Advances in Blueberry (Vaccinium spp.) In Vitro Culture: A Review
Round 1
Reviewer 1 Report
Comments and Suggestions for AuthorsDear Authors,
I reviewed your manuscript titled "Advances in blueberry in vitro culture: a comprehensive review" and I find that your review is solid and sufficiently informative. I think it is a helpful resource for researchers starting micropropagation of blueberry in vitro.
I have detected only minor issues, which are listed below.
Title - I would suggest you include the Latin name of the taxon already in the title. E.g. "Advances in blueberry (Vaccinium sp.) in vitro culture: a comprehensive review"
Somewhere you use the term "advancement", and somewhere it's "advances". Better use advances throughout the text.
L104 - maybe add more photos of micropropagated blueberries? Perhaps from your own lab, too?
L107 - "However, alternative approaches have been explored, such as supplementing WPM with Murashige & Skoog medium (MS)" you mean, substituting, not supplementing?
L186 - what about other growth regulators? If there is no sufficient research data available, perhaps include that too, as a comment and further development.
L212 - this is a little bit imprecise. Both ethanol and bleach are bactericides (and fungicides and anti-viral disinfectants). Bleach is actually more effective against spores. Re-phrase this, please. Perhaps briefly explain the mechanisms of action of these disinfectants.
L213 - it's not so much to ensure purity, but to minimize longer exposure to toxic compounds like ethanol or bleach?
L212 - maybe instead of 0.0% contamination say "contamination was eliminated"?
Author Response
Good afternoon,
Please see the attachment.
Best Regards.
Author Response File: 
Author Response.pdf
Reviewer 2 Report
Comments and Suggestions for AuthorsThe topic proposed by the author is interesting, but unfortunately, the document is not informative and does not present anything new that is needed for an updated review. I have suggested some points to be addressed in this review - when we talk about in vitro culture/micropropagation, we mean all phases within a protocol, from the selection of a mother plant to provide an explant and its physiological conditions, to the phases in a micropropagation protocol, i.e. in vitro establishment, propagation, rooting and acclimatization. My suggestion is to have the first topic about the mother plants that will provide explants for micropropagation, and then you can work on the different phases within a micropropagation system, the main parameters that influence each phase, and finally work on the main applications of micropropagation systems. Please find below suggestions/comments:
Title: It is not necessary to have the word "comprehensive" in the title - it will be judged by the readers.
Next time, please include the line numbers to make the review process easier.
The abstract should be a total of about 200 words maximum. See the journal guidelines in the Instructions for Authors.
Pag 2: Although Table 1 provides great information on anthocyanin concentration in some cultivars, it is not the main focus of this manuscript - so please consider deleting it. The author can add this information in the previous paragraph "Notably, blueberries rank high in anthocyanin content among red fruits ranging from x to x (Kalt et al., 2019).
Pag 2: How about seed propagation?
Pag 2: It is necessary to add a reference to support the following sentence "Blueberries are traditionally propagated by stem cuttings". I also suggest that a paragraph be devoted to the traditional methods of propagation and their limitations - also have references to support this information. There are many papers on this topic - I am sharing some examples below
https://doi.org/10.3390/ijms25010544
https://www.actahort.org/books/626/626_58.htm
https://www.actahort.org/books/715/715_59.htm
https://doi.org/10.5539/jas.v10n9p450 https://doi.org/10.36560/16220231667
Pag 2: It is necessary to add a reference to support the following sentence "Micropropagation via axillary shoot production has been documented in lowbush, highbush, and rabbiteye blueberries."
Pag 2: "Studies have shown that blueberry plants propagated through micropropagation not only yield increased fruit production but also exhibit enhanced rooting ability in cuttings compared to traditionally propagated plants" - This is great, but since it is a review, it is necessary to further explore the information, why is it happening? does micropropagation support the production of high-health plants? virus eradication?
Pag 2: Internodal?
Pag 2: It is necessary to review the paper's objective - the main objectives of a review are to outline and document the progress made in a given topic and to discuss it, as well as to give consideration to what will come next, rather than to "establish reliable protocols".
Pag 2: last paragraph - shoot multiplication - That is fine, but what about the age of the source mother plants, the physiological state and growth stage of the mother plants, in vitro establishment, rooting, and acclimatization? All of these guys are part of a micropropagation system.
Pag 3: My suggestion is to have the first topic about the mother plants that will provide explants for micropropagation, and then you can work on the different phases within a micropropagation system, and lastly to work on the main parameters that influence each phase.
Pag 3: "Several studies indicate that the most effective culture medium for blueberry in vitro culture is the McCown Woody Plant Me-dium (WPM)" This is great, but since it is a review, it is necessary to further explore the information, why? Authors can even have the WPM medium formulation as a supplementary material.
Pag 3: All abbreviations must be described the first time they are used in the text.
Pag 3: Table 2 - I have no idea what the point of this information would be.
Pag 5: 4. Effective Disinfection Methods for Micropropagation -
"The subsequent step involves the collection of plant material followed by disinfection." This is the first step after harvesting the explant from the mother plant!! There are many topics that need to be covered before talking about media, such as (a) In vitro establishment [physiological state and growth stage of mother plants, explant selection, sterilization, contamination x oxidation, etc.] (b) Shoot micropropagation, (c) shoot elongation?, (d) rooting, and (e) acclimatization.
After discussing all these points, I suggest having a topic on the advances in the application of tissue culture to blueberries - perhaps discussing the production of high-health plants, conservation of plant genetic resources, breeding, etc.
Author Response
Good afternoon,
We appreciate your comments and sugestions, please see the attachment.
Best regards.
Author Response File: Author Response.pdf
Reviewer 3 Report
Comments and Suggestions for AuthorsMicropropagation in blueberries is a useful tool for accelerating the clonal propagation of this economically important fruit species.
The article is structured appropriately and the described micropromotional techniques are correctly cited. I suggest minor corrections to the article. This is, for example, writing a decimal point, as it should be in English, and not a decimal comma in the tables.
At the end of the article, I would recommend the authors to think about the use of automation and robotization in the field of micropropagation techniques, including blueberries. Similar automation systems already exist, and I see this as potentially economically beneficial for the further development of the entire area in the case of blueberries, a species that can be propagated well in vitro and is commercially important.
I see the sequence of the two consecutive sentences below as illogical:
The addition of IBA as an auxin has been found to induce higher rooting percentages and promote root development compared to other auxins tested in vitro [18]. These studies reported that the best results were observed using NAA in combination with other cytokinins, although the use of these two auxins does not lead to significant differences [18].
Authors should clarify claims about the appropriateness of IBA and NAA for rooting.
Comments on the Quality of English LanguageEnglish used is correct and readable. I recommend minor changes stated in comments for authors.
Author Response
Good afternoon,
Please see the attachment.
Best regards.
Author Response File: Author Response.pdf
Round 2
Reviewer 2 Report
Comments and Suggestions for AuthorsI see a significant improvement in the paper compared to the previous version - the important issues that were recommended have been included in this version. However, a further revision covering the following points (below) is highly recommended. The detailed review with comments and suggestions is below, and I would also like to emphasize that authors need to check whether a figure adapted from another paper requires permission to be reused.
Title - Please double-check the use of sp. or spp.- The designation “sp.” after a genus refers to a single unnamed species, while the designation “spp.” after a genus refers to more than one unnamed species.
L 100: Do references 6, 7, and 8 support the use of tissue culture for virus eradication? There are many nice and recent papers on this topic that the author could add to support this application. I suggest some references on this topic below:
https://doi.org/10.1094/PHYTO-07-23-0232-KC
https://doi.org/10.1111/ppa.13565
L 116: "several factors regarding" - please add the factors
L 121-123: This sentence is quite similar to the one in lines 113-115. It requires revision - Please have a look at these references to have an idea and can contribute to your paper.
https://doi.org/10.1016/j.biotechadv.2010.02.008
https://doi.org/10.1007/s00425-019-03100-x
https://colostate.pressbooks.pub/clonalcryopreservation/chapter/introduction-of-plants-into-tissue-culture/
L 143: "...... sodium hypochlorite." Add a reference to support this sentence
Table 1: describe the abbreviation at the table footer
L 176: "When the time of NaClO increased, contamination was eliminated" poor sentence - please explore further....
L 177: Are there factors to consider when disinfecting field or ex vitro grown plant material? Consider adding a comment. - these references can help:
https://doi.org/10.1007/s00425-019-03100-x
https://doi.org/10.3390/plants13081089
https://colostate.pressbooks.pub/clonalcryopreservation/chapter/introduction-of-plants-into-tissue-culture/
L 204: "The optimal treatment" - add the seed germination percentage
L 205: However, they also observed that V. corymbosum seeds were terminated at xxxx % on ...
L 208: Please check to see if a figure adapted from another paper requires permission to reuse.
L 210-211: shorter durations of stratification using xxxxx (add the treatment and duration)
L 213: "..... V. uliginosum." Add a reference to support this sentence
L 219: "This stage" - Which stage? You have two stages here, so please specify if you are referring to one or both.
L 223: phase or stage?
L 227: "Cytokinins, such as zeatin, are often" - add other cytokinins examples
L 247: Does this technique produce clonal material or not? Consider adding a comment on this
L 255: Double check if in vitro should be italicized
L 257: "......and humidity" (Add references to support this sentence)
L 267: "the best root formation" add the rooting percentages
L 269: same comment as L 255
L 271 "and for V. floribundum with 2.0 mg/L of IBA" add the values as added to the other species.
L 273: Description of Figure 2 needs improvement - on what media and what was the auxin concentration? Do not use abbreviations and add a scale to the pic. Again, please check to see if a figure adapted from another paper requires permission to reuse.
L 296: How about sucrose?
L 309: Consider adding the major difference between WPM medium to MS or DKW
L 311: same comment as L 273
L 317: All abbreviations must be described the first time they are used.
L 323: describe the abbreviation at the table footer
L 336: The information on the sucrose concentration in the medium is in Table 2 - consider adding a comment on this as well.
L 349: The abbreviation for this medium has been added to L 327.
L 354: same comment as L 255
Table 3 is incomplete because the use and concentration of growth regulators are related to each phase within the micropropagation protocol - so I strongly suggest the author add an extra column indication in which situation (initiation, multiplication, rooting, etc.) the regulator and its concentration was used. Also, describe the abbreviations at the table footer.
L 368-369: Poor sentence - it needs review
L 372-373: "Among the growth regulators, zeatin and 2- iP are the most used." for what??
L 373-376: Sentences need references
L 377-378: Poor sentence - it needs review
l 379: "Additionally, other cytokinins such as BAP, kinetin (KIN), and TDZ can also be used" for what??
L 385: add the concentrations
Figure 4 - same comment as L 208
L 411: Why are these regulators described here? It has been described before.
L 420-241: delete "particularly those propagated through tissue culture or micropropagation techniques"
L 428: "..... and development." - Sentences need references
L 430: same comment as above
Figure 5: same comment as L 208
L 449-450: Poor sentence - it needs review
L 467: Sentence needs references
L 518: same comment as L 255
L 518: consider adding cryopreservation - some references for your consideration
https://doi.org/10.1007/s11627-023-10399-5
https://doi.org/10.5511/plantbiotechnology.16.1231b
L 596: Consider adding a comment on the importance of tissue culture for the production of high-quality plant material and the conservation of plant genetic resources.
The reference list must be edited - for example, scientific names must be italicized and authors must follow journal guidelines - https://www.mdpi.com/journal/horticulturae/instructions#preparation
L 822: ? Comments on the Quality of English Language
Suggestions for grammar improvements have been added to the review report.
Author Response
Good afternoon,
Please see the attachment.
Thank you for your time and attention.
Author Response File: Author Response.pdf
