corrected it.
Reviewer 2 Report
comments
Comments for author File: 
Comments.pdf
Author Response
Thanks for your comments on our works. We have checked this manuscript again, including the English language improvement by MDPI editing service. We have supplemented necessary explanation and references for introduction, methods and discussion. Please refer to the revised version.
Reviewer 3 Report
The manuscript presents results on salinity-resistant dandelion varieties developing, phytochemical and genetical assessment, as well biological activity investigation.
However, results are interesting the manuscript requires substantial improvements:
1) English language requires substantial improvement.
2) Latin names should be written using Italic font.
3) The Materials and Methods section requires substantial correction, e.g. 2.2.1. is unclear and confusing, the sequence of carried out investigations should be clear;
The whole section requires corrections and re-writing:
it is unclear what kind of media was used: liquid or solid, what were the conditions of cultivation: dark or light? What mean “good performance”?
Further it is not clear how many lines of plants were obtained, were they propagated separately or not?
”…The soil salt content was averaged at 0.3%.” – how?
Phytochemical analysis details should be given, it is not enough to write in reference…, and not in the section named: Field evaluation.
4) Results and Discussion:
3.1. “While some wanted traits caused by DNA variation could be inherited stably was important for breeding purpose.” - what does it mean?
3.2.1. - this section presents results of callus or plants investigations?
3.3. – what means “effective”?
“…their harvest was mainly relied on artificial way.” – why this is mentioned?
“..Y2was better performance..” – what it means?
How the total flavonoid content was determined and expressed? [mg/g] of what kind of compound: catechin? Quercetin?
Author Response
Thank you very much for your valuable comments, we have revised this manuscript, as follows:
1) English language requires substantial improvement.
Reply: we have improved the English language by the service of MDPI editing.
2) Latin names should be written using Italic font.
Reply: we have checked all and corrected these Latin names.
3) The Materials and Methods section requires substantial correction, e.g. 2.2.1. is unclear and confusing, the sequence of carried out investigations should be clear;
Reply: the sequence of carried out investigations in this section involves explant selection, callus induction, tissue plantlet regeneration etc. Of these, shift salt treating callus is a complex and time-consuming process, we have added some details for better understanding how we step by step carry out this process.
The whole section requires corrections and re-writing:
Reply: we have checked the whole section and supplemented some necessary details, please refer to the revised version.
it is unclear what kind of media was used: liquid or solid, what were the conditions of cultivation: dark or light? What mean “good performance”?
Reply: here we mainly use solid medium. Generally MS medium has a relatively fixed formula, but for some purpose, we can adjust parts of constituents, e.g. we add agar, that was called MS agar medium, is a solid medium because agar liquid turns into a solid condition after cooling down. “good performance” means “good development or growing conditions”, we have changed this confusing term. In addition, we give the details of cultivation conditions. Related corrections refers to the revised version.
Further it is not clear how many lines of plants were obtained, were they propagated separately or not?
Reply: Related details have been added to this section, refer to the revised version. Here simply describe our works: we selected only one callus from each saline treatment (we set 6 NaCl treatments), then, each callus produced 2-5 available tissue culture plantlets. We separately propagate each tissue culture plantlet over 3 generations (by end of year 2021). During this propagation, we repeatedly observe their various characteristics (mainly the morphological variations), until picked out the plant with stable characteristics as dandelion line. We finally selected 6 lines (Y1-Y6) for later evaluation. This field selection work was also time-consuming.
”…The soil salt content was averaged at 0.3%.” – how?
Reply: Our expressing is confusing. Actually we checked the soil content and found the average value was around 0.3%. Now we have corrected it and gave the details.
Phytochemical analysis details should be given, it is not enough to write in reference…, and not in the section named: Field evaluation.
Reply: we have given details and adjusted this section subtitle.
4) Results and Discussion:
3.1. “While some wanted traits caused by DNA variation could be inherited stably was important for breeding purpose.” - what does it mean?
Reply: our express is confusing. We have edited it. Because some new variant traits were not stable, especially through some mutant breeding methods. Actually we want to express the opinion that breeders hope to obtain some ideal traits and these traits can be stably maintained in the next generation.
3.2 - this section presents results of callus or plants investigations?
Reply: yes, here we have addressed the result from the plants investigations.
3.3. – what means “effective”?
Reply: we have changed this words. It means some bioactive substances, here refers to phenolic acids and flavonoids for dandelion.
“…their harvest was mainly relied on artificial way.” – why this is mentioned?
Reply: because here we discussed the trait of “plant shape”. The advantage of plant shape (upright and half-upright growth) was favored for the mechanized harvest of fresh leaf. It can greatly decrease the production cost comparing to manual harvesting.
“..Y2 was better performance..” – what it means?
Reply: we have changed this expression. We would like to say Y2 had high contents of bioactive compounds.
How the total flavonoid content was determined and expressed? [mg/g] of what kind of compound: catechin? Quercetin?.
Reply: Total flavonoid content is a regular index for checking some plants quality. It involves many substances, including you mentioned chemicals. Normally in lab using UV spectrophotometer to check its content, expressed by mg/g, µg/g, %, etc. We have added the determination details in the method section.
Round 2
Reviewer 3 Report
Comments:
1) Materials and Methods section has to be improved: expressions like “with MS agar..” -solid MS medium;
“saline MS” – what is this? The scheme for NaCl treatment is unclear, it is not clear what Y1 etc. means;
“Half of the Above obtained salt-treated calluses obtained above were identified by
random amplified polymorphic DNA (RAPD) markers. The other half was for the regeneration of tissue culture plantlet.” – about what is this?
Culture conditions are unknown;
Did Authors obtain plants that gave seeds which were sow in greenhouse?
2) Results and discussion:
Section 3.1 – what about is this section? It must be improved.
Author Response
Thank you very much for your valuable comments, we have revised this manuscript, as follows:
- Materials and Methods section has to be improved: expressions like “with MS agar..” -solid MS medium;
Reply： we have changed all related terms.
“saline MS” – what is this? The scheme for NaCl treatment is unclear, it is not clear what Y1 etc. means;
Reply：here we used NaCl to treat callus (each solid MS medium adding different concentrations of NaCl, and “NaCl to free-NaCl” periodically treating all calluses). Now we have edited this section and specified the related terms including the annotation of Y1-Y6.
“Half of the Above obtained salt-treated calluses obtained above were identified by random amplified polymorphic DNA (RAPD) markers. The other half was for the regeneration of tissue culture plantlet.” – about what is this?
Reply：we want to express that parts of calluses were taken for the use of RAPD identification, and remaining ones were used to produce tissue culture plantlet. We have deleted these redundant expressions.
Culture conditions are unknown;
Did Authors obtain plants that gave seeds which were sow in greenhouse?
Reply：We have supplemented the cultivation conditions. About dandelion lines selection, we got plants from that gave seeds which were sow in greenhouse, but not very more at the beginning. However, we continued propagating these plants at least 3 generations (someone has over 6 generations up to date), that means we have got abundant of progenies of dandelion tissue culture seedlings. In order to improving the efficiency, we repeatedly investigated some characteristics and eliminated those plants with unstable characteristics. For example, we found the plant shape of half-upright-growth was dominant in some group, so we just keep these plants for this group. Finally we got 6 dandelion lines from each group. Here “Each group” means dandelion progenies of tissue culture seedlings that originated from the callused treated under NaCl concentrations of 85 mM (Y1), 136 mM (Y2), 170 mM (Y3), 205 mM (Y4), 256 mM (Y5), and 307 mM NaCl (Y6), respectively.
2) Results and discussion:
Section 3.1 – what about is this section? It must be improved.
Reply：We have improved this section. Here we showed the RAPD identification results and discussed this variation for future breeding.
Round 3
Reviewer 3 Report
Interesting work. Congratulations
