Early Withering of Enlarged Ovules in Pollinated Fruits of Bananas (Musa spp.) Suggest Abortion after Fertilization

Round 1

Reviewer 1 Report

For authors:

I have found your work „Early withering of enlarged ovules in pollinated fruits of bananas (Musa spp.) suggest abortion after fertilizationˮ very interesting. You have done extensive work of importance in my opinion and I think you’re obtained results and conclusions could interest many researchers and readers. You presented an innovative approach and there are fine well-documented observations, but I think that you should take a count a little modification of this article. I recommend publishing it in "Horticulturae" after correcting listed below suggestions:

Line 7-8: Delete initial A.W. and S.B.M., no need to add them here.

Abstract:

This part of the article is not exactly written according to the instruction for Authors of "Horticulturae", there are no Methods, and it should be included according rules:

https://www.mdpi.com/journal/horticulturae/instructions.

Introduction:

Line 34-35: Musa acuminata and M. balbisiana - state either the full name or abbreviated for both species.

Line 35: designated as AA and BB respectively - please indicate that this refers to genomic constitution.

Line 108-109: Explain why you are 5 min. rinsed in a solution of aniline blue, and then kept in the same solution for 2h? Modify this part.

Results

I suggest deleting Figure 1, it is totally unclear, as you stated in the text.

Line 190: Delete - days after pollination, no needed, only DAP.

Line 221-222: Delete meaning of abbreviation for DAA, Control and +PGM you already mentioned that in Material and Methods.

I hope my comments will be helpful.

With best regards,

Author Response

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Author Response File: Author Response.docx

Reviewer 2 Report

All my recommendations were inserted in the manuscript

Comments for author File: Comments.pdf

Author Response

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Reviewer 3 Report

The subject of the article and the questions posed at the end of the introduction are relevant for breeding purpose.

The overall quality of the paper should however be improved. Some data (from littérature and also from study results) have to be checked ; in general the authors have to improve data presentation and data interpretation to make the outcomes of their study more convincing. The authors have also to take care not to « over-interpret » their results  (see detailed comments for strained interpretation).

Detailed comments

1. Introduction

• 39-40: reference [4] is not available online, and natural banana tetraploid varieties are extremely rare ; so do they observed pollination involving natural or « human-bred » 4x ?
• 43-44: are you talking about 3x hybrid only ? this must be stated clearly because some banana prebreeding programs are also producing quite fertile 2x or 4x hybrids (or at least with a sufficient residual fertility)
• 49-51: please ckeck the number of seeds per bunch for references 9 and 12 ; the data in your paper seem very high (227 and 219) compared to data I found in the paper or in the abstract 26.4 and 21.7 respectively). Moreover, in this part you focused on maximum number of seeds that can be obtained, but the mean number or the observed range of seed/bunch is more important for banana breeding purpose. Moreover, you didn’t talk about the « quality » of the obtained seeds i.e. the nb of seeds with embryo and/or the % of germinated seeds.

2. Materials and Methods

• 139-140: the sentence « Only ‘Calcutta 4’ ovules remained fresh and continued to develop whereas in 139 EAHBs, large and small ovules had withered by 14 days after pollination. » seems to be part of results rather than Materials and methods.
• 147-150: same thing for the sentences « For ‘Calcutta 4’, 1 DAA had the highest numerical fertilization rates in the two hand pollination technique. The two hand pollination techniques were therefore compared with open pollination in the ANOVA. », could be placed in the Results part, as a preamble.
• 157: it could be interessant not to use only temperature at 15:00h, but also at the time of pollination

3. Results

• 1 is not useful at all as it is. If the authors want to keep it, they need to improve it by adjusting the brightness or contrast (if possible), and/or by adding some details (arrow to show focus point/cell details,…) to help readers
• Table 1: if possible, put all the lines of the table on the same page. Some parts/sections of this table are not really useful (or at least used by the authors) and could thus be removed to reduce the table’s size.
• Table 2: In my opinion, it would make more sense to move this table above table 2, to start from general data to move on to the more detailed. This table could perhaps be replace by a bar graph/histogramm for ease of reading.
• 182-183: this result is not well « tapped » in the discussion, whereas it is cited in conclusion
• 200-201: « there was a bias of seed 200 development at the distal end of Matooke hybrid ‘222K-1’ fruits » ; this affirmation is not clear for me (not enough seeds in the fruit), what results in a strained interpretation. Same observation for lines 271-274.
• 209 « Differences in ovule fertilization 208 rates in the different hands were detected in ‘Enzirabahima » ; l.212 « ‘Nakitembe’ had a significant difference in 212 ovule fertilization rates after pollination on different DAA » : due to the small number of observed seeds (and so the small sample size for statistical analyses), the authors have to remain cautious in their interpretation of these results
• 225-226 « The highest average ovule fertilization rate in ‘Calcutta 4’ for controlled pollination 225 on different DAA was on 1 DAA » ; results from day -1 to day 1 are quite similar for me
• 228-229: data from open-pollination are not presented, and the authors don’t explain how they obtain them ; data from control pollination (43.3%) are not consistent with data in table 1
• 244-246 (and 323-324, 344-345): temperature data should be added to convincingly presentate the (lack of) effect of temperature. With no data on the range of temperature during this period, it’s difficult to conclude on this point ;  it could be argued that you don’t have sufficient contrasting temperatures
• 298-299 : i did not see data on ovule number for distal vs proximal hands ? or reference is missing ?
• 308-309 : it’s for me a strained interpretation of the results ; there are a wide variety of factors which differ from open-pollination to control pollination (pollinators efficiency, time and length of pollination period, …)

Author Response

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Reviewer 4 Report

Dear authors, while I think your submission could be relevant to the crop, and some of your findings are of interest, I’m afraid the submission is a compilation of observations carried out on different plant material, and not the result of a well-planned experimentation with design and procedures carefully followed. I suggest reject but resubmission not including all observations but selecting those experiments result of more careful designs.

I am attaching the pdf with comments and corrections, but I also detail some more important questions below. Since the procedures followed gave me no confidence I stopped somewhere in the sections of Results. So please, apply to the entire manuscript the suggestions made.

INTRODUCTION

The second part of the Introduction needs clear improvement. The presentation of pollination and fertilisation processes contains several mistakes and the writing is imprecise. I detail some examples.

L 49. Define bunch for readers non-familiar with bananas. You switched from flower to bunch and the meaning and proportions are not clear.

L57-60 On the other hand, seed set bias towards the distal end of the fruit was thought to be a 57 mechanism affected by changes in the environment [11]. But bias of seed in the mid-section of the bunch and towards the distal end of the fruit may suggest that pollen tubes do not reach the ovules.

Omit these sentences. They do not add anything clear, and the last sentence is confused and probably wrong: if polen tubes do not reach ovules, then no seed can be formed. So, better remove this sentence too.

L61. Rephrase again. This sentence is unclear.

L65. whether pollen tubes reach all parts of the ovaries of different hands …

The ovary has no different parts, unless you referred to locules (that it seems not the case). You probably mean different sections as in crucifers and fabaceae, families with long ovaries.

L66 and the position of ovule development in ovaries. Meaning again unclear. Do you mean the pollen tubes do not reach basla ovules, or on the contrary, that some ovules do nor fully develop. Rephrase.

Material and methods

M&M redaction needs a clear improvement too, especially the pollination experiments.

Not clear to me if a liquid media was added free of pollen to wet the stigmas, with pollen grains included in the media. “on pollen on stigmas to enhance receptivity” Not clear at all. Besides liquid media may prolong, but no enhance, stigma receptivity. If pollination is performed in the right moment no need to prolong stigma receptivity in this kind of experiments (different situation would be in the field when short SR compromises fruit set as in many fruit crops). In Line 130 you wrote “with pollen, not on pollen, and again not for enhancing nor prolonging SR (since Pollen grains are deposited) but probably for facilitating pollen-stigma interaction.

Sampling flowers just 48 h after hand pollination based on average PTG rate is risky and probably wrong. I do not understand why not sampling for instance 4 days after HPollination, to be sure that PT had plenty of time to reach ovules in all positions.

First time I heard of peeling ovaries. We are looking for what happened within the ovary, the peel causes no conflict processing the samples. I might understand taking longitudinal sections of the ovaries if the are too thick and you do not see other mean to process the flower, but peeling…

I have not worked on bananas but 10 M NaOH for 2 weeks is extraordinary long and high (0.8 M Na OH for a few hours is common in many flowers). The removal of the fixative with only 10 minutes of washing is clearly inadequate.

Section 2.3 I do not know of any ovule of Angiosperms surviving 20 days after anthesis (well, maybe hazelnut), but I think you are observing seed development (or, on the contrary, senescent ovules). If such a survival could be possible, (that is not), for sure the pollen tubes at that time are useless since the degenerated synergids cannot permit fertilization of, for sure, a dying egg cell. Please, modify.

The assessment of Effective Pollination Period (Williams 1970) implies frequent, consecutive pollination in successive days and determination of the corresponding seed and fruit set and/or the determination of SR, PTG and ovule longevity. Any of these parameters were determined.

L134-136 Ovules that distinctly increased in size were presumed to have been fertilized and those that remained small were presumed to be unfertilized. You must understand that ovules develop until anthesis and often beyond, and their development always implies growth. So an enlarged ovule does not confirm fertilisation. Certainly if the increase in ovule size is great, then you may assume fertilization took place and you have growing seed. Said that, you need to probe that the increase in size is the result of fertilization.

Results.

Florescence microscopy Figure 1 is poor. Nothing can be distinguished as you recognised in the first lines of this section. On the contrary, figure 2 is nice and suggest you really found out something. I suggest repeating some experiments more carefully with selected material.

Pollen tubes reaching the ovules do not mean fertilization taking place subsequently. As hypothesized in your paper, it could be impossible due to ovule sterility or due to ovule senescence (that can be assessed by fluorescence microscopy).

Discussion.

You mentioned parallel studies carried out by you and your colleagues that seem to probes some of the hypotheses here in study. It seems to be that you have split the results in two papers. While based on images of fluorescence microscopy that are not shown (or are poor) reader cannot be confident on your statements. This apply to your main conclusion that is that pollen tubes reached all sections of the ovary and then fertilization is presumed. Seedlessness in grape do not necessary apply to banana unless you demostrate it.

Comments for author File: Comments.pdf

Author Response

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Round 2

Reviewer 2 Report

The authors performed all adjustments and the manuscript has improved a lot.

Author Response

Dear Reviewer,

Thank you for your valuable comments.

Reviewer 3 Report

Most of the comments were taken into account, and modifications respond to suggestions. The overall quality of the paper (including figures and tables) have thus been improved.

However, lots of the comments on the  over-interpretation of results have been ignored by the author (l. 194-197 ; l.261-262 ; l.269 "there was the effect of season" should be replaced "could be the effect of season"), as well as a comment on data l.216-218.

I also found some minor errors that have to be corrected :

• Reference [9] is not cited in the text, and ref. [27] is not listed in the references
• l.260 : "uNpollinated fruits"

Author Response

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Author Response File: Author Response.docx