Influence of Polyamines on Red Beet (Beta vulgaris L. ssp. vulgaris) Gynogenesis

Round 1

Reviewer 1 Report

Polyamine effects on gynogenesis have been little explored so far. In this study, Kiszczak's group reports the effects of two polyamines, putrescine and spermidine, on gynogenesis induction and regeneration in some red beet cultivars. The results reported in this manuscript are novel and could be helpful for further research on genetic improvement in this species and other crops. The manuscript is generally well-written and organized, and the study justification is clear. Data are correctly presented and well organized. Discussion may be improved. 

 

-Please, check the editorial rules for the manuscript's title. The binomial scientific name has to be written in italic. Same case to the abstract (line 14).

-Please, check the spacing between characters in the text. In some cases, it seems there is a double spacing, i.e. lines 135, 136, 150, 221, 303.

-Lines 19, 20. Use superscript for L-1, please.

-Line 60. Check to spell for similarities, please.

-In M&M section, please, indicate the company and country for all reagents used in this study.

-Lines 80-84. Please, indicate what time (days, months) the plants were maintained in the growth chamber. Were the two  POLAN's lines also cultivated under these conditions? I do suppose these plants were used as donors for the explants used for the in vitro assays. This information is not clear.

-Lines 96-97. Were the ovules in a liquid medium placed under agitation or stirring?

-Lines 100-101. Please, move the sentence ¨24 ovules…as replication¨ after the sentence …below (Table 1) (line 97). In this way, the message about gynogenesis induction efficiency is not broken. 

-Lines 105-109: Any references supporting the PGRs doses used in this study? 

-Line 127. Basal MS media?

-Line 152. Kaptan solution? Captan is a fungicide. Please add this information about this product used.

-Line 155. Check the symbol used for 18 C. 

-Lines 182-184. Check the font size for the section. It is different from the rest of the text.

-Table 10. Check to spell for isoenzymes. A suggestion: following the title format for the last column (number of plant homozygous/heterozygous to isoenzymes), maybe it is better to present the numbers with the following format: 11/69, 5/31, 16/100, 0, etc.

-Discussion. Add to the discussion, what about the combination Spd and PGRs on the gynogenesis induction efficiency, and compare and discuss the efficiency of Spd and Put on this same parameter. Any idea about Spd is more efficient than Put? Why did Put work better when combined with auxins and cytokinins than with only auxins?

 

 

 

Author Response

Dear Reviewer,

The response to Your revision is attached in the manuscript in the appendix below.

Thank You for Your contribution

Author Response File: Author Response.doc

Reviewer 2 Report

Review of manuscript "Influence of polyamines on red beet (Beta vulgaris L. ssp. vulgaris) gynogenesis" submitted for publication in Agronomy journal.

Overall, the manuscript is presented on a very relevant topic, as there are very few publications on the induction of doubled haploids in red beet. The use of PAs as an inducer of gynogenesis is extremely interesting, as these substances can immediately and stimulate the transition of the resulting haploids into diploids. The data presented are very interesting, but the manuscript needs considerable improvement.

Main remarks and recommendations:

L 51 - Amaranthaceae family must be written in non italics

L50 - Full Latin name of the red beet Beta vulgaris L. ssp. europaea Krass. var. atrorubra Krass.

The full Latin name of the sugar beet must also be given

L 62-63 "Barański [16] managed to obtain several red beet haploid plants ..." - was not the only work, in 2021 there were other publications, which reported the induction of gynogenesis and obtaining haploid red beet plants 

L 87 - "heterozygotic breeding lines" is not the correct term as "line" implies homogeneity, better replaced by breeding accession

L 91 – “Green, immature flower buds 3 mm long with undivided petals were collected" - it is highly doubtful that all three genotypes had the same bud size and it is not indicated within what range it fluctuated ±. It is well known that bud size will depend on the genotype of the donor plant, cultivation conditions, size of the parent root as well as position on the inflorescence (main, lateral) and timing of flowering (early flowering or late flowering). For Beta vulgaris L. the most frequently used indicator is the position of the bud on the spike inflorescence in relation to the flower that has opened.

L 93 - "disinfected with 70% ethanol for 10 min" - did the authors really use this sterilization regime? Is this not a misprint? Beet buds are quite sensitive to such a long exposure to 70% ethanol.

L 95 - "use of a stereoscopic microscope" - indicate the model of a stereomicroscope and the magnification used. 

L 166 - "incubated in ice for 2-3 hours in darkness" - check if the time is correct (usually less than 10 minutes).

L 168 - "CyFlow Ploidy Analyser (Partec Germany) - indicate model, city, country of manufacture

 

The experiment is extremely confusing. It begs the question, in what years were the studies carried out? If you look carefully at Fig.1A, it was 2008, wasn't it? Or is this data from several repeated experiments? Perhaps this would explain why such combinations of growth regulators were chosen.

 

It should be explained in the text, on what basis were the nutrient media chosen as a control? Also it is not clear why nutrient media with 2.4 D 0.1 mg L^-1 with a mineral base of B-5 were not used? 

 

The statistical validity of the effect of adding PAs to the induction nutrient media on the induction of gynogenesis should also be done.

 

Also, the text talks about induction of gynogenesis through embryoid formation, but judging from the photos (not very clear), the process was through callus formation? Are there any better photos of embryoids?

L 209, 217 - should write "significant difference at p ≤ 0.05"

 

L 221-222 - "The complete plants, 0.12 per embryo" - what does this value mean? Out of 100 induced embryoids only 12 plants were formed? How can this be? At the regeneration stage, propagation usually takes place, the beet is perfectly micropropagated and produces many additional shoots and 2, 3, 4.... plants should form from 1 embryo.  Table 4 is an extremely unfortunate representation of the data obtained. Please explain better. 

 

The data for section "3.2. Plant regeneration" is extremely poorly presented. Reduce the number of tables and try to present the data better. 

 

When evaluating the ploidy of the plants, it is desirable to represent how the histogram of the control sample looks like and to overlay the resulting peaks in the analyzed plants of the regenerants on it.

In table 9 the number of haploid plants formed in the 'Czerwona Kula' genotype is incorrect: 11 instead of 61.

In section "3.5. Homozygosity evaluation", an illustration of how homozygotes and heterozygotes looked in the analysis would be desirable. 

 

From the title of the manuscript, the authors should have analyzed which nutrient medium produced homozygous doubled haploids and which predominantly haploids. Did the addition of PAs affect the ploidy of the resulting plants?

Author Response

Dear Reviewer,

The corrected version and the responses to Your remarkas are attached in the appendix below. Thank You for Your contribution.

Author Response File: Author Response.doc

Round 2

Reviewer 2 Report

The authors have improved the manuscript "Influence of polyamines on red beet (Beta vulgaris L. ssp. vul-48 garis) gynogenesis", but it still requires major revision.

L 152  - is not the full Latin name for sugar beet

L 234 -235 - the authors' arguments do not look very convincing, the sterilization regime of 70% alcohol for 10 minutes is very damaging to the plant explants. It looks like a fixation of plant material. Alcohol causes tissue dehydration unlike sodium hypochlorite.

In section 2.3 the authors write: «Embryos of ‘Czerwona Kula’ obtained in gynogenesis process were transferred for plant regeneration on MS media [2326] with the addition of sucrose 30 g L-1, solidified with 6.5 g L-1 agar and pH adjusted to 5.6., applied by Zayachkovskaya et al. [18] for the regeneration of plants from the gynogenetic embryos of red beet» - However, this information is not correct because in their work Zayachkovskaya et al. [18] used a nutrient medium not with agar, but with Phytogel 3.5 g/l and pH 5.8. In addition, the sucrose concentration in the article was 2 g/L.

There is a big question about the correctness of the data presented, since they were conducted in different years. Were the 2008 experiments repeated later? Since a huge number of factors influence the process of gynogenesis induction, independent replications are necessary. The data for the different years of the studies should be put in an appendix.

 

The authors' answer is confusing:«BA and IAA were used in liquid media based on the preliminary research presented in a poster at the 56th Congress of  the PBS in Olsztyn. The information was included in the article.

Also it is not clear why nutrient media containing 2.4 D  0.1 mg L-1 and mineral base of B-5 were not used? - This medium proved to be less effective in relation to the media supplemented with BA and IAA media (results of the study in the poster described above)» .

This publication studied the process of androgenesis, not gynogenesis!!!(24. Górecka, K.; Kowalska, U.; Kiszczak, W.; Krzyżanowska, D.; Górecki, R.; Fornal, L. 1264 Induction of androgenetic embriogenesis in red beet. 56th Congress of the PBS in Ol-1265 sztyn, Interdisciplinary and application significance of botanical sciences, 2013, 24-30 1266 June, English poster summaries, K205).

 

L 476 –477 – «Put or Spd did not influence the number of obtained gynogenetic embryos.»  - The conclusions made by the authors must be statistically valid. Please provide two-way ANOVA (genotype and polyamine addition) data in the Appendix.

The authors' answer and the caption to the picture are confusing: «…Photo B and C was changed - growing androgenetic embryo in ovule cultures…». How could an androgenetic embryo develop into ovule cultures?

The data in Table 5 look extremely confusing. It turns out that microclonal multiplication did not occur at all (coefficient of regenerated plants per plated embryo in two genotypes is less than 1)?  How can this be? Beets are perfectly capable of micropropagation. Apparently, the authors did not count the plants formed correctly.

The answer of the authors is not satisfactory... «Unfortunately, obtained photographic material was lower quality, the bands on the gels were visible to the naked eye, but blurred in the photographs. Probably the available photographic equipment at that time had too low image resolution». The manuscript in this case should include a description of the results obtained and a description of the spectra obtained with molecular weight data.

In general, the manuscript should be substantially revised. It deals with a very relevant topic, but in view of the fact that the authors used data from experiments conducted more than 10 years ago, perhaps it makes sense to repeat these experiments and verify the data obtained? The statistical treatment of the data needs to be improved. The manuscript is called "Influence of polyamines on red beet (Beta vulgaris L. ssp. vul-48 garis) gynogenesis", so the authors need to statistically prove the effect of adding polyamines to the nutrient medium on the induction and regeneration of red beet gynogenesis.

Author Response

The response to the remarks is attached in the appendix below.

Author Response File: Author Response.doc

Round 3

Reviewer 2 Report

The authors corrected typos discovered by the reviewer. Nevertheless, the authors failed to provide statistically valid data and constructive responses to the reviewer's comments.

For example:

Main remark: There is a big question about the correctness of the data presented, since they were conducted in different years. Were the 2008 experiments repeated later? Since a huge number of factors influence the process of gynogenesis induction, independent replications are necessary. The data for the different years of the studies should be put in an appendix.

Manuscript authors' reply: «The overriding goal of the research was to obtain homozygous plants (dihaploids) and transfer them to breeders. This task is difficult, laborious and expensive. Due to the fact that dihaploids were obtained, in this type of applied research, repeating the same experiments is not justified. Nevertheless, in these studies, a clearly beneficial effect of polyamines on the generation of red beet gynogenetic plants was revealed.

The repetition of this studies is the second experiment, where embryos were plated and in this case shoots (without roots) were obtained (shorter shoots < 1 cm and shoots > 1 cm).»

- As a reviewer, I understand how difficult and time-consuming it is to obtain doubled haploids in red beets, yet for a scientific article in a Q1- journal, this response seems unsatisfying.

In addition, I would like to point out that it is not correct to use the term "dihaploids" for red beets (this is a diploid crop), you should use the term "doubled haploids"

 The manuscript is marked as an "article," it would make sense to change the category to "short communication," in case the editor approves it.

Author Response

Dear reviewers

Thank You for your valuable comments.

Answer is attachment.

Best Regards

Author Response File: Author Response.docx