High Frequency Direct Organogenesis in Five Romanian Tomato (Lycopersicon esculentum Mill.) Cultivars
Round 1
Reviewer 1 Report
The MS entitled “High frequency direct organogenesis in tomato (Lycopersicon 2 esculentum Mill.)” with authors Adela Halmagyi, Ana Coste, Constantin Deliu and Ioan Băcilă presents new data and is interesting but needs minor improvement to be published.
The tomato (Lycopersicon esculentum Mill) is one of the most widely cultivated economically important vegetable crops, with almost 4 million hectares planted worldwide. In recent years, biotechnological approaches have been used to improve tomato yields. In vitro methods are widely applicable to obtain virus-free plants, genetic transformations, etc. In this regard, the research done in the presented manuscript merits attention.
The article follows the order required by the journal: Introduction, Materials and Methods, Results, Discussion, Conclusions. Abstract is informative; the Introduction is good and to the point to the purpose; the purpose of the study is clearly stated, but some of the text after it should be moved to a more appropriate place (see below the comments). Over 53% of the cited literature is from the last 10 years. However, the quality of the manuscript will improve if the authors use clearer and more precise sentences, as they are difficult to understand. It is essential to minor improve Material and Methods, and data presentation.
I have some comments on the text:
L14 - Throughout the manuscript ex vitro and in vitro should be in italics
L59-61 – “The tomato genotypes…..valuable traits.” - to be moved to Material and method
L61-69 – this text to be move in Results
L71 - Do the authors have data on the age of the seeds?
L112 - To avoid repetitions of the type of: “The pH of the medium was adjusted to 5.7 before autoclaving (20 min at 121 °C)”, it is sufficient to specify it once, but in an appropriate place
L127 - nowhere in the text is it stated why the authors cultivated the explants on this medium (zeatin + IBA)
L133 – „apical explant“? Authors may use „apical buds explant“
L242 - Is it a callus or callus-like formations?
L310 - The authors incorrectly compared average number of shoots with their variants in percentages. Do you have some data for mean number of shoots per explant?
L351 - The authors conclude that they have developed a protocol. When a protocol is published it is correct to describe it step by step in the Results. Please, indicate how many plants are produced from one seed?
The authors should carefully and critically review publications on the subject and highlight clear their novel contribution.
Author Response
Dear Editor and dear Reviewers,
We appreciate your precious time in reviewing our paper and providing relevant and constructive comments. Your valuable and insightful comments led to possible improvements in the current version.
We have carefully considered the comments and tried our best to address every one of them. We hope that after a careful revisions the manuscript meets your high standards. The authors welcome further constructive comments if any.
Below we provide the point-by-point responses. All modifications and the missing information were introduced in the manuscript with track changes.
Please note that the initial line numbering is changed.
Reviewer 1
Comments and Suggestions for Authors
The MS entitled “High frequency direct organogenesis in tomato (Lycopersicon 2 esculentum Mill.)” with authors Adela Halmagyi, Ana Coste, Constantin Deliu and Ioan Băcilă presents new data and is interesting but needs minor improvement to be published.
The tomato (Lycopersicon esculentum Mill) is one of the most widely cultivated economically important vegetable crops, with almost 4 million hectares planted worldwide. In recent years, biotechnological approaches have been used to improve tomato yields. In vitro methods are widely applicable to obtain virus-free plants, genetic transformations, etc. In this regard, the research done in the presented manuscript merits attention.
The article follows the order required by the journal: Introduction, Materials and Methods, Results, Discussion, Conclusions. Abstract is informative; the Introduction is good and to the point to the purpose; the purpose of the study is clearly stated, but some of the text after it should be moved to a more appropriate place (see below the comments). Over 53% of the cited literature is from the last 10 years. However, the quality of the manuscript will improve if the authors use clearer and more precise sentences, as they are difficult to understand. It is essential to minor improve Material and Methods, and data presentation.
I have some comments on the text:
L14 - Throughout the manuscript ex vitro and in vitro should be in italics
Ex vitro and in vitro have been changed in italics throughout the manuscript
L59-61 – “The tomato genotypes…..valuable traits.” - to be moved to Material and method
Was moved to Materials and methods
L61-69 – this text to be move in Results
The text was moved
L71 - Do the authors have data on the age of the seeds?
The seeds were two years old. This information was added to Section 2.2.
L112 - To avoid repetitions of the type of: “The pH of the medium was adjusted to 5.7 before autoclaving (20 min at 121 °C)”, it is sufficient to specify it once, but in an appropriate place
The repetition was removed.
L127 - nowhere in the text is it stated why the authors cultivated the explants on this medium (zeatin + IBA)
Zeatin+IBA was selected based on previously tests and is mentioned in the text that the data was not published.
The tested plant growth regulators were: 1.5 mg L-1 BAP + 0.2 mg L-1 IBA; 1.5 mg L-1 KIN + mg L-1 IBA; 1.5 mg L-1 TDZ + mg L-1 IBA; 1.5 mg L-1 Z + mg L-1 IBA, and the highest regeneration was obtained on medium with Z + IBA.
L133 – „apical explant“? Authors may use „apical buds explant“
‘Apical explant’ was replaced with ‘apical bud’ throughout the manuscript.
L242 - Is it a callus or callus-like formations?
It was a compact callus.
L310 - The authors incorrectly compared average number of shoots with their variants in percentages. Do you have some data for mean number of shoots per explant?
The comparison was made between the percentages of explants showing shoot regeneration, not regarding the number of shoots per explant.
L351 - The authors conclude that they have developed a protocol. When a protocol is published it is correct to describe it step by step in the Results. Please, indicate how many plants are produced from one seed?
Some parts of the Results were rewritten after recalculating statistics and comparing the data within each cultivar as required. Because tomato is a species with ‘orthodox’ seeds that germinate relatively easy without the need of special treatments, we did not track the exact number of shoots obtained after many subcultures.
The authors should carefully and critically review publications on the subject and highlight clear their novel contribution.
The entire Discussion was rewritten, some recent publications were added and the novel contribution was underlined.
On behalf of all the co-authors
Yours sincerely,
Ana Coste
Author Response File: 
Author Response.pdf
Reviewer 2 Report
The work intends to define a protocol for the induction of organogenesis in already marketed tomato cvs. It is not very clear what the novelty of the work is. Much has been written about in vitro culture of tomato since the 80s and huge experimental evidence on the most efficient tissue, media, dosage and hormone ratios is available. In some cases, with high efficiency.
The work has several problems and does not appear suitable for publication.
The title is too general and makes one think of a general review/study on tomatoes. It should be specified that it is limited only to cvs. widespread in Romania. M&M should be carefully reviewed. So does the presentation of the results. The bibliography is not very up-to-date and there are some inaccuracies. The English need to be revised very well.
The main observations can be found in the attached pdf.
Comments for author File: Comments.pdf
Author Response
Horticulturae - MDPI
High frequency direct organogenesis in five Romanian tomato (Lycopersicon esculentum Mill.) cultivars
Dear Editor and dear Reviewers,
We appreciate your precious time in reviewing our paper and providing relevant and constructive comments. Your valuable and insightful comments led to possible improvements in the current version.
We have carefully considered the comments and tried our best to address every one of them. We hope the manuscript after careful revision meets your high standards. The authors welcome further constructive comments if any.
Below we provide the point-by-point responses. All modifications and the missing information were introduced in the manuscript with track changes.
Please note that the initial line numbering is changed.
Reviewer 2
Comments and Suggestions for Authors
The work intends to define a protocol for the induction of organogenesis in already marketed tomato cvs. It is not very clear what the novelty of the work is. Much has been written about in vitro culture of tomato since the 80s and huge experimental evidence on the most efficient tissue, media, dosage and hormone ratios is available. In some cases, with high efficiency.
The work has several problems and does not appear suitable for publication.
The title is too general and makes one think of a general review/study on tomatoes. It should be specified that it is limited only to cvs. widespread in Romania. M&M should be carefully reviewed. So does the presentation of the results. The bibliography is not very up-to-date and there are some inaccuracies. The English need to be revised very well.
Response and clarifications to the observations are included in the attached file:
In vitro and ex vitro was changed in italics throughout the manuscript.
Line 2: The title was change in: High frequency direct organogenesis in five Romanian tomato (Lycopersicon esculentum Mill.) cultivars
Line 40: ‘characters’ was replaced with ‘traits’
Line 40-42: the sentence was reformulated
Line 42-43: the sentence was removed. There was no connection between the previous and following information.
Line 44-57: All paragraph (lines 44-56) is little bit confused. Micropropagation is a form of clonal multiplication useful for some applications, while somatic embryogenesis, protoplast culture and haploids are tools more suitable for breeding purposes
The paragraph was rewritten as follows:
In vitro tomato cultures have been successfully used in some biotechnological applications [12-14], for breeding purposes by somatic embryos [15] and to obtain virus-free high-value commercial cultivars [16]. In vitro regeneration of tomato shoots was induced by direct and indirect organogenesis [17-19] or through somatic embryogenesis [20]. There are many factors that affect the in vitro regeneration capacity of tomato plants, of which the most important are: the genotype, explant type, culture media composition, concentration of plant growth regulators, intensity and quality of light, photoperiod and temperature [14]. Considering the diversity of parameters, optimization of tissue culture system key factors is essential to achieve high efficiency and reproducibility of a certain approach.
Line 55: Regeneration of shoots can be induced directly from explants [21,22] or indirectly via a callus phase [23]. In this case it could be somatic embryogenesis?
In vitro regeneration of tomato shoots was induced by direct and indirect organogenesis [17-19] or through somatic embryogenesis [20].
Line 60: ‘homologated’ was replaced with ‘approved’ throughout the manuscript
Line 65-68: the paragraph was removed due to the redundant information
Material and methods
Every word or sentence that was marked for removal was removed throughout the manuscript.
Line 100: Seed germination. The usefulness of this test is not clear. Did it serve to provide explants for subsequent tests? Please, specify
Ex vitro germination of seeds aimed only to determine if germination was affected by the 2 years of storage period.
Shoots resulted by in vitro seed germination served as stock plants for excision of explants: (a) cotyledons, (b) cotyledonary nodes, (c) hypocotyls, (d) leaf explants, (e) internodes, (f) stem nodes and (g) apical buds.
Line 100-114: The entire section 2.2. Seed germination. Establishment of in vitro cultures was reformulated.
Line 110: All exponents have been revised
Line 119: (2.3. Morphogenic response of various explants) The section 2.4. Morphogenic response of various explants was modified. The following text was moved at the section beginning.
‘Based on a preliminary survey related to the efficiency of various plant growth regulators on shoot regeneration from various explants, a MS medium supplemented with 1.5 mg L-1 zeatin (Z) and 0.2 mg L-1 indole-3-butyric acid (IBA), 20 g L-1 sucrose and 7.6 g L-1 agar (the same medium pH as mentioned above) (unpublished data) was selected for further studies’.
Line 121: What was the age of the seedlings from which the explants were taken? How had they been produced? What are apical explants? the apical meristems? If so, how did you make to isolate them? Did you make shoot-tip culture (meristem with leaf primordia) or isolate just the meristem?
The seedlings (resulted from in vitro germinated seeds) were 25 days at the time they were used for explant excision. Apical explant was actually the apical bud with approx.1-1.5 cm in length (please see the newly introduced figure). It was not just the apical meristem. Due to their size, the excision of all types of explants was performed without the need of a stereomicroscope.
For more accuracy a new Figure was introduced with the presentation off all explant types used in regeneration experiments.
(Fig. 3. Explants used for in vitro regeneration experiments. (a) 1-hypocotyl; 2-cotyledon; 3-cotyledonary node; (b) 1-stem node; 2-leaf explant; 3-internode; 4-apical bud. Bars 1 cm.)
Line 124: Someone use the central part of cotyledons by removing the apical and basal part. In this case?
Cotyledons were excised from the central part. The information was added in text.
Line 125: Cotyledons too have two sides. Which side was exposed to the medium?
Cotyledons were placed with the abaxial side towards the medium surface. The information was added in text.
Line 130: The work should be start from these test for demonstrating the efficiency of the system. Was the substrate suitable for all the explants?
The information was added at the paragraph beginning: Based on a preliminary experiments related to the efficiency of various plant growth regulators (in different concentrations) on shoot regeneration from various explants, a MS medium supplemented with 1.5 mg L-1 zeatin (Z) and 0.2 mg L-1 indole-3-butyric acid (IBA), 20 g L-1 sucrose and 7.6 g L-1 agar (the same pH as mentioned above) (unpublished data) was selected for further studies.
Line 135: From which seedlings such explants (stem nodes and apical buds) were taken?
Seedlings resulted from in vitro germinated seeds were micropropagated (two successive subcultures each at 25 days) to serve as explant source. This information was added in text.
Line 137: pH value was added
Line 138: Each cytokinin would require a separate study. On the basis of what reasoning were the doses of hormone chosen? What is it Z? Zeatin riboside?
We used zeatin (Z) from Sigma-Aldrich not zeatin riboside.
Line 147: They referred to different test. Should be distinguished according to the test they refer to.
Line 152: Callus formation to generic
The entire section 2.5. Experimental design and statistical data analysis was restructured and rewritten.
For more accuracy, the section 2.5. has been separated into two distinct sections: 2.5. Experimental design and 2.6. Statistical analysis.
Line 153: ISTA 2016 -Please, include in the references
Was included in references.
Line 157: How much seedling per replicate? five? Which is the experimental design?
For each explant type and culture medium variant, 15 samples were used in each of the three replicates per treatment.
Line 165: the text was removed due to redundant information.
Results
3.1. Seed germination. Establishment of in vitro cultures
Line 176-179: ‘A variation in germination timing (the time elapsed between the first and the last germinated seed) among genotypes was observed. For example, in cv. ‘Capriciu’ ….. nevertheless the evaluation was made after 30 days for all cultivars’.
I would suggest to include in one table the data on germination and on timing
A table (Table 1) was introduced including data from Figure 3 (total germination percentages) and new data about the mean germination time (MGT).
Line 201: ‘frequencies’ was replaced with ‘percentage’.
3.2. Morphogenic response of various explant categories
Line 208: Table 1. In vitro shoot regeneration of various explants after 45 days in culture.
I would find more correct to compared the cultivar within each explants and to calculate the mean of each type of explant in order compared the percentage of organogenesis
The statistical analysis was corrected as required.
Line 214: One of the main effects of cytokinins is the production of multiple shoots. The information is of remarkable interest for micropropagation purposes. Did you assess the number of shoots produced per explant?
The number of shoots per explant was not assessed.
Line 221-226: ‘Pearson’s correlation analysis between height of shoot and length of roots developed from stem nodes showed moderate positive correlation (0.8) in cvs. ‘Darsirius’ and ‘Kristin’ and week negative correlation (-0.3) in cv. ‘Pontica’. In shoots and roots developed from apical buds the Pearsons correlation coefficient showed week negative correlation (-0.5) in cv. ‘Darsirius’ and moderate positive correlation (0.9) in cvs. ‘Capriciu’ and ‘Kristin’’.
The statement needs a specific table.
A new Table with the correlation coefficients was added.
Table 5. Correlation coefficients on shoots height and roots length (from stem nodes and apical buds) of analysed tomato genotypes.
Line 227-230: the unclear paragraph was rewritten.
Line 254-270: The utility about a description of callus formation is not so clear. It could be important for embryogenesis from callus, but this was not the case.
Description of callus was realised because it was a spontaneous reaction of the in vitro cultures and not in the context of somatic embryogenesis. Due to the fact that it was present in all cultivars, we considered appropriate to analyse callus formation as well.
Discussion
Line 281: Discussion appears a little bit confuse
The entire Discussion section was rewritten.
Line 316: Are you sure that the position (phyllotaxis) of the explantation is mentioned in this publication?
‘It was shown that the induction and growth of in vitro tomato shoots was dependent on both the explant type as well their phyllotactic position on the culture medium [29]
The sentence was corrected in: ‘It was shown that high germination rates are important to obtain homogenous sets of shoots used for the induction of tissue cultures [26].
Line 282-288: The concepts have been always expressed previously.
To avoid repetition the text was removed.
Line 331-334: However, the media with cytokinins also contained IBA, which may have influenced rooting. In some cases tomatoes can root even in the absence of auxins.
The paragraph was reformulated.
Line 341: ‘Plant regeneration from callus is not suitable for transformation and/or genetic editing, where the genetic uniformity of the plants must be maintained’. Genetic uniformity is of paramount importance especially for micropropagation to preserve all the traits of the cultivar.
The sentence was modified in: To preserve all the traits of the cultivars genetic uniformity is of paramount importance especially for micropropagation.
Line 336: Somatic embryogenesis?
The authors reported indirect organogenesis via callus, not somatic embryogenesis. The sentence was reformulated.
Line 341: Genetic uniformity is of paramount importance especially for micropropagation to preserve all the traits of the cv.
The sentence was rewritten.
Line 347: ‘In the present study callus formation at the explants base was a spontaneous secondary effect which did not influence shoot regeneration’.
Apparently, it may not have influenced shoot production, but to be sure of not having induced morphological changes the seedlings had to be observed at least also in vivo till ripening.
The following sentence was added: ‘However, further studies are necessary to assess if morphological changes arise after ex vitro acclimatization seedlings till ripening’.
Line 348-349: ‘Direct organogenesis or somatic embryogenesis is preferred for genetic engineering research, due to the genetic identity of regenerated plants, in which the avoidance of genetic variation is desirable’. To my knowledge, passing through a callus phase with stages of dedifferantiation and de novo differentiation, somatic embryogenesis can induce different rates of variability.
The above statement was replaced with: Direct organogenesis from explants is the best option for rapid multiplication as it leads to the generation of true-to-type plants (Verma et al. 2021).
Conclusions
Line 358: The statement seems in contradiction with the previous at lines 348-349
The sentence was removed and the Conclusion section was rewritten.
References
Line 428: reference 22: The link was removed because it is not functional.
Line 483: reference 48: a functional link was added (now reference 42): https://doi.org/10.31018/jans.v12i3.2233
Line 489: reference 51. This reference was removed by rewriting some paragraphs.
On behalf of all the co-authors
Yours sincerely,
Ana Coste
Author Response File: Author Response.pdf
Reviewer 3 Report
The experimebt design is appropriate. The tables and figures are appropriate
The cited references are relevant but not upto date. It's better if the author has to add new references within 5 years and omit the old references that have similar results.
The manuscript has some deficiencies that need to be corrected accordingly:
The presentation is not clear enough. After analysing the data it's necessary to, clearly, draw conclusions about which genotypes and which parts of plants give the highest regeneration rate. Moreover, it's necessary to clarify how the protocol of plant regeneration dirctly from stem nodes and appical plant is specific and what is more advanced than previously published. In addition, it's necessary to specify what of the five genotypes studied can be used for further research using tissue culture technique.
- The conclusion is, also, not clear.
- The manuscript can only be accepted after major revision based on reviewer's comments and suggestions.
Author Response
Horticulturae - MDPI
High frequency direct organogenesis in five Romanian tomato (Lycopersicon esculentum Mill.) cultivars
Dear Editor and dear Reviewers,
We appreciate your precious time in reviewing our paper and providing relevant and constructive comments. Your valuable and insightful comments led to possible improvements in the current version.
We have carefully considered the comments and tried our best to address every one of them. We hope the manuscript after a careful revision meets your high standards. The authors welcome further constructive comments if any.
Below we provide the point-by-point responses. All modifications and the missing information were introduced in the manuscript with track changes.
Please note that the initial line numbering is changed.
Reviewer 3
Comments and Suggestions for Authors
The experiment design is appropriate. The tables and figures are appropriate
The cited references are relevant but not up to date. It's better if the author has to add new references within 5 years and omit the old references that have similar results.
The manuscript has some deficiencies that need to be corrected accordingly:
The presentation is not clear enough. After analysing the data it's necessary to, clearly, draw conclusions about which genotypes and which parts of plants give the highest regeneration rate.
We mentioned in Conclusion which genotype and which explant revelead the highest regeneration rate.
Moreover, it's necessary to clarify how the protocol of plant regeneration directly from stem nodes and apical plant is specific and what is more advanced than previously published.
The novelty consists of the studied cultivars. These are autochthonous, economically important cultivars which were not studied before. It is well known that the regeneration response is highly influenced by the genotype and medium culture composition and conditions. In this context, our results may be valuable to the scientific community.
In addition, it's necessary to specify what of the five genotypes studied can be used for further research using tissue culture technique.
It was specified that: ‘Based on the promising results in all mentioned parameters we anticipate that these five Romanian tomato genotypes will represent new resources for improved breeding varieties and approaches and research towards preserving local cultivars’.
- The conclusion is, also, not clear.
The conclusions were rewritten.
- The manuscript can only be accepted after major revision based on reviewer's comments and suggestions.
All reviewer's comments and suggestions have been thoroughly revised throughout the manuscript.
On behalf of all the co-authors
Yours sincerely,
Ana Coste
Author Response File: Author Response.pdf
Round 2
Reviewer 3 Report
The MS has been revised accordimgly and now can be accepeted for publication.
Author Response
Thanks very much for your precious time and comments.
