In Vitro Evaluation of the Effects of BAP Concentration and Pre-Cooling Treatments on Morphological, Physiological, and Biochemical Traits of Different Olive (Olea euorpea L.) Cultivars

Round 1

Reviewer 1 Report (New Reviewer)

To my opinion, the work lacks some originality.

Methods are not always described exhaustively, and statistical evaluations are not reported clearly. 

Results (including tables and figures) are not always presented clearly, and conclusions are not of great impact. 

Here I reported my comments.

 

Introduction

Line 38: Fully describe the purposes of in vitro culture of olive, and the reasons of low success rate for the traditional propagation methods.

Line 44: Among the issues for a successful establishment of in vitro protocol, please mention “culture medium composition”.

Line 55-58: the word “growth” is reported too many times

Line 57: as the paper deals with growth regulators, to my opinion it would be better if author insert a dedicated paragraph in the “Introduction” thoroughly describing the effects of the various plant growth regulators more commonly used in tissue culture in general, and more specifically in olive.

Line 59-64: The sentences express very similar concepts in a redundant way Please, simplify and re-write a more concise phrase.

Line 64-66: Please reconsider the concept expressed in this sentence, and supply more bibliographic references.

Line 79: Please, more deeply describe the opposite actions of ABA and vernaline molecules in activating/releasing bud dormancy

Line 71: “Comparatively” respect to what? Please, better complete the sentence.

Line 78: Please, re-write as follows: “In this context, pre-cooling treatment before shoot induction during in vitro tissue culture…”.

Line 79: Add some more bibliographic references, and shortly describe the mode of action of vernalin in contrasting the effect of ABA. Did the authors try to measure variation in ABA and Vernalin concentration during each pre-cooling treatment (respect to time 0)?

Line 82-83: Please, better explain what kind of technical limitations arise from the high phenolic content in woody plants. List (with more abundant references) and describe what kind of pre-culturing treatments have demonstrated successful in facing the practical constraints of woody explants shooting.

A huge number of protocols for in vitro regeneration of polyphenols-rich explants (especially in grape) report the addition of activated charcoal to the growth medium, to adsorb phenol-derived oxidated compounds. Why did the authors not add such a component to the shooting substrate? Please, add your comments on this, also referring to some protocols including the recent one by Giancaspro et al. 2022.

Line 85: To my opinion, it would be interesting to read some examples of proper durations of pre-cooling treatments in different woody plant species other than olive (with the relative bibliographic reference).

 

Materials and Methods

Line 96: Please, re-write “shoot induction and proliferation”.

Line 110: Specify why Rugini Olive Medium was used for tissue culture.

Line 110-113: Paragraph 2.2 should contain details on the preparation of BAP solution at the different concentrations used for the experiments. Authors wrote that OM medium was prepared, supplemented with different BAP concentrations, and then autoclaved.  Being a thermolabile compound, authors should specify that BAP solution was previously filter-sterilized (which pore diameter?) and added to liquid OM medium following autoclaving and cooling at 50° C (which temperature was used?).

Line 115: Pre-cooling treatment was conducted in a chiller at 4 to 8 °C. Please, in “Results” or “Discussion” supply more details or discussion about the choice of chilling temperature (a little range is allowed, rather than a fixed temperature), also referring to literature.

Line 115-116: Were 3 to 4 nodal segments cultured in each glass bottle? Clarify how many explants were used for each treatment, and how many explants were cultured for each olive cultivar in the whole experiment.

Line 119: Paragraph 2.2 should better detail the protocol followed for in vitro regeneration of olive plantlets, so, please, address the following points:

·         Report the composition of OM, also indicating the pH value.

·     Clarify if shoots were cultured on the same medium from induction to plantlet development, or there was any modification of substrate composition?  

    Were plantlets at 50 and 60 days kept in the growth chamber or were they acclimated? Was any sub-culturing practiced to restore nutrients in the growth medium?

Line 123: Was sub-culturing onto fresh growth medium systematically applied to growing shoots? or only to browning explants?

Line 126-127: Please, better detail how growth-related parameters were calculated:

·         how many nodal explants were sampled for each olive cultivar and subjected to each treatment?

·         as multiple shoots could originate from a single explant, was PIS (Percentage of Induced Shoots) calculated as the percentage of nodal segments (on the total segments cultured for each cultivar, in each treatment) showing some (one or more) shoot formation?

·         Specify if LPS (Length of Primary Shoot) was averaged on the total number of primary shoots originated from all the nodal explants cultured for each cultivar and in each treatment.

·         Specify if NLPS (Number of Leaves per Shoot) was calculated only on the primary shoot or also on the secondary shoots of each single nodal explant (and then averaged on the total number of explants cultured for each cultivar, in each treatment)

 

Line 127-128: Please, re-write correctly: “number of leaves per shoot (NLPS)” and “number of shoots per explant”. (NSPE).

Line 127: Please, describe how the primary shoot of each nodal explant was identified.

Line 130: Replace “chl” with “chlorophyll (chl)”

Line 140: Specify that metabolites were quantified in the supernatant.

Line 141: Specific in detail the protocol used for starch extraction and quantification, also supplying bibliographic references

Line 153: In paragraph 2.6 please detail the meaning of tri-factorial and tri-replicate arrangement of the experimental design (as reported at Line 97). Were the three nodal segments the biological replicates for each cultivar in each treatment? How many technical replicates were conducted for each parameter (growth-related traits, primary and secondary metabolites) measurement?

 

 Results

Line 165, 172: The authors should clarify what they mean by “statistically distinct”. Maybe it would be better to write “statistically different” or “statistically significant”.

Line 166-168: Authors reported that cv. Arbosana recorded the maximum improvement in all the growth-related traits with increasing regimes of pre-cooling and BAP concentration, as indicated in Table 1. To my opinion, it is not possible to derive such a consideration from Table 1, as what emerges from the Table is just that V4 revealed the highest mean values for the growth traits, respect to the other 3 cultivars (as reported in Lines 169-173). The Table fails at showing the increasing trend of LPS, PIS, NLPS and NSPE for each cultivar with increasing values of PCT and BAP.  If necessary, refer to Tables 5 and 6. Please check the same also in Tables 2, 3, and 4.

Line 171: replace “SIP” with “PIS”

Line 194: Table 1: To my opinion, to be better understandable by readers, the order of the evaluated growth parameters in columns should be the following:  PIS, NSPE, LPS, NLPS. Please, modify accordingly also in Table 5 and 6.

Table 1: Please, better explain what the average numbers reported in columns of Tables 1-4 refer to:

·         Are the numbers in the PCT field the averages of all the 4 genotypes at all the 4 BAP concentrations?

·     Are the numbers in the V field the averages of all the 3 pre-cooling treatments at all the 4 BAP concentrations?

·         Are the numbers in the BAP field the averages among all the 3 pre-cooling treatments on all the 4 genotypes?

Uniform all the values should show 1 or 2 decimals (in some cases there are three decimals; at PIS-T3 I cannot understand the numbers.

Check the number of decimals in LSD, too.

In the title of Table 1, Table 2, Table 3 and Table 4: replace “olive” with “olive (Olea euorpea L.)”.

In the food notes of Table 1, Table 2, Table 3 and Table 4: replace “treatment” with “variable”

In the title of Table 3 and Table 4, please replace “metabolic” with “metabolites”

Line 204-205: How the “increase” can be deduced from?

Line 205: in all physiological traits…Add “except for carotenoid content”…

Line 208-209, Line 210-211: please, better explain where these considerations come from. At line 211 the value in brackets (relative to carotenoid content of V4) is not reported in Table 2.

Line 259: Data reported in Table 3 depict that the four sugars showed a different behaviour in response to varying PCT, cultivars, or BAP concentration; Sucrose was the most variable metabolite, followed by glucose, fructose, and starch. This aspect should be reported and better commented in the manuscript. Could the authors give any explanation for this?

Line 260: please, better clarify the meaning of “statistically distinct”.

Line 262: Please replace “rise” with “mean value”; .…in all primary metabolites…add “except for glucose”

Line 263-267: Better re-write this paragraph. I found it a bit redundant with some repetitive information. Authors could simply report the cultivar showing the lowest value for each trait (number in brackets).

Table 3: please uniform the number of decimals.

Table 5: Do the mean values reported in each column - for each BAP concentration - refer to the average among the 4 olive genotypes? Table 6: Do the mean values reported in each column - for each BAP concentration - refer to the average among the 3 pre-cooling treatments?  Please, the authors should detail this in Material and Methods, or in the Table Title. Why did the authors not provide a similar Table reporting the interaction effects of varieties and Pre-cooling treatments?

Line 352: Table 1 or Table 7?

Line 370: Rectify the number of the Table (n. 7, not n. 1). The meaning of *** should be reported in the footnotes. Anyway, the Table looks not very informative as the reported paired correlations show the same significance level. Maybe the Table could be removed and replaced by a comment in the text.

 

Conclusions

Line 403-404: Authors reported that the use of zeatin at high concentration (which one? ) usually give better results in shooting induction. Please, better clarify if the use of a combined BAP/pre-cooling treatment can reach the same shooting percentage of zeatin. If yes, why BAP should be used as an alternative growth regulator to zeatin, in the OM?

Line 418-427: Comment made in this paragraph are in contrast with data reported in Table 2 (there is an effective decrease in proline with increasing pre-cooling treatment and BAP concentration). Please, reconsider this paragraph and clarify how can this be explained?

Line 439: Please, add some bibliography. It would be better to report the current concentration (or range) of zeatin and BAP usually added to OM and compare their efficiency with the shooting percentages observed in the present work.

 

Figures

In Figures 1, 2 and 3 please comment the different contribute of each dimension to variability. Better comment and explain the negative and positive correlations observed among the different variables. If possible, optimize the images as the names of parameters are almost completely overlapped.

 

 

Author Response

 

Author Response File: Author Response.docx

Reviewer 2 Report (New Reviewer)

Authors evaluated effect of in vitro techniques on olive plants under different treatments.

Title: what means naturally enriched? You sprayed potato plants with solutions of mineral fertilizers.

ABSTRACT:

Line 23: it is not common to put which statistical program you use in study, remove. You can add some other  methods data.

Line 25: simplify, remove however and starat new senetence with This increase

 

INTRODUCTION:

It is well writen providing sufficient information about topic, but please divide text  in some subparagraphs (of course without numbering) so that it is easier to read.

MATERIALS:

Line 102: …of one year old olive plants…. not cultivars if I properly understand, or you were thinking on one year old shoots taken from trees of different cultivars.

Line 109: not beside, maybe parallel with ….

Line 130: not chl, put full name and abbreviation and later put only abrv.

For 2.4, and  2.5, please include how you took leaf samples for analyses, what leaves, how many

                                                                                                                                                                           RESULTS:

Table 1. in table title you have genotypes and in table varieties, please unify terms trough the manuscript. You also put PCT in table but you do not have abbreviation in title, please check all manuscript for this kind of mistakes.

Table 5. is corrrect (T×L) in title, not (V×L). Why you did not put significance letters in table?

 

DISCUSSION

Line 440: current study showed… not recorded. Please check manuscript with English speaker

 

Line 449: different genetic architect !?, maybe architecture, it is not scientific, please check mansucript, and use proper terms.

Author Response

 

Author Response File: Author Response.docx

Reviewer 3 Report (New Reviewer)

Suggestions

Page 1, line 4: …traits of four olive (Olea europaea L.) cultivars

Page 1, line 14: … (Olea europaea L.)

Page 1, line 15: oxidation of its tissues.

Page 1, line 20 : leaves per shoot

Page 1, line 36: … (Olea europaea L.)

Page 1, line 41 : one of the best

Page 1, line 42: Delete ‘round the season’

Page 1, line 43: Replace ‘owe’ with ‘due’

Page 2, line 49: Walnut Medium

Page 2, line 52: found that

Page 2, line 68: apart from this, the choice of explant type for olive

Page 2, line 75: primordia

Page 2, line 80: Replace ‘due’ with ‘thanks’

Page 2, line 81: Although high phenolic contents content is an obstacle

Page 2, line 82: woody plants, their effects

Page 2, line 84: rid of

Page 2, line 84: Delete ‘properly’

Page 2, line 89: Delete ‘different’

Page 2, line 91: BAP concentrations.

Page 2, line 95: Which were the criteria to choose these olive cultivars?

Page 2, line 95: In the current study, four olive cultivars (‘Leccino’ (Italy), …(Spain))were evaluated…

Page 3, line 98: …(RCBD) at Jilin Agricultural University, China.

Page 3, line 102: Replace ‘branches’ with ‘stems’

Page 3, line 116: Delete ‘under laminar flow chamber’

Page 3, line 127: leaves per shoot

Page 3, line 131: proline

Page 4, line 146: with rutin used as standard.

Page 4, line 176: related to traits

Page 4, line 180: and the three-way interaction

Page 5, line 208: of A_CO2 at

Page 6, line 210: Moreover, under analogous

Page 7, line 267: Replace ‘contents’ with ‘concentrations’

Page 7, line 272: Replace ‘however’ with ‘while’

Page 12, line 265-267: The meaning here is not clear. Rewrite better these 3 lines

Page 16, line 400: As regards BAP, the present study

Page 17, line 425: micropropagation

Page 17, line 455: cooling conditions

Page 19, ref. 13: Chatzissavvidis

Author Response

 

Author Response File: Author Response.docx

Round 2

Reviewer 1 Report (New Reviewer)

Please remove "(chl)" from the text. Leave "chlorophill" only, both in the text and in Tables.

You should use alternatively the terms "cultivars" and "genotypes".

Author Response

Thanks for your worthy comments. 

 

said changes have been incorporated 

This manuscript is a resubmission of an earlier submission. The following is a list of the peer review reports and author responses from that submission.

Round 1

Reviewer 1 Report

General comments:

In general, this manuscript has a valuable topic. The topic is scientifically sound. Experimental design is adequate. Unfortunately, the manuscript is not written with due care. There are errors in the editing of the text, in the processing of graphics. I have some comments that may improve the quality of the manuscript.

 

Detailed comments:

Title:

The author should include the Latin name of the species (Olea europea L.)

 

Abstract:

This section is well written. The aim of the study is clearly stated.

Line 13 - „In vitro” should be written in italics. The author did not always follow this rule. I am asking for improvement

Line 16 - OM medium - no information on composition and company.

Line 16 - BAP- no information about the company. On first use, there should be a complete form and an explanation of the abbreviation., when mention for the first time the abbreviation is given in brackets after the abbreviated word.

In this mnuscript, chlorophyll is always abbreviated. There is no information whether it is total chlorophyll?

Line 27 -29 - the author writes: „Among cultivars, Arbosana responded more dramatically to changing pre-cooling and BAP treatments on morphological, physiological and biochemical levels as compared to compared to Moraiolo, Gemlik and Leccino with in vitro system”. I am asking for a linguistic proofreading

 

Key words:

Some of the keywords presented by the authors also appear in the title of the manuscript. Authors should consider changing keywords.

 

Introduction:

The topic is very important and has a great value. I see that the introduction didn’t provide enough background about the topic and needs to be enriched.

Line 37 - „In vitro” should be written in italics. The author did not always follow this rule. I am asking for improvement

Line 64 abbreviation ABA, no full name, when mention for the first time the abbreviation is given in brackets after the abbreviated word

 

Materials and Methods:

The design of the experiment was relevant and relevant to the current study, but I have a few problems:

- There is no information on the number of explants inoculated onto the medium

- Please let me know about the course of the culture for 50 - 60 days. Was there a transfer of the explants to fresh medium at that time?

- Why did the author not include photographs documenting the course of the experiment in the manuscript?

- Proline content was estimated following what method? Literature data.

Line 82-83 - no information on the origin of the olive varieties tested

Line 89 - no spaces

Line 103 - I am asking for a correction

Line 106 - the author has put the abbreviation (PIS) here and the abbreviation (SIP) is used in the following parts of the chapter.

Line 118 - no detailed information about the centrifuge.

Line 131 - no spaces.

Line 133 - no detailed information on the programs applied. Please state the creator, and the location (city, country) from where the software was sourced.

 

Results:

I found that the results department could still be improved. The author should describe the results for Fig 6-8 in more detail. No justification for the use of the method.

All the figure resolution should be improved. Especially fig. 3 and figs. 6-8 have little legible inscriptions, no explanations of the charts.

 

Discussion

The discussion needs substantial improvement.

 

The authors should write the Conclusion part separately, highlighting the study's significant findings.

References:

The authors provided a sufficient number of citations, but maybe they will find new citations (works from the last 5 years), they need to be included.

There are no DOI numbers. DOI numbers are not mandatory but highly encouraged.

Line 379 - no boldface for the year

Line - 346, 347, 349, 352, 356, 364, 367, 387, 392 please correct („in vitro” and species names should be written in italics).

Authors should check that all References items are describe in accordance with the requirements of the Editorial Board. References should be described depending on the type of work.

Author Response

 

Author Response File: Author Response.docx

Reviewer 2 Report

Dear Authors,

 

The manuscript is well-structured and brings important information concerning the issues of the micropropagation of olive.

I suggest you improve the introduction with a paragraph about the use of pre-cooling treatments in the micropropagation of olive and other species.

Author Response

 

Author Response File: Author Response.docx

Reviewer 3 Report

Dear authors,

 

The manuscript focus on the optimization of in vitro propagation through axillary buds of four cultivars of olive, a recalcitrant species to in vitro culture.  The effect of precooling treatments in combination with different concentration of 6-benzylaminopurine (BAP) was evaluated on the in vitro development of shoots. Furthermore, they quantified different parameters, such as the chlorophyll content, assimilation rate of CO2 as well as the content of carbohydrates and secondary metabolites (alkaloids, phenols, tannins and flavonoids).

They found that the 48h pre-cooling treatment in combination with the use of 2,5 mg/L of BAP in the culture medium improved all the analyzed parameters. The principal component analysis revealed a genotype-dependent response to the treatments.

In general, the manuscript is well written and provides relevant information to improve micropropagation of olive genotypes.

I have some minor comments that I think could help to improve the final version of the manuscript which should be addressed before publication.   

-          Some abbreviations first appear in the text are not use the full name such as BAP, Chl, PCA, ABA.. Please include the explanation of abbreviations.

-          Line 28- the second “compared to” should be removed from the sentence

-          “Auxilary buds” should be changed by axillary buds (lines 62, 63, 67, 287, 289)

-          In material and methods, authors described that they used nodal segments, which are supposed to bear axillary buds that are induced to form shoots. However,  in the discussion section, line 297, it appears “internodal segments” which do no bear axillary buds and they are commonly used for adventitious shoot regeneration.

-          Line 103: t0 should be to

-          Subsection 2.3: Does the term “Percentage of the induced shoots” mean the percentage of shoot induction from initial explants?

-          Subsection 2.5.1: Lines 118-120: The sentence should be rewritten: Afterward, metabolites were quantified using…

-          Subsection 2.5.2:  semicolon should be removed in line 128

-          Line 138 and 145: SIP should be replaced by PIS

-          Caption of Figure 1: Line 156: “tri-replicate”

-          Results  section: Primary metabolites

-          I agree that the best treatment was BAP 2.5 mgL^-1 in combination with 48h precooling treatment since they provided the best results and the highest values. However, I am not sure regarding to the meaning of  “maximum incline”, at least in the case of sucrose and 0,5 mgL^-1 of BAP (Figure 2)

Line 196: It should be capital letter (In). I also think there is some misinterpretation of the described data, since at 2.5 mgL^-1 BAP plus 48h precooling, V4 reach the maximum of glucose content (1mg g^-1DW) and V1 reach about 0.9 mg g-1DW. V1 reach 0.75mg  g-1DW in the control.

 

Page 236. I have a similar concern to the description of data related to phenols content. V4 reach the maximum value (1.5) under BAP 2.5 mgL-1 plus 48h precooling treatments (the increase with respect to control was from about 1,2 to 1.5). However, according to the data shod in Fig 4,  V1 seems to experience a greater increase in phenols content regarding to the control, when explants were cultured on the same conditions as V4. Please revise these data.

Caption of Figure 4: please insert a space after genotypes (line 232)

-Lines 293, 296: Please change SIP by PIS

 I also recommend the authors proofread the manuscript to correct editorial errors

Author Response

 

Author Response File: Author Response.docx

Reviewer 4 Report

The manuscript “In vitro Evaluation of the effects of BAP concentration and pre-cooling treatments on morphological, physiological and biochemical traits of different olive cultivars” by Khatoon, et al., talks about the experiments conducted to optimize shoot induction and proliferation protocol for different olive cultivars. The article is well written and thoroughly described and it can find interest among the researchers in this field. However, there are some major concerns that I feel should be explained.

First, the authors have mentioned they used RCBD design to analyze the results. For this kind of experiment conducted under controlled conditions, you cannot used RCBD design, in this case it makes the whole results unreliable and cannot be true.

Secondly, the authors have not mentioned the methods how they treated the explants for pre-cooling treatment. As far as I know, when the olive shoots are stored more than 24 hours after sterilization, they release different phenolic compounds (e.g oleuropein, tyrosol and hydroxytyrosol etc.), that I feel should have been studied as well.

Moreover, the shoot induction and proliferation protocols are already well established in olive by using zeatin, whereas, both references (ref 6 & 8) quoted by the authors have always used zeatin in their experiments. In this scenario the authors also did not provide any solid justification for the use of BAP instead of zeatin.

Figures: In the figures 1,2,3 & 4, also missing the legends explanation for the different colors used for the results

Author Response

 

Author Response File: Author Response.docx

Round 2

Reviewer 4 Report

Dear Authors, 

I really appreciate your efforts. Anyway, I think a manuscript were there is lack of statistical analysis is not acceptable. 

Furthermore, the protocol you described have no improvements respect the existing protocols. 
Best regards

Author Response

 

Author Response File: Author Response.docx