Indirect Somatic Embryogenesis: An Efficient and Genetically Reliable Clonal Propagation System for Ananas comosus L. Merr. Hybrid “MD2”

Round 1

Reviewer 1 Report

The manuscript entitled "Indirect somatic embryogenesis: an efficient and genetically re- 2 liable clonal propagation system for Ananas comosus L. Merr 3 hybrid "MD2"" sets out to develop an efficient and genetically re- 2 liable clonal propagation system for Ananas comosus L. Merr 3

hybrid "MD2" through Indirect somatic embryogenesis. The submitted manuscript is written clearly and provides new insights concerning somatic embryogenesis of Ananas comosus and general interest to the readers. However, I have several concerns that should be addressed.

Make sure that species names are in italic format and the family names are in Roman type, on lines 3 (Ananas comosus) and 43 (Bromeliaceae) , there are only the example, please check the whole manuscript.

Make sure that the concentration units are correct, on lines 124 to 136. The “mg.L” should be “mg·L^-1”, check the whole manuscript.

Line 150 to 151: “2:1:10:7” instead of “10:5:50:35”

In the material and methods section, all the sources of chemicals and equipment need to be added or completed (add city and country).

Figure 5 should be more clear.

Author Response

RESPONSE TO REVIEWER 1

The authors analyzed the suggestions made by the reviewer and made the pertinent corrections. All modifications are marked in the text with the track changes tool.

Suggestions of the reviewer 1:

The manuscript entitled "Indirect somatic embryogenesis: an efficient and genetically reliable clonal propagation system for Ananas comosus L. Merr 3 hybrid "MD2"" sets out to develop an efficient and genetically reliable clonal propagation system for Ananas comosus L. Merr hybrid "MD2" through Indirect somatic embryogenesis. The submitted manuscript is written clearly and provides new insights concerning somatic embryogenesis of Ananas comosus and general interest to the readers. However, I have several concerns that should be addressed:

1. Make sure that species names are in italic format and the family names are in Roman type, on lines 3 (Ananas comosus) and 43 (Bromeliaceae), there are only the example, please check the whole manuscript.

R/ all the species names were written in italics and the family name in Roman type (lines 3, 43, 546).

2. Make sure that the concentration units are correct, on lines 124 to 136. The “mg.L” should be “mgL^-1”, check the whole manuscript.

R/ the concentration units were corrected throughout the document.

Line 150 to 151: “2:1:10:7” instead of “10:5:50:35”

R/ the ratio of distilled water was corrected.

3. In the material and methods section, all the sources of chemicals and equipment need to be added or completed (add city and country).

R/ All the sources of chemicals and equipment were completed

4. Figure 5 should be more clear.

R/ Figure 5 was improvement and replaced in the text

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments concerning manuscript agriculture-1680826, entitled “Indirect somatic embryogenesis: an efficient and genetically reliable clonal propagation system for Ananas comosus L. Merr 3 hybrid "MD2"”

 

In this paper, the authors described establishment of an efficient protocol for in vitro multiplication of pineapple cultivar MD2 and obtaining genetically stable regenerants through process of somatic embryogenesis. Multiplication efficiency (shown in Figure 6) is really impressive, and these results may contribute to the improvement of in vitro regeneration of this species.

The used methodology is appropriate; however, the research design is not described adequately. More importantly, the presentation of the results concerning evaluation of different treatments used for in vitro regeneration as well as the accompanying histological analysis is poor, making it hard to estimate if the interpretation and the conclusions are sound and justified by the data. These parts need to be re-written for clarity.

To make this paper publishable, the authors need to respond to the suggested corrections, and also to the comments to the text listed below.

 

 

General comments:

 

Concentration units

Plant growth regulator concentrations should be expressed as mg L^-1 (and not as mg. L), throughout the manuscript.

Non-standard abbreviations

Should only be introduced if necessary, but then must be used CONSISTENTLY throughout the manuscript!!!

I would strongly recommend against the usage of abbreviations such as NERM, TRM, NB/NSPE etc., since they only appear in the table and nowhere else in the text. Furthermore, ‘number of shoots formed per explant’ is designated as NB in Material and Methods (line 142), but NB is absent from the Table 1, where (I suppose) it was replaced with NSPE, which is then in the footnote explained differently, as ‘number of multiple shoots per explant’. This alone is sufficient to confuse the reader, not to mention an incorrect use of ISE (instead of DSE) in the subtitle referring to Direct Somatic Embryogenesis.

 

Specific comments:

 

Title

The name of the species should be italicized.

 

Abstract

Lines 31-33: “As a result, M7 (2 mg. L 2,4-D and 2 mg. L BAP) was selected as the most efficient treatment, where indirect somatic embryogenesis occurred, with an average of 120 somatic embryos per explant....” should be replaced with:

“The medium containing 2 mg L^-1 2,4-D and 2 mg L^-1 BAP, where indirect somatic embryogenesis occurred, was selected as the most efficient treatment with an average of 120 somatic embryos per explant....”

 

Introduction

Lines 97-98: “in the differentiation process (callus formation) and dedifferentiation (seedling regeneration)”

Here (and elsewhere in the text) the terms ‘differentiation’ and ‘dedifferentiation’ are used incorrectly – callus formation implies dedifferentiation of (previously differentiated) cells, whereas seedling regeneration implies cell differentiation.

 

Materials and Methods

Line 124: what is DM?

Murashige and Skoog’s medium has an exact formulation, and unless there were some modifications to it, there is no need to specify individual compounds and their concentrations.

Line 127: Naphthaleneacetic Acid (NAA) is properly abbreviated only here – throughout the rest of the manuscript, NAA is designated as ANA!?! This must be corrected throughout the manuscript.

Treatments (media formulations) should better be presented in a separate table (instead in the text), just as it was presented e.g. in the work of Paola et al. (2020), which was cited in this manuscript as ref [30].

It would be desirable to specify the exact amount of medium per one magenta box. More importantly, the number of explants per one magenta box should be specified. But most importantly, the total number of explants used for each treatment must be specified!!!  Especially, since the results are presented as ‘number of [X]‘ instead of proportions, or percentages. 

The position/orientation of the initial explants on/in the culture medium should be specified!

Line 133: “Callus-forming treatments were incubated in the dark...”

1. Not treatments, but cultures are incubated.
2. If only callus-forming cultures were incubated in the dark, what culture conditions were other cultures exposed to? If all the cultures were incubated in the dark, why specify only callus-forming ones?
3. The exact sequence of different media must be specified! The authors should clearly state here what happened to the cultures after 90 days of incubation in the dark, to which media they were transferred later, how long they were maintained on these media, etc.
4. There is a huge information gap between obtaining any morphogenic response of initial explants, and obtaining 6-8-cm high seedlings. Were those seedlings obtained from isolated somatic embryos only? If so, were the embryos removed from the callus/leaf tissue and then transferred to separate medium? Which one? For how long were they cultured, until they grew to be 6-8 cm high? Etc.

The term ‘morphogenic/morphogenetic structures’ should be used with caution, since e.g. leaves or roots are also morphogen(et)ic structures. Perhaps it would be more appropriate to use the term ‘morphogenic response [of explants]’

 

Results

 

Table 1

NERM, TRM, NE, NSPE and NP should be replaced with the phrases that were abbreviated. Is NE number of embryos per one explant, or total number of embryos in all explants?? What does the phrase “same letters: not significant” mean?? The footnote should state whether the letters refer to the column, or to the row. How come that for NSPE there are no ‘a’ and ‘b’ in the column?!

Table 1 presents the sheer number of specific structures, without stating what proportion of the total number of explants that is.

 

Evaluation of the treatments established for in vitro regeneration of the pineapple hybrid ‘MD2’

Line 231: Direct Somatic Embryogenesis is abbreviated with ISE!!!

Lines 232-234: “... the treatments in which BAP and ANA were used favored the morphogenic response in pineapple, which varied depending on the BAP/ANA balance to which the explants were exposed”. From this statement, the reader might think that no morphogenic response was obtained in cultures treated with 2,4-D or P. I suppose that the authors probably referred to direct morphogenic response, but that is merely a speculation.

Lines 236-237, Figure 2: “formation of globular structures directly from explant” – these so-called globular structures are not denoted in the figure, and they should be, e.g. by arrows. In that sense, Figs. C and D are totally non-informative, since they appear the same as A and B, and do not point out to different stages of embryo (?) development. Furthermore, inspection of such an explant with the naked eye could hardly show whether these structures originate directly from the explant, nor would it allow us to discern their nature (i.e. whether these are adventitious embryos or adventitious buds). Perhaps a higher magnification is also needed.

Line 243: what does the phrase ‘multiple shoots’ mean?

There is certain time incongruence between the statements concerning indirect SE and the legend to Figure 3, depicting ISE. For example, the appearance of the callus at 30 days of culture (lines 264-265) referring to Figure 2 A-B, is followed later by the statement “After the first subculture, a semi-friable callus proliferated on the primary callus (Figure 3B)” – after the first subculture should signify the subculture after 90 days. At least, that is stated in Material and Methods. In lines 270-271, it is said “In this treatment, the callus started in the basal zone of the leaf and, six weeks later, the first embryogenic structures (globular) became visible” – six weeks later from which time point?? It is not even clear when the callus appeared!? Etc.

Figures 3 C, D, F allegedly show embryogenic structures (at different stages of development), but those structures are not marked in those figures, rendering at least two of them obsolete.

Line 297: Abbreviation IO was previously used to denote indirect organogenesis, whereas here it denotes adventitious organogenesis, which is incorrect since ‘direct’ organogenesis can also be adventitious.

Line 307: What does the phrase “shoots multiply adventitiously” (after the first generation of shoots is formed) mean?!?

Lines 315-318: “Shoots obtained in any of the treatments evaluated and subsequently transferred to M4 medium for development, at 4 weeks, reached an average height of 6-8 cm, ready for transfer to the acclimatization stage (Figure 2D)”. This statement belongs to the section Materials and Methods. However, it raises a concern about the very title of this manuscript: if the shoots obtained in any of the treatments were used for acclimatization, what about the embryos?!? And if shoots were in fact used, did they develop roots (and how?) to be successfully established in the soil?!

Figure 4. Missing legend for E (denoted as D) and F. Figures 4C/4E practically show the same thing, as well as 4D/4F.

Histological analysis is inadequate, as the figures hardly show what the authors claim that they do show. For example, legend to the Fig 5 A says “direct somatic embryogenesis in leaf explants grown in M9 treatment”. However, in fig. 5A neither leaf tissue nor embryos are discernible; not a single structure is marked in the figure, by arrow, asterisk or anything. But more importantly, in Table 1, M9 treatment is presented as one that only induced indirect organogenesis, and not direct somatic embryogenesis.

The bars in Figure 5 also seem to be inadequate.

Successive divisions cannot be seen in the figure; only the result of successive divisions can be observed in a still micrograph.

Legend to the figure 5 does not explain marks such as TCA (in 5B); ECDC, CP, MP etc. (in 5C), GSVC (in 5D), etc.

Line 348: what is rounded, and what is an acute pole?

Lines 354-355: “Figure 5 D-E also shows very early stages of embryo development (quadrant, octant) giving rise to globular structures”. Not only these ‘very early stages’ are not marked in these pictures, but this is an untrue statement – these are multicellular structures, and no ‘quadrant and octant giving rise to globular structures’ can be seen in them, at least not in this magnification.

Author Response

RESPONSE TO REVIEWER 2

The authors analyzed the suggestions made by the reviewer and made the pertinent corrections. All modifications are marked in the text with the track changes tool.

Suggestions of the reviewer 2:

In this paper, the authors described establishment of an efficient protocol for in vitro multiplication of pineapple cultivar MD2 and obtaining genetically stable regenerants through process of somatic embryogenesis. Multiplication efficiency (shown in Figure 6) is really impressive, and these results may contribute to the improvement of in vitro regeneration of this species.

The used methodology is appropriate; however, the research design is not described adequately. More importantly, the presentation of the results concerning evaluation of different treatments used for in vitro regeneration as well as the accompanying histological analysis is poor, making it hard to estimate if the interpretation and the conclusions are sound and justified by the data. These parts need to be re-written for clarity.

To make this paper publishable, the authors need to respond to the suggested corrections, and also to the comments to the text listed below.

General comments:

Concentration units

1. Plant growth regulator concentrations should be expressed as mg L-1 (and not as mg. L), throughout the manuscript.

R/ The concentrations of plant growth regulators were changed throughout the manuscript and are expressed in mg L^-1

2. Non-standard abbreviations. Should only be introduced if necessary, but then must be used CONSISTENTLY throughout the manuscript!!! I would strongly recommend against the usage of abbreviations such as NERM, TRM, NB/NSPE etc., since they only appear in the table and nowhere else in the text. Furthermore, ‘number of shoots formed per explant’ is designated as NB in Material and Methods (line 142), but NB is absent from the Table 1, where (I suppose) it was replaced with NSPE, which is then in the footnote explained differently, as ‘number of multiple shoots per explant’. This alone is sufficient to confuse the reader, not to mention an incorrect use of ISE (instead of DSE) in the subtitle referring to Direct Somatic Embryogenesis.

R/ throughout the manuscript the non-standard abbreviations were revised and corrected.

 Specific comments:

Title. The name of the species should be italicized.

R/ it was corrected

 Abstract

Lines 31-33: “As a result, M7 (2 mg. L 2,4-D and 2 mg. L BAP) was selected as the most efficient treatment, where indirect somatic embryogenesis occurred, with an average of 120 somatic embryos per explant....” should be replaced with:

“The medium containing 2 mg L-1 2,4-D and 2 mg L-1 BAP, where indirect somatic embryogenesis occurred, was selected as the most efficient treatment with an average of 120 somatic embryos per explant....”

R/ this sentence was replaced

 Introduction

Lines 97-98: “in the differentiation process (callus formation) and dedifferentiation (seedling regeneration)” Here (and elsewhere in the text) the terms ‘differentiation’ and ‘dedifferentiation’ are used incorrectly – callus formation implies dedifferentiation of (previously differentiated) cells, whereas seedling regeneration implies cell differentiation.

R/ these terms were corrected

 Materials and Methods

Line 124: what is DM? Murashige and Skoog’s medium has an exact formulation, and unless there were some modifications to it, there is no need to specify individual compounds and their concentrations.

R/ the formulation of MS medium was eliminated, because it was not modificated.

Line 127: Naphthaleneacetic Acid (NAA) is properly abbreviated only here – throughout the rest of the manuscript, NAA is designated as ANA!?! This must be corrected throughout the manuscript.

R/ this term was corrected throughout the manuscript.

Treatments (media formulations) should better be presented in a separate table (instead in the text), just as it was presented e.g. in the work of Paola et al. (2020), which was cited in this manuscript as ref [30].

R/ Table 1 with all treatments was included in the manuscript.

 

It would be desirable to specify the exact amount of medium per one magenta box. More importantly, the number of explants per one magenta box should be specified. But most importantly, the total number of explants used for each treatment must be specified!!!  Especially, since the results are presented as ‘number of [X]‘ instead of proportions, or percentages.

R/ It was included in the material and methods section (line 144).

The position/orientation of the initial explants on/in the culture medium should be specified!

R/ It was specified in lines 117 and 124.

 

Line 133: “Callus-forming treatments were incubated in the dark...” Not treatments, but cultures are incubated.

R/ Redaction was corrected (line 135)

If only callus-forming cultures were incubated in the dark, what culture conditions were other cultures exposed to? If all the cultures were incubated in the dark, why specify only callus-forming ones?

R/From line 126 to line 151 the conditions for all treatments are described.

 

The exact sequence of different media must be specified! The authors should clearly state here what happened to the cultures after 90 days of incubation in the dark, to which media they were transferred later, how long they were maintained on these media, etc. There is a huge information gap between obtaining any morphogenic response of initial explants, and obtaining 6-8-cm high seedlings. Were those seedlings obtained from isolated somatic embryos only? If so, were the embryos removed from the callus/leaf tissue and then transferred to separate medium? Which one? For how long were they cultured, until they grew to be 6-8 cm high? Etc. The term ‘morphogenic/morphogenetic structures’ should be used with caution, since e.g. leaves or roots are also morphogen(et)ic structures. Perhaps it would be more appropriate to use the term ‘morphogenic response [of explants]’

R/ These clarifications are in lines 246 to 259, and 279 to 305 where the text were clarified and y the rest of the manuscript the term morphogenetic was replaced.

 

Results

Table 1

NERM, TRM, NE, NSPE and NP should be replaced with the phrases that were abbreviated. Is NE number of embryos per one explant, or total number of embryos in all explants?? What does the phrase “same letters: not significant” mean?? The footnote should state whether the letters refer to the column, or to the row. How come that for NSPE there are no ‘a’ and ‘b’ in the column?!

Table 1 presents the sheer number of specific structures, without stating what proportion of the total number of explants that is.

R/ This table and its foot were corrected and now is Table 2.

 

Evaluation of the treatments established for in vitro regeneration of the pineapple hybrid ‘MD2’

Line 231: Direct Somatic Embryogenesis is abbreviated with ISE!!!

R/ it was corrected in text

 

Lines 232-234: “... the treatments in which BAP and ANA were used favored the morphogenic response in pineapple, which varied depending on the BAP/ANA balance to which the explants were exposed”. From this statement, the reader might think that no morphogenic response was obtained in cultures treated with 2,4-D or P. I suppose that the authors probably referred to direct morphogenic response, but that is merely a speculation.

R/ it was clarified in lines 314-336. The document refers to the fact that, depending on the balance of BAP and ANA, the morphogenic response varied, due to the fact that direct embryogenesis was observed in one of the treatments, treatment (M3), so it was considered, in our working conditions, that these concentrations, as a suitable balance to induce direct embryogenesis

 

Lines 236-237, Figure 2: “formation of globular structures directly from explant” – these so-called globular structures are not denoted in the figure, and they should be, e.g. by arrows. In that sense, Figs. C and D are totally non-informative, since they appear the same as A and B, and do not point out to different stages of embryo (?) development. Furthermore, inspection of such an explant with the naked eye could hardly show whether these structures originate directly from the explant, nor would it allow us to discern their nature (i.e. whether these are adventitious embryos or adventitious buds). Perhaps a higher magnification is also needed.

R/Figure was corrected and explanation in the text was clarified

 

Line 243: what does the phrase ‘multiple shoots’ mean?

R/ This refers to the fact that there was a high frequency of appearance of shoots as it seemed in Figure 4, but this phrase was eliminated from the text.

 

There is certain time incongruence between the statements concerning indirect SE and the legend to Figure 3, depicting ISE. For example, the appearance of the callus at 30 days of culture (lines 264-265) referring to Figure 2 A-B, is followed later by the statement “After the first subculture, a semi-friable callus proliferated on the primary callus (Figure 3B)” – after the first subculture should signify the subculture after 90 days. At least, that is stated in Material and Methods. In lines 270-271, it is said “In this treatment, the callus started in the basal zone of the leaf and, six weeks later, the first embryogenic structures (globular) became visible” – six weeks later from which time point?? It is not even clear when the callus appeared!? Etc.

R/ it was clarified in the results and figures as we mentioned before when we followed another suggestion.

 

Figures 3 C, D, F allegedly show embryogenic structures (at different stages of development), but those structures are not marked in those figures, rendering at least two of them obsolete.

R/Figure was corrected and explanation in the text was clarified

 

Line 297: Abbreviation IO was previously used to denote indirect organogenesis, whereas here it denotes adventitious organogenesis, which is incorrect since ‘direct’ organogenesis can also be adventitious.

R/ it was change in order to correct it

 

Line 307: What does the phrase “shoots multiply adventitiously” (after the first generation of shoots is formed) mean?!?

R/ This phrase was eliminated

 

Lines 315-318: “Shoots obtained in any of the treatments evaluated and subsequently transferred to M4 medium for development, at 4 weeks, reached an average height of 6-8 cm, ready for transfer to the acclimatization stage (Figure 2D)”. This statement belongs to the section Materials and Methods. However, it raises a concern about the very title of this manuscript: if the shoots obtained in any of the treatments were used for acclimatization, what about the embryos?!? And if shoots were in fact used, did they develop roots (and how?) to be successfully established in the soil?!

R/ In lines 315 to 329, where this part is now, was clarified this issue. The wording in lines 315 -318 (in the results) was improved, explaining that due to the rapid evolution of the shoots in the M4 treatment, it is suggested to incubate the plants obtained in the different regeneration protocols, in this medium, until reach the appropriate height, to move on to the acclimatization phase. In addition, it should be noted that the plants were regenerated in all the treatments during the acclimatization phase

 

Figure 4. Missing legend for E (denoted as D) and F. Figures 4C/4E practically show the same thing, as well as 4D/4F.

R/Figure was corrected and explanation in the text was clarified

 

Histological analysis is inadequate, as the figures hardly show what the authors claim that they do show. For example, legend to the Fig 5 A says “direct somatic embryogenesis in leaf explants grown in M9 treatment”. However, in fig. 5A neither leaf tissue nor embryos are discernible; not a single structure is marked in the figure, by arrow, asterisk or anything. But more importantly, in Table 1, M9 treatment is presented as one that only induced indirect organogenesis, and not direct somatic embryogenesis.

The bars in Figure 5 also seem to be inadequate. Successive divisions cannot be seen in the figure; only the result of successive divisions can be observed in a still micrograph. Legend to the figure 5 does not explain marks such as TCA (in 5B); ECDC, CP, MP etc. (in 5C), GSVC (in 5D), etc. Line 348: what is rounded, and what is an acute pole?

Lines 354-355: “Figure 5 D-E also shows very early stages of embryo development (quadrant, octant) giving rise to globular structures”. Not only these ‘very early stages’ are not marked in these pictures, but this is an untrue statement – these are multicellular structures, and no ‘quadrant and octant giving rise to globular structures’ can be seen in them, at least not in this magnification.

 

 

R/ Figure 5 was enhanced to show specifically how cell differentiation occurred in treatment M7.

 

 

 

Author Response File: Author Response.pdf

Round 2

Reviewer 2 Report

Comments concerning manuscript agriculture-1680826-peer-review-v2

 

The authors have significantly improved the manuscript, especially Materials and Methods section. There are some minor corrections that the authors need to respond to, in order to make this paper publishable. In addition to corrections and comments/suggestions listed below, there are also some inserted in the pdf version of the manuscript.

 

Materials and Methods

 

Line 117: adaxial position [in “was used in adaxial position as plant material”] should be deleted, and the position should be added in a separate sentence, following the description of initial explants: “Leaf explants were placed horizontally, with their adaxial surface on the culture medium.”

Explant position can be omitted from the Figure 1 legend, as marked in the pdf version of the manuscript (Lines 122-123).

 

Lines 126-127, 131: specify the carbon source that was used for culture media, and its concentration [e.g. “supplemented with sucrose (30 g L^-1), Myoinositol (100 mg L^-1) and Thiamine (5 mg L^-1)”]

 

Lines 128-129: “The shoots obtained were subcultured every 45 days, until seedlings were obtained.” Does this mean that the shoots have rooted on the same BAP+NAA-supplemented medium? If so, it should be stressed out. If another medium was used to obtain adventitious roots, then that should be stated too.

 

Lines 134-141: “the calluses [...] were transferred to conditions of a photoperiod of 16 h of light and 8 h of darkness, and a temperature of 27-30 °C, to induce the formation of morphogenic structures (embryos or shoots) to a culture medium without auxin, which contained BAP at different concentrations (0.5, 1 and 2 mg L^-1), in which they remained 145 days, with refreshing passes every 45 days, until reaching seedlings of 6 to 8 cm in height.”

What is ‘they’ here? This sentence implies that the calluses were subcultured every 45 days, so that the calluses remained on the particular medium until the seedlings reached 6-8 cm. Seedlings imply roots, and adventitious roots develop only after the adventitious shoot has been excised from the callus tissue (and cultured on ‘rooting’ medium, where it will subsequently develop adv. roots). Therefore, the observed structures should be designated as shoots, whereas the rooting process should also be described (which medium was used, for how long, etc.)

This entire procedure remains even more elusive when it comes to somatic embryos: could they be released from the callus at any point (which?)? If so, why transfer the entire callus? If not, how could they germinate, let alone grow up to 8 cm?!?

 

 

Results

 

Table 1

1. Numbers in the column “Number of explants with morphogenic response” should better be expressed as percentages of responsive explants. The total number of explants used in each treatment must be clearly stated in the footnote, along with an explanation of the numbers presented in the table [e. g. Values represent XXX of three independent experiments with 45 explants per treatment. Within columns, means followed by the same letter did not differ significantly according to XYZ], where XXX could be means (preferably followed by ± SD or SE, if the given numbers are averages)

 

2. What exactly do the numbers in columns Number of embryos and Number of shoots represent? Are they total number of embryos/shoots obtained for each treatment (sum of embryos/shoots in all responsive explants), or an average number of embryos/shoots per explant?? From the abstract and occasionally from the results section, it is clearly ‘per explant’. But then, it is unclear to me how come that in some of the treatments (M2, M5, M6, M8-10), average number of embryos/shoots per explant is equal to the exact number of responsive explants?? Are you saying that in 22 explants cultured on M8, you obtained exactly an average of 22 shoots per explant, while in 25 explants on M9, you obtained exactly an average of 25 shoots per explant?!?

 

Scale bars are missing from the Figures 2, 3 and 4. They should be inserted.

 

I still hold my view that the inspection of an explant with the naked eye could hardly show whether the newly formed morphogenic structures originate directly from the explant, or would allow us to discern their nature (i.e. whether these are adventitious embryos or adventitious buds).

Comments for author File: Comments.pdf

Author Response

Response to Reviewer 2

The authors analyzed the new suggestions made by the reviewer and made the pertinent corrections. All modifications are marked in the text with the track changes tool.

“Comments concerning manuscript agriculture-1680826-peer-review-v2:  The authors have significantly improved the manuscript, especially Materials and Methods section. There are some minor corrections that the authors need to respond to, in order to make this paper publishable. In addition to corrections and comments/suggestions listed below, there are also some inserted in the pdf version of the manuscript.”

Materials and Methods:

 Line 117: adaxial position [in “was used in adaxial position as plant material”] should be deleted, and the position should be added in a separate sentence, following the description of initial explants: “Leaf explants were placed horizontally, with their adaxial surface on the culture medium.”

R/ It was corrected in manuscript lines 116-117

Explant position can be omitted from the Figure 1 legend, as marked in the pdf version of the manuscript (Lines 122-123).

R/ This sentence was eliminated from the Figure 1 legend.

 Lines 126-127, 131: specify the carbon source that was used for culture media, and its concentration [e.g. “supplemented with sucrose (30 g L-1), Myoinositol (100 mg L-1) and Thiamine (5 mg L-1)”]

R/ It was corrected in manuscript lines 126 and 131.

 Lines 128-129: “The shoots obtained were subcultured every 45 days, until seedlings were obtained.” Does this mean that the shoots have rooted on the same BAP+NAA-supplemented medium? If so, it should be stressed out. If another medium was used to obtain adventitious roots, then that should be stated too.

R/ It is correct, the shoots have rooted on the same medium, it was clarified in line 135.

 Lines 134-141: “the calluses [...] were transferred to conditions of a photoperiod of 16 h of light and 8 h of darkness, and a temperature of 27-30 °C, to induce the formation of morphogenic structures (embryos or shoots) to a culture medium without auxin, which contained BAP at different concentrations (0.5, 1 and 2 mg L-1), in which they remained 145 days, with refreshing passes every 45 days, until reaching seedlings of 6 to 8 cm in height.” What is ‘they’ here? This sentence implies that the calluses were subcultured every 45 days, so that the calluses remained on the particular medium until the seedlings reached 6-8 cm. Seedlings imply roots, and adventitious roots develop only after the adventitious shoot has been excised from the callus tissue (and cultured on ‘rooting’ medium, where it will subsequently develop adv. roots). Therefore, the observed structures should be designated as shoots, whereas the rooting process should also be described (which medium was used, for how long, etc.)

This entire procedure remains even more elusive when it comes to somatic embryos: could they be released from the callus at any point (which?)? If so, why transfer the entire callus? If not, how could they germinate, let alone grow up to 8 cm?!?

R/ In lines 140 to 142 was clarified all about the rooting medium used in order to obtain seedlings

 

Results

 Table 1

1. Numbers in the column “Number of explants with morphogenic response” should better be expressed as percentages of responsive explants. The total number of explants used in each treatment must be clearly stated in the footnote, along with an explanation of the numbers presented in the table [e. g. Values represent XXX of three independent experiments with 45 explants per treatment. Within columns, means followed by the same letter did not differ significantly according to XYZ], where XXX could be means (preferably followed by ± SD or SE, if the given numbers are averages)

 R/ Table 1 was modified with suggestions and all SD were included.

2. What exactly do the numbers in columns Number of embryos and Number of shoots represent? Are they total number of embryos/shoots obtained for each treatment (sum of embryos/shoots in all responsive explants), or an average number of embryos/shoots per explant?? From the abstract and occasionally from the results section, it is clearly ‘per explant’. But then, it is unclear to me how come that in some of the treatments (M2, M5, M6, M8-10), average number of embryos/shoots per explant is equal to the exact number of responsive explants?? Are you saying that in 22 explants cultured on M8, you obtained exactly an average of 22 shoots per explant, while in 25 explants on M9, you obtained exactly an average of 25 shoots per explant?!?

R/ It was clarified in lines 272 to 274. From the shoots obtained in all the treatments, the same number of plants was obtained in the rooting medium, this means that there was a 100% conversion of shoots to plants.

 Scale bars are missing from the Figures 2, 3 and 4. They should be inserted.

R/ Scale bars were inserted in Figures 2, 3 and 4

 

 

I still hold my view that the inspection of an explant with the naked eye could hardly show whether the newly formed morphogenic structures originate directly from the explant, or would allow us to discern their nature (i.e. whether these are adventitious embryos or adventitious buds).

R/ When we say that it is direct formation of the explant, we mean that the formation of structures occurred without prior callus formation, which was not observed.

Author Response File: Author Response.pdf