Polyploidy Induction of Wild Diploid Blueberry V. fuscatum
Round 1
Reviewer 1 Report
Comments and Suggestions for AuthorsThe topic of this paper is highly relevant for the introduction of wild blueberries into intensive cultivation and for expanding genetic diversity in agriculture. The research has been conducted thoroughly. However, the paper requires minor revisions before it can be considered for publication. You can find my comments and suggestions in the attached PDF.
Comments for author File: 
Comments.pdf
Author Response
Reviewer 1
The topic of this paper is highly relevant for the introduction of wild blueberries into intensive cultivation and for expanding genetic diversity in agriculture. The research has been conducted thoroughly. However, the paper requires minor revisions before it can be considered for publication. You can find my comments and suggestions in the attached PDF.
Thank you!
At line 177-178 “Once axillary shoots grew to approximately 20 mm long 177 in about a month, they were harvested and defoliated.” Why?
Explanation added at line 181-182 as below
Harvesting the new shoots growing in the lab minimized the contamination from the field.
At line 202 (https://www.bootstrapfarmer.com) . I can't access this link.
The weblink is activated at line 210
At line 232, is this your formula or did you copy it from someone else?
Reference [49] for the formula was added at line 232
At line 248 Three leaves per genotype were recorded. In how many repetitions?
At line 256-257 the following sentence was added:
“Each leaf was considered as a biological replication.”
At Line 321 to 332
This part belongs to material and methods. In this part, it seems as if there is both material and method, results and discussion. The suggestion is to somehow break down and explain the material and method in more detail so that this segment is not dragged into the results.
The following description in the results section was moved to materials and methods at line 201 to 205
“To ensure recovery of sufficient polyploidy induced materials, in a separate experiment, seventy-two nodal segments of ‘Rebel’ and ‘Emerald’ were treated with 500 µM colchicine whereas 200 nodal segments each were treated with 5000 µM colchicine. As for V. fusca-tum, seventy-nine nodal segments of were treated for both colchicine levels..”
At line 336-338 Where is presented this result?
This result is presented in table 1
At line 419 to 436, Please standardize the font and font size of this part of the text.
Font size was adjusted.
Reviewer 2 Report
Comments and Suggestions for AuthorsPlease make minor revisions, including enriching and expanding the discussion section.
Comments for author File: Comments.pdf
Author Response
Reviewer 2
General Comments:
This manuscript investigates colchicine-induced polyploidy in wild diploid V. fuscatum, evaluating induction efficiency relative to two southern highbush cultivars ('Emerald' and 'Rebel'), and characterizes resultant morphological alterations. The study addresses a critical bottleneck in blueberry breeding—ploidy barriers between wild diploid germplasm and cultivated tetraploids—offering significant potential for broadening the genetic diversity of commercial blueberries. The experimental design is methodologically sound, protocols are replicable, and results are generally well-supported by data. However, key aspects require clarification and expansion to strengthen scientific rigor and interpretative depth.
Thank you!
Major Strengths:
- The research focuses on V. fuscatum, a wild blueberry species with underutilized breeding potential, making the work innovative and relevant for blueberry genetic improvement.
- The use of colchicine treatment combined with flow cytometry for polyploidy identification is a reliable approach, and the detailed description of tissue culture and treatment procedures ensures reproducibility.
- The characterization of morphological traits (stem thickness, leaf size, stomata features) provides valuable insights into the phenotypic effects of polyploidization, which is important for understanding the potential agronomic value of the induced polyploids.
- The observation that V. fuscatum exhibits higher tolerance to colchicine and more efficient shoot induction after treatment is a notable finding, offering practical guidance for polyploidy induction in similar species.
Thank you for the summary.
Major Concerns:
- Representativeness of the V. fuscatum clone: The study uses a single clone ('FL 21-1423') of V. fuscatum, and the authors mention that "additional research will be needed to determine the representativeness of this clone for V. fuscatum". However, this limitation is not sufficiently discussed in the context of the study's conclusions. It is unclear whether the results can be generalized to other V. fuscatum genotypes.
An explanation was added at line 170 to 171
Due to the presence of a high level of genetic diversity in V. fuscatum [19], additional research will be needed to determine the representativeness of this clone for V. fuscatum.
- The observed significant reduction in leaf size in synthetic octoploid 'Rebel' (contrary to the typical gigas effect) warrants deeper analysis. Authors must explore whether this represents stochastic experimental variation, a genotype-specific response to polyploidization, or an artifact. Statistical re-evaluation (e.g., outlier assessment) or discussion of potential biological mechanisms (e.g, gene dosage effects, epigenetic reprogramming) is essential.
Revision was made at line 439-444
In our study, the opposite effect of polyploidy induction on the leaf width and length in ‘Rebel’ indicates genotype specific response to polyploidization. This finding conformed with the previous report on perennial giant grasses (Miscanthus) in which one out of five polyploidy induced genotypes had reduced plant size [68]. This negative response of ‘Re-bel’ could be caused by the retardation of growth development associated with chromo-somal doubling [65].
The proposed exceptional colchicine resilience in V. fuscatum presents a compelling opportunity to hypothesize on underlying mechanisms (e.g., differential expression of detoxification enzymes, altered tubulin binding affinity, enhanced DNA repair). The discussion should be expanded to propose testable mechanistic hypotheses, elevating the work's conceptual contribution.
Revision was made at line 370 to 377
Recently, colchicine cytotoxicity on mammalian cell division and toxin production was reported and a NEDD8-activating enzyme was shown to mitigate the toxicity effect of colchicine by maintaining cellular integrity [53]. The mechanism of detoxification against colchicine in V. fuscatum may involve differential expression of detoxification enzymes, altered tubulin binding affinity, and enhanced DNA repair. These potential underlining mechanisms should be further studied since they may provide an alternative therapeutic option for colchicine poisoning.
- Ploidy identification of 'Rebel' and: The table (Table 1) shows that for 'Rebel' and 'Emerald', the number of 2x shoots is marked as "N/A". It should be clarified whether this is because the original cultivars are tetraploid (4x) and 2x shoots are not expected, or due to other reasons, to avoid confusion.
N/A was changed to N/D. The following legend was added to the end of the table.
N/D: Not detected. These ploidy levels were not detected, and they were not expected from the testing genetic materials and
- Practical breeding: The conclusion mentions that synthetic tetraploid V. fuscatum is expected to produce viable gametes for interspecific hybridization, but there is no data on fertility (e.g., pollen viability, seed set) to support this, which limits the practical significance of the study.
Revision was made at line 486 to 489.
As a perennial bush, blueberries have one to two years of juvenile stage before flowering. We will continue to study the synthetic tetraploid V. fuscatum on flowering, fruiting, seed setting, and its crossing efficiency with highbush blueberries.
Minor Corrections
- P9, Figure 4 "The '500M' and '5000M' labels on the X-axis of Figure 4 should be positioned directly below the center of the three germplasm bar graphs to avoid misalignment."
Correction was made in the figure.
- P10, L340 "Fucatum" should be a spelling error.
Correction was made.
- P14, Table 2 Header: “Stomata/mm²” “Stomatal density (no./mm²)” is much clearer.
Correction was made.
Conclusion:
This manuscript presents valuable research on polyploidy induction in V. fuscatum, with potential applications in blueberry breeding. Addressing the above concerns and revising accordingly will significantly improve the manuscript's scientific rigor and impact. I recommend conditional acceptance, pending revisions.
Reviewer 3 Report
Comments and Suggestions for AuthorsGiven my experiments, I rate it as successful and have no serious comments. I have several notes and questions in the text of the contribution aimed at clarifying some of the professional statements.
Comments for author File: Comments.pdf
Author Response
Reviewer 3
Given my experiments, I rate it as successful and have no serious comments. I have several notes and questions in the text of the contribution aimed at clarifying some of the professional statements.
At Line 130: Was the use of another polyploidizing agent (e.g. oryzalin) not considered? and why?
Revision made at line 128 to 131 as follow:
Several antimitotic agents including trifluralin, amiprophos methyl, and oryzalin were used for polyploidy induction [30]. Among these, colchicine was the most widely used for multiple Vaccinium species, therefore, it was chosen to induce polyploidy in V. fuscatum.
At line 167: different font – size
Font size adjusted
At line 187: origin of colchicine ?
(Sigma Aldrich) added at line 193
At line 223: please explain the abbreviation RE for control, usually a representative of a given species with known ploidy is used
(rabbiteye, 2n=6x=72) was added at line 230.
At line 441: It's missing that it's a picture F
Added at line 467
At line 462: Is it expected or will an evaluation of fruits and yields, or possibly content substances, be carried out in the obtained polyploid individuals? Is it realistic for these individuals/genotypes to enter commercial fruit production?
Revision made at line 489 to 491 as below.
In addition, there is a potential fruit size increase in the synthetic V. fuscatum from the chromosomal doubling. Evaluating the fruit size, yield, and quality will further inform the breeding value of the synthetic V. fuscatum.
