Review Reports - High-Temperature Induction of 2n Female Gametes to Produce Triploid Birches: Timing, Parameters, and Growth Outcomes

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

Dear Authors,

This manuscript presents a practical protocol for producing triploid birch via short high-temperature treatments of female inflorescences—an approach with clear breeding value. Before the work can be fully assessed, please address the following essentials:

Major comments (substantive revisions)

1. Define the induction-rate denominator + report Ns per cell.

Table 3 shows “induction rate (%)” but the denominator isn’t specified. Is it (triploid seedlings / total seedlings analyzed) or (triploid seedlings / seeds harvested), or per female inflorescence? Similarly, Appendix Table A1 shows germination rates strongly affected by heat. If induction is calculated only among surviving seedlings, the rate may be inflated; if by total seeds, it may be deflated. Please (i) add sample sizes per (day × temperature × duration × clone), (ii) unambiguously define the denominator in Methods, and (iii) provide binomial 95% CIs for each rate.

2. Statistical framework and consistency.

Methods state ANOVA + Tukey HSD (p < 0.05), Table 2 note uses Duncan’s test, and Figure 3 captions use paired t-tests. Please standardize: (a) Use one multiple-comparison method (Tukey HSD is fine); (b) For proportional outcomes (induction, germination), fit a GLM/GLMM (logit) with fixed effects (day, temperature, duration) and random effects (clone, inflorescence) to account for hierarchical structure and avoid pseudoreplication; (c) For growth traits, treat plant as the unit, include clone/family as random effects, and avoid paired tests unless true pairing exists.

3. Calibration and validation of the heating treatment.

The “Tree Non-In Vitro Branch Bud Heating Treatment Device” is cited, but no evidence is provided that ovules reached the target temperature. Please report: (i) thermocouple measurements inside catkins (target vs ambient; time to equilibrium), (ii) spatial variability across positions, and (iii) day-to-day environmental conditions (humidity, greenhouse temp). Without these, reproducibility is limited.

4. Morpho–developmental timing: generalize beyond “day 15”.

You nicely correlate ovary L/T ratio and embryo sac stages. Consider modeling development vs thermal time (growing degree hours/days) to accommodate inter-year/clone variation and make the protocol portable. Provide a field key (e.g., L/T ratio threshold + bract angle + seed-wing onset) to trigger treatment timing.

5. Terminology and cytology clarity.

Use standard terms: megasporocyte (MMC), megaspores, megasporangium; replace “macrospore.” Ensure consistency in figure labels (MMC, functional megaspore, 1-,2-,4-,8-nucleate embryo sac, etc.). Add scale bars to all micrographs and detail staining protocols (hematoxylin concentration, fixation times).

6. Flow cytometry reporting standards.

Specify: internal standard (diploid Betula), nuclei extraction buffer composition, CV% of peaks, gating strategy, events counted, and replicate runs per sample. Include representative histograms with axes labeled and peak CVs.

7. Chromosome counts: clarify pipeline.

The root-tip squash pipeline routes through leaf-derived callus/regeneration; justify why, or simply collect root tips from seedlings directly. Specify number of cells counted per plant and present at least one metaphase spread image with scale.

8. Germination protocol volume likely misstated.

Methods list 80 mL water in a 9-cm petri dish—this seems physically implausible (flooding). Likely 8 mL. Please correct and report substrate hydration checks.

9. Growth comparison: effect sizes + visualization.

Figure 3: report Cohen’s d (or Hedges’ g), show individual points (jitter + mean ± SEM), and confirm equal variance or use Welch’s test. Clarify whether n=19 triploids comes from multiple families and whether diploid controls are matched by family.

10. Potential confounding by embryo lethality.

High temperature reduces germination; embryo abortion may be non-random across treatments. Discuss whether triploids might have different survival probabilities than diploids under heat, and state how you accounted for post-treatment selection in estimating induction efficiency.

11. Novelty claim and context.

The Abstract/Introduction suggests this approach “has not been proven in birch.” Please temper or support this with either (i) specific birch citations (if any) or (ii) rephrase to “systematically defined optimum timing and conditions in birch.” Also, quantify the breeding-cycle shortening (e.g., “reduces X years compared with generating tetraploids and waiting for reproductive maturity”).

12. Data availability.

“Data available in the article” is insufficient for reproducibility. Please deposit raw ploidy files (FCM), staging images, per-inflorescence treatment logs, growth raw data in a public repository (Zenodo/OSF) and provide a DOI.

Minor comments (clarity, style, presentation)

Title: Consider “High-temperature induction of 2n female gametes to produce triploid birches: timing, parameters, and growth outcomes” (crisper, avoids “optimizing” unless you run formal optimization).
Abstract: Add Ns (seeds/seedlings analyzed), clearly define induction rate denominator, and report germination impacts alongside induction. Shorten last sentence and quantify “significantly shortened.”
Species names: At least once the text says “Betula purpurea” when the study species is B. pendula—correct this.
Inconsistent tests: Methods (Tukey), Table 2 note (Duncan), Figure 3 (paired t-test). Unify.
Units & symbols: Use non-breaking spaces between numbers and units (e.g., 42 °C, 5–8 µm). Replace “10×40 magnification” with “400×”.
Table 1: Clarify that “Angles between the bracts and the female flowers” refers to bract–rachis angle; revise “Less than30” → “< 30”.
Table 2: Heading “Ratio of longitudinal and transverse in diameter” → “Longitudinal:transverse diameter ratio.” Ensure letters for post-hoc groups match the stated test.
Table 3: Use a consistent missing-data symbol (“—”), and include N for each cell. Consider a heatmap visualization of induction rate vs (day × condition).
Figure 1: Add scale bars to all panels, ensure labels (F–U) match text, and annotate key structures (MMC, tetrad, functional megaspore).
Figure 2: Label axes (relative fluorescence), indicate internal standard peak, and list CVs.
Figure 3: Add units to axes (cm, mm²); clarify time since transplant; note that leaf area n=15 is leaves, not plants—avoid pseudoreplication (analyze as plant means or mixed model with plant random effect).
English polishing: A few phrases to fix—e.g., “burst through the bud scales occurred” → “female inflorescences emerged from bud scales,” “sinergies” → “synergies,” “Macrospore” → “Megaspore,” “Benzidine blue–hydrogen peroxide” → “benzidine–hydrogen peroxide.”
Safety note: Benzidine is carcinogenic; consider citing and/or switching to a safer peroxidase assay (e.g., guaiacol- or DAB-based) and mention lab safety measures.

Suggested additions to strengthen the paper

Power and precision: Provide a simple power analysis (post hoc is fine) for induction-rate comparisons to justify sample sizes.
Optimization surface: Fit a response-surface or logistic regression model (induction ~ day + temperature + duration + interactions) to formally justify “optimum,” and include contour plots.
Generality across clones/species: You used two maternal clones for treatment; present clone-specific results and discuss transferability to other Betula species.
Phenology key: Offer a concise practical key for field timing (e.g., “bract angle > 60°, L/T ≈ 0.8–0.9, seed wings faintly visible → treat now”).
Breeding impact: Add a paragraph estimating time saved vs tetraploid routes (with cited averages for time to first flowering).

Recommendation

Major revision. The core result is valuable and publishable after clarifying experimental design and statistics, standardizing terminology and reporting, and improving figures/data availability.

Author Response

We are grateful for your valuable comments and suggestions on our manuscript. We have revised the manuscript accordingly and provide a point-by-point response to all the comments, please see the attachment.

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

Review of

Optimizing chromosome doubling in female gametes using 2 high temperature for production of triploid birches

Jingnan Li, Yijie Hu, Shaoqing Duan, Jie Feng, Ying Cui, Guifeng Liu, Jing Jiang, and Huiyu Li

General Comment:

It is refreshing to read a paper involving tree improvement using cytogenetic manipulations. Research in this potentially fruitful area has been largely ignored in recent years, aside from studies involving Populus. This paper is well-written with appropriate methodology and conclusions. I have made only minor editorial suggestions.

Specific Comments:

Line 20 – triplogenesis is not an accepted term. Suggest rewording.
Line 46 – a reference is needed after “…forest industry.”
Line 51 - references to the authors’ previous studies are needed.
Line 124 – change “Pollens were…” to Pollen was….
Line 126 – change pollens to pollen
Line 381 – author needed for the scientific name.
Line 384 – author needed for the scientific name.
Line 392 - author needed for the scientific name.
Line 426 – germplasms should be changed to germplasm.
Line 444 – Extensive research…. I don’t think that two studies on different species warrant extrapolation to suggest this is true in all species. Suggest that the authors mention the specific species instead of making a blanket statement about all species.

Author Response

Author Response File: Author Response.pdf

Reviewer 3 Report

Comments and Suggestions for Authors

Dear Authors,

The manuscript is well-organized and presents a comprehensive and methodologically sound study. The objectives are clearly defined, and the results are effectively supported by well-prepared figures and tables. The discussion demonstrates a clear understanding of the topic and provides a strong connection with relevant literature.

Some sections could be made more concise, particularly by shortening long sentences to improve readability. Minor technical and formatting corrections are also recommended. Overall, this work represents a valuable and meaningful contribution to the field of triploid birch breeding.