Micropropagation Protocols for Three Elite Genotypes of Stevia rebaudiana Bertoni

Round 1

Reviewer 1 Report

The present article describes clonal Stevia rebaudiana production through different concentrations of hormones in vitro propagation. The aim is to identify the system with the highest micropropagation rate in three different clones. This article is interesting and well-written, but there are a few points that need clarifying.

Therefore, it needs a revision.

Line 48: DW, write the full name the first time.

Line 57: specify the type of conventional propagation (for example: conventional sexual seed propagation).

Line 61: Why “In contrast”? Do you always talk about vegetative propagation? Choose another adverb (e.g. in addition, moreover…).

Line 135: change the word inoculated with transferred, placed, cultured, …, throughout the text.

Line 136: Murashige e Skoog 1962, missing bibliographical reference.

Line 136: replace “culture medium” with “basal salt” if present only Gambor's vitamins. Or are there vitamins MS and Gambor in the same culture medium?

Line 136: missing pH and sterilization.

Line 141: Add cultivation conditions, also to “Shoot multiplication” and “Root Induction”.

Line 166: replace “culture medium” with “basal salt”.

Line 178: delete MS in “an MS culture medium for root induction” and replace culture medium with basal salt in “The MS culture medium with Gamborg′s”.

Line 232: change the word inoculated with transferred, placed, cultured, …, throughout the text.

Line 232: delete Murashige Skoog.

Line 249: replace “AIB” with “IBA” throughout the text.

Line 249: replace “ANA” with “NAA” throughout the text.

Line 249: replace “AIA” with “IAA” and “AIB” with “IBA” throughout the text.

Line 447: review the title missing letters.

Line 504: replace “bioassay” with “bio assay”.

February 10, 2024

Author Response

in an attached file.

Author Response File: Author Response.pdf

Reviewer 2 Report

The author proposed a micropropagation protocol for Stevia rebaudiana Bertoni genotypes, optimizing each phase within the micropropagation system. After reading/reviewing the manuscript, I can say that the authors have made impressive efforts in testing variations in each phase and interesting results have been obtained. However, some points need to be clarified and additional information is needed in the materials and methods - I have detailed these points below.

Discussion: I strongly suggest the authors to highlight the main advantages/differences of the present micropropagation protocol compared to previous protocols for this plant species and also the future use of the optimized protocol - germplasm conservation.

It is necessary to check the grammar before resubmitting the revised version. I have suggested some grammatical improvements, but a thorough review is recommended.

Please see my comments/suggestions for improving the paper:

L 24-26: rewording - The results showed that optimal shoot multiplication (17.3 shoots per explant) for L020 was achieved when cultures were grown on a basal medium MS supplemented with 1 μM 6-benzylaminopurine (BAP).
L 26-28: rewording - optimal shoot multiplication (18.5 shoots per explant) was achieved in MS supplemented with 1 μM BAP and 0.5 μM naphthalene acetic acid (NAA), while for Morita II, the best
L 31-33: on tissue culture, particularly in shoot multiplication. The development of a successful micropropagation system is crucial for the conservation and improvement of S. rebaudiana, with significant implications for its future use and performance.
L 34: Keywords - organize alphabetically
L 37: in Paraguay (add a reference to support this sentence).
L 40-42: rewording - Stevia species were used primarily as a sweetener and source of medicine by the Guarani tribes of Paraguay and Brazil. These days, however, its cultivation has spread to numerous nations worldwide.
L 62: vegetative cuttings or tissue culture??
L 63-64: Authors were talking about cutting propagation and suddenly started a sentence about micropropagation. I suggest adding a sentence in between highlighting the use of tissue culture.
L 70: on shoot formation than ??
L 71: noted or found?
L 74: 25% when cultivated in a
L 75-76: rewording - However, since rebaudioside A has the most desirable flavor characteristics, improved genotypes with high levels of rebaudioside A need to be developed and propagated
L 78: describe
L 78: mass propagation using xxxx
L 95: land or field??
L 96-98: Hypothesis iii is confusing - please review it.
L 104-112: If you have this information from a published paper, it would be nice to reference it.
L 115: Double-check the grammar for "mother-plants". Is it necessary to use "-" between words? Review this throughout the manuscript
L 115: add vessel capacity
L 115: established
L 115: I am thinking about hypothesis i - please double-check it.  It seems that in this hypothesis the authors collected shoots from the plants under field conditions.
L 116: under 50% shade
L 116: How about the photoperiod?
L 117: netting or net??
L 119: Were fertilization and phytosanitary treatment carried out on the basis of recommendations? If yes, please add the reference

L 122: Check the grammar in the Figure 1 description - established, mother plants, etc.
L 132-133: have a look at these 2 lines - there are the words "min" and "minutes" - standardize this throughout the paper
L 134: The explants were then rinsed twice with
L 135: finely??
L 136: PGR-free medium composed of Murashige-Skoog (MS) salts (add a reference for MS medium) supplemented with
L 139: How about the medium pH?
L 140: How many shoots per container? So the initiations were in Magent containers instead of test tubes? Just think, if you have an explant that shows contamination, you have lost everything in the container.
L 144: Is it not easy to use days instead of hours? It would make reading easier.
L 162: how old were the explants in the multiplication phase? Add this information.
L 177: same inquiry as above - add this information. Also size, number of leaves, etc,
L 183: how many shoots per container?
L 186: add the brand of the commercial product
What were the growing conditions at each phase? Temperature, photoperiod, etc. Add this information
L 187: How many days do cultures take to root?
L 190: Describe the acclimation period in detail so that others can replicate it.
L 196: How many shoots are within each replicate? Was it the same for all stages?
L 201: Add data-numbers - I can't see results for the three varieties, only one. Is this data for what material?
L 232: Table 1 should be included in the initiation topic.
L 232: Are these data in percentage? Add this information
L 236: "n=5" - It is necessary to add this information to the statistical part. Is 5 enough?
L 242: "combined with" or "without NAA"??
L 260: Letters have been added to the table, but their meaning has not been described in the description.
L 260: ANA??
Figures and tables: It is necessary to describe all abbreviations in the description.
L 262: small case letters should be lowercase letters - review this throughout the paper
L 268: "Best treatment" - it is necessary to describe it. There is also an unspecified scale in the figure.
L 272-274: Include this information in the material and methods section. So most of the information on this topic is materials and methods - please rewrite it.
Discussion: I strongly suggest the authors to highlight the main advantages/differences of the present micropropagation protocol compared to previous protocols for this plant species and also the future use of the optimized protocol - germplasm conservation.
L 279: Instead of transferring to ex vitro conditions, why not say acclimatization?
Please move figures 4, 5, and 6 to Results.
L 318: seedling rooting?? How many days does it take to root the plant in each figure? Include this information here and in the Materials and Methods section.

It is necessary to check the grammar before resubmitting the revised version. I have suggested some grammar improvements, but a thorough review is recommended.

Author Response

in an attached file

Author Response File: Author Response.pdf

Round 2

Reviewer 2 Report

I can see a significant improvement in the paper compared to the previous version. A further minor revision covering the following points is recommended.

L 71: have also been used and documented in the literature

L 74: agar medium supplemented. 

media: plural

medium: singular

L 81-82: months, which should be increased by 25% when cultivated in a 

L 135: from x-month old mother-plants (add the age)

L 179: All treatments were repeated five times.

Please double check that this information is correct. The initiations were performed in Magent containers with 5 nodal explants per container and the multiplication phase was performed with only 1 explants per container.

L 207: totaling 144 treatments? Please double check that this information

L 220: It is necessary to describe in detail how the acclimatization was performed. For example, was the root pruned? Were the seedlings kept under saturated humidity conditions? etc.

L 240-245: It must be moved to the footer of the table - Values followed by lowercase letters denote statistical differences between NaOCl exposure time in the same sodium hypochlorite (NaOCl) concentration, and uppercase letters denote statistical differences between NaOCl concentration at the same exposure time. The values denote a mean (± SE; n=5) of the notes attributed by the Likert [45] scale. 

Figures 5 and 6: Information about the age of the plant must be added.

Author Response

In attached letter

Author Response File: Author Response.pdf