Micropropagation of Tulip via Somatic Embryogenesis

Round 1

Reviewer 1 Report

The Manuscript entitled: “Micropropagation of tulip via somatic embryogenesis” contains a new and original contribution but some given information should be improved.

In the Abstract please change the superscript index in l^-1 units.

In Tables and Figures, the letter designation of the significance of differences should be from the highest to the lowest ("a" at the highest value).

The subject falls within the general scope of the journal and the title clearly reflects the contents. This article contains a new and original contribution. The interpretation of the results is correct.

Author Response

Responses to the reviewers’ comments

Thank you very much for your critical reviewing and the valuable comments. According to the comments, we have modified and corrected our manuscript. We believe this revised version will be acceptable for publication in Agronomy.

Response to the comments by Reviewer 1

The Manuscript entitled: “Micropropagation of tulip via somatic embryogenesis” contains a new and original contribution but some given information should be improved.

Comment 1: In the Abstract please change the superscript index in l^-1 units.

Response to the comment 1:

The index has been corrected in Line 12

Comment 2: In Tables and Figures, the letter designation of the significance of differences should be from the highest to the lowest ("a" at the highest value).

Response to the comment 2:

The letter designation of the significance of differences has been corrected  from the highest to the lowest.

Comment 3: The subject falls within the general scope of the journal and the title clearly reflects the contents. This article contains a new and original contribution. The interpretation of the results is correct.

Reviewer 2 Report

“Micropropagation of tulip via somatic embryogenesis” has been thoughtfully reviewed and some comments are pointed:

Major concerns

1. In this work, the authors aimed to develop a protocol for somatic embryogenesis of tulip and performed a histological analysis of the process. The theme of the MS is interesting and relevant, however, some points needs to be considered and revised.
2. One of the major concerns is the somatic embryogenesis concept. The author seems a little bit confused regarding the somatic embryo development. There are some sentences, such as “(…) further development of an embryo into shoots (…)” that demonstrates the confusion. Somatic embryos, when formed, are able to convert (germinate) into a bipolar structure (somatic plantlet). In the figure 7c it is possible to clearly observe the somatic embryo conversion into plantlet. Despite the apparent inexistence of root mentioned by the authors, this green structure is a somatic plantlet. I think that the authors should revise the MS correcting this issue.
3. The introduction also needs to be revised. This section is too extensive, being similar to a review. The authors should edit it for a more concise and to the point introduction. In addition, there are some grammar issues and problems with the connection between the sentences and paragraphs. I think that a much better version of the introduction could be presented.
4. The topic 2.4. of the M&M needs to be clarified. I strongly suggest to the authors to formulate a graphical representation of the implemented experiments. A flowchart indicating the substances evaluated (PGRs, amino acids, additives). It will be easier to follow the methodology used.
5. The discussion topic “SE induction and embryo development” also needs to be revised. This topic is very informative; however, the authors are very speculative regarding the molecular basis of the SE process in tulip. The results presented are associated to dose-response experiments, in which several compounds were evaluated to stimulate the callus/somatic embryos formation and somatic embryos conversion. Thus, it is impossible to speculate regulation of specific genes, such as ARFs, LEC, PRC and etc, without a gene expression analysis. I strongly suggest to the authors to revise it and keep in the discussion only the correlated and compatible studies.
6. The authors performed a series of experiments, testing several compounds in different steps of SE. Thus, to better elucidate the main findings, I suggest a scheme summarizing the indicated PGRs/additives and its concentrations in each step of SE, to be included in the conclusion. This figure could be very helpful.

Minor corrections

1. Please convert all the units of measure of PGRs from the mg/L to µM. The molarity of these substances can greatly vary, so it is important to represent it in molarity.
2. Line 142: Please correct for “callus clumps”.
3. Figure 1: Picloram is wrongly written. Please correct it.
4. In the graphs (Figs 2, 3, 5 and 6) and tables the statistical representation by the letters should be corrected. The convention is represent the higher values by the “a”, and the lower levels by the subsequent letters. In all these data the representation is inverted. Please correct it.
5. It is not necessary repeat the city and country of the manufacturer of “Tytanit®” in the figure caption. Please only indicate in the M&M topic.

Author Response

Response to the comments by Reviewer 2

Major concerns

Comment 1: In this work, the authors aimed to develop a protocol for somatic embryogenesis of tulip and performed a histological analysis of the process. The theme of the MS is interesting and relevant, however, some points needs to be considered and revised.

Comment 2: One of the major concerns is the somatic embryogenesis concept. The author seems a little bit confused regarding the somatic embryo development. There are some sentences, such as “(…) further development of an embryo into shoots (…)” that demonstrates the confusion. Somatic embryos, when formed, are able to convert (germinate) into a bipolar structure (somatic plantlet). In the figure 7c it is possible to clearly observe the somatic embryo conversion into plantlet. Despite the apparent inexistence of root mentioned by the authors, this green structure is a somatic plantlet. I think that the authors should revise the MS correcting this issue.

Response to the comment 2:

I would like to thank the reviewer for important comments. We incorrectly named these leafy structures as shoots. In fact, these long green structures are cotyledons. The shoots or bulbs can develop from opposite pol, which is a stolon. We have changed “the shoot” into cotyledon, longer embryo or plantlets throughout, depending on context.

Comment 3: The introduction also needs to be revised. This section is too extensive, being similar to a review. The authors should edit it for a more concise and to the point introduction. In addition, there are some grammar issues and problems with the connection between the sentences and paragraphs. I think that a much better version of the introduction could be presented.

 Response to the comment 3:

The introduction has been revised. We tried to shorten the introduction and focus more on the merits.

Comment 4: The topic 2.4. of the M&M needs to be clarified. I strongly suggest to the authors to formulate a graphical representation of the implemented experiments. A flowchart indicating the substances evaluated (PGRs, amino acids, additives). It will be easier to follow the methodology used.

Response to the comment 4:

We have prepared a scheme of our investigations in a form of Table 4. It will help to follow the methodology.

Comment 5: The discussion topic “SE induction and embryo development” also needs to be revised. This topic is very informative; however, the authors are very speculative regarding the molecular basis of the SE process in tulip. The results presented are associated to dose-response experiments, in which several compounds were evaluated to stimulate the callus/somatic embryos formation and somatic embryos conversion. Thus, it is impossible to speculate regulation of specific genes, such as ARFs, LEC, PRC and etc, without a gene expression analysis. I strongly suggest to the authors to revise it and keep in the discussion only the correlated and compatible studies.

Response to the comment 5:

All fragments on molecular mechanisms of SE have been removed.

Comment 6: The authors performed a series of experiments, testing several compounds in different steps of SE. Thus, to better elucidate the main findings, I suggest a scheme summarizing the indicated PGRs/additives and its concentrations in each step of SE, to be included in the conclusion. This figure could be very helpful.

Response to the comment 6:

For better understanding, we have prepared a scheme of experiments: Table 4. Conclusion has been corrected, lines 528-538

Minor corrections

1. Please convert all the units of measure of PGRs from the mg/L to µM. The molarity of these substances can greatly vary, so it is important to represent it in molarity.
   - Due to the fact that this work is of an application nature and was not physiological research, we tend to leave the PGR concentrations in mg/L.
   
   
2. Line 142: Please correct for “callus clumps”.

- The correction has been made.

1. Figure 1: Picloram is wrongly written. Please correct it.

- The spelling of Picloram has been corrected

1. In the graphs (Figs 2, 3, 5 and 6) and tables the statistical representation by the letters should be corrected. The convention is represent the higher values by the “a”, and the lower levels by the subsequent letters. In all these data the representation is inverted. Please correct it.

- The letter designation of the significance of differences has been corrected  from the highest to the lowest

1. It is not necessary repeat the city and country of the manufacturer of “Tytanit®” in the figure caption. Please only indicate in the M&M topic.

- In the caption to Figure 5, the information on the city and country of the manufacturer of “Tytanit®” has been removed.

Reviewer 3 Report

General comments for authors:

The paper entitled Micropropagation of tulip via somatic embryogenesis represents nicely performed work. However, there are two major issues regarding the materials and methods:

1. The quantification of a light source. It is not essential which light source we use, the important thing when comparing different light sources is the quantification of the wavelengths, i.e. the quantification of the wavelength composition. Here authors noted that two light treatments were conducted: white light (Philips TLD 36W/95) or red light (Philips TLD 36 W/15). However, we do not have information about peak wavelength (nm). Authors can measure this using spectrometer (for example, https://www.licor.com/env/products/light/spectrometer.html?gclid=Cj0KCQjwoaz3BRDnARIsAF1RfLczHcZONBXP06Oz03U0CHD0BvF5xCFfYwxb2EiQc8z-GzfLMqQOygkaAlrCEALw_wcB), or they can provide information from the lamp producer (Philips).
2. Statistical analysis of the data. Namely, it seems that in all cases one-way ANOVA is conducted. For this reason it is not possible to compare results on effect of auxins on callus induction between different cultivars which is very important in this case since you stated that Blue Parrot produced the highest amount of embryogenic calli. The same applies to data presented in Figure 2, Figure 3, Figure 6, Table 1 and Table 2. I suggest to conduct two way ANOVA for these data.

Specific comments on individual passages:

Line 138-145: Please, define how long this phase lasted (how many supcultivations) and harmonize it with results (Line 246): The embryogenic capacity of callus was followed for four years.

Line 195, 196: The addition of TDZ to the medium with auxins significantly stimulated callus formation in 'Apeldoorn' irrespective of the auxin type, and in ‘Prominence’, on media containing picloram and NAA.

From this sentence I understand that addition of TDZ to the medium with picloram and NAA significantly stimulated callus formation in ‘Prominence’. However, if compared this statement with Figure 2, it is not correct conclusion.

Line 197: In ‘Blue Parrot’, the highest amount of callus was observed for all auxins applied alone, as well as for picloram and NAA combined with TDZ at a lower concentration.

When compared this statement with Figure 2 Most of treatments do not differ significantly

Line 207: In Figure 2, major revision shoud be made. It seems that you have used one way ANOVA. In this case you can not compare results from different cultivars. Moreover, it is stated that Blue Parrot produced the highest amount of embryogenic calli which can not be seen in Figures 2 and 3. I suggest to conduct two way ANOVA for data presented in Figure 2, Figure 3, Figure 6, Table 1 and Table 2. In the results, you should show ANOVA tables and comment each variable as well as interaction effect of variables.

Line 231: Add information on callus mass on which somatic embryos were counted.

In Line 245 it is stated that: ˝The intensive proliferation of ‘Blue Parrot’ embryogenic callus was achieved only on media containing 2.5 mg l-1 2,4-D in combination with 0.1 and 0.5 mg l-1 TDZ.˝ However, from Figure 5 it is not easy to understand if presented values of callus production originate from medium 2.5 mg l-1  2,4-D + 0.1 mg l-1 TDZ or 2.5 mg l-1  2,4-D + 0.5 mg l-1 TDZ.

In Line 259 it is stated that: Tyrosine and red light had an inhibitory effect. However, from Figure 6 it seems that number of SE did not significantly differ in treatment with Tyrosine under red light when compared to treatment with Tyrosine under white light.  

As I have suggested before, you should conduct two way ANOVA for this data and in the results you should show ANOVA tables.

Line 260: Add information on callus mass on which somatic embryos were counted.

In line 276 it is written:˝ At higher concentrations, gibberellin significantly decreased shoot growth compared to other PGR combinations that (Table 1, 2).˝ Check this sentence. It seems it is not finished. Moreover,  from Table 1, it can be concluded that there was not significant difference in total number of embryos between lower and higher concentrations of gibberellin. When you conduct two way ANOVA, effect of each subcultivation can be presented and discussed.

In line 270 it is written:˝ On the other hand, the best quality embryos with shoots longer than 10 mm … ˝ however this is in collision with the Table 2 in which are presented data on ˝the effect of PGRs on the production of somatic embryos longer than 10 mm…˝

Author Response

Response to the comments by Reviewer 3

The paper entitled Micropropagation of tulip via somatic embryogenesis represents nicely performed work. However, there are two major issues regarding the materials and methods:

Comment 1

The quantification of a light source. It is not essential which light source we use, the important thing when comparing different light sources is the quantification of the wavelengths, i.e. the quantification of the wavelength composition. Here authors noted that two light treatments were conducted: white light (Philips TLD 36W/95) or red light (Philips TLD 36 W/15). However, we do not have information about peak wavelength (nm). Authors can measure this using spectrometer (for example, https://www.licor.com/env/products/light/spectrometer.html?gclid=Cj0KCQjwoaz3BRDnARIsAF1RfLczHcZONBXP06Oz03U0CHD0BvF5xCFfYwxb2EiQc8z-GzfLMqQOygkaAlrCEALw_wcB), or they can provide information from the lamp producer (Philips).

Response to the comment 1

At present, we cannot exactly describe the wavelength picks for white lamps because three years ago all lighting of our growing rooms were changes for LED lamps, although we have such instrument (GL SPECTROLUX, GL-POTIC). We fished most of the experiments on tulip SE earlier, 4 years ago. Therefore, we could add only the information that the lams emitted “cool white light”.  For the monochromatic red light lamp (Philips TLD 36 W/15), the pick was about 660 nm (according to lamp characteristic). Philips TLD 36W/95 is an old lamp type and the information is not available now in the internet.

Comment 2

Statistical analysis of the data. Namely, it seems that in all cases one-way ANOVA is conducted. For this reason it is not possible to compare results on effect of auxins on callus induction between different cultivars which is very important in this case since you stated that Blue Parrot produced the highest amount of embryogenic calli. The same applies to data presented in Figure 2, Figure 3, Figure 6, Table 1 and Table 2. I suggest to conduct two way ANOVA for these data.

Response to the comment 2

Many thanks to reviewer for these comments.

We have added a detailed description of ANOVA for each experiment in MM (lines 192-201 of corrected version).

We have analysed again the experiment 2.2 “Callus induction and direct somatic embryo formation” as three-way ANOVA (cultivar x auxin type x TDZ concentration),  previously the ANOVA was done as two-way analysis (auxin x TDZ separately for each cultivar). In case of callus growth (Figure 2) all the effects and their interactions were highly significant according to F ANOVA statistic with P < 0.001 and for one interaction, auxin x TDZ,  P = 0.02. We have given this information under the Figure 2 (lines 226-228). Similarly, the ANOVA results have been added under the corresponding Figure 3, 5 and 6. In all of the tables, we have added P (probability) of ANOVA for all effects and thei interactions. In addition, we add the means (averages) for each PGR treatment and subculture obtained from the two-way analysis (Table 1 and 2). These averages can help to interpret the results.

All changes we have introduced are marked in red.

The letters following the means were changed according to reviewer suggestions (“a” at the highest value).  

The statement that “Blue Parrot produced the highest amount of embryogenic calli” referred to the fact that following initial regeneration of callus and somatic embryos, we started to produce embryogenic callus to the amount which would sufficient for further experiments. That was described by us not clearly. We improved this description on callus stabilization and production to provide material for further research (lines 136-137).

Specific comments on individual passages:

Comment 3

Line 138-145: Please, define how long this phase lasted (how many supcultivations) and harmonize it with results (Line 246): The embryogenic capacity of callus was followed for four years.

Response to the comment 3

There were two subcultures and each subculture lasted one month. This has been corrected (lines 142-145).

Comment 4

Line 195, 196: The addition of TDZ to the medium with auxins significantly stimulated callus formation in 'Apeldoorn' irrespective of the auxin type, and in ‘Prominence’, on media containing picloram and NAA.

Response to the comment 4

This mistakes have been corrected and the result were reinterpreted according to three-way ANOVA (lines 207-214)

Comment 5

From this sentence I understand that addition of TDZ to the medium with picloram and NAA significantly stimulated callus formation in ‘Prominence’. However, if compared this statement with Figure 2, it is not correct conclusion.

Response to the comment 5

These results have been also reinterpreted as for Appeldorn and Blue Parrot (lines 207-214).

Comment 6

Line 197: In ‘Blue Parrot’, the highest amount of callus was observed for all auxins applied alone, as well as for picloram and NAA combined with TDZ at a lower concentration.

Response to the comment 6

This was also reinterpreted (lines 207-214).

Comment 7

When compared this statement with Figure 2 Most of treatments do not differ significantly.

Response to the comment 7

Following the three-way ANOVA the separation of homogenous groups have been slightly changed and we could better interpret the results.

Comment 8

Line 207: In Figure 2, major revision shoud be made. It seems that you have used one way ANOVA. In this case you can not compare results from different cultivars. Moreover, it is stated that Blue Parrot produced the highest amount of embryogenic calli which can not be seen in Figures 2 and 3. I suggest to conduct two way ANOVA for data presented in Figure 2, Figure 3, Figure 6, Table 1 and Table 2. In the results, you should show ANOVA tables and comment each variable as well as interaction effect of variables.

Response to the comment 8

Response to this comment is summarized and given together with response to Comment 2.

Comment 9

Line 231: Add information on callus mass on which somatic embryos were counted.

Response to the comment 9

“Somatic embryo number per initial explant” has been inserted. That was direct embryo regeneration on initial explants (flower stem fragments)

Comment 10

In Line 245 it is stated that: ˝The intensive proliferation of ‘Blue Parrot’ embryogenic callus was achieved only on media containing 2.5 mg l-1 2,4-D in combination with 0.1 and 0.5 mg l-1 TDZ.˝ However, from Figure 5 it is not easy to understand if presented values of callus production originate from medium 2.5 mg l-1  2,4-D + 0.1 mg l-1 TDZ or 2.5 mg l-1  2,4-D + 0.5 mg l-1 TDZ.

Response to the comment 10

Our description for the obtaining a stabilized embryogenic calliin  was really unclear. So we tried to improve this issue explaining that from the primary callus, formed on initial explants, we had to obtain the callus amount which would be sufficient to perform a series of experiment. We obtained such amount of callus following several subcultures but only for Blue Parrot and only when callus cultures were maintained on medium containing 2.5 mg l^-1  2,4-D + 0.1 mg l^-1 TDZ or 2.5 mg l^-1  2,4-D + 0.5 mg l^-1 TDZ. These data were not presented. We tried to explain this issue in lines 138-139.

Comment 11

In Line 259 it is stated that: Tyrosine and red light had an inhibitory effect. However, from Figure 6 it seems that number of SE did not significantly differ in treatment with Tyrosine under red light when compared to treatment with Tyrosine under white light.  

Response to the comment 11

This statement has been corrected as: “Tyrosine had no significant effect on embryo number. Red light revealed generally an inhibitory effect on SE. Based on two-way ANOVA, the embryo number obtained under red light was on average significantly lower (4.0) compared to white light treatment (5.2). The significantly lower embryo numbers under the red light compered to white light were observed for proline treatments at 100 and 400 mg l^-1. (Lines 280-284) 

Comment 12

As I have suggested before, you should conduct two way ANOVA for this data and in the results you should show ANOVA tables.

Response to the comment 12

If it would be necessary we can provide small tables of ANOVA with P values confirming significance of the effects for Figure 2, 3, 5 and 6. (perhaps as supplementary materials?). But to safe a place, we have given such information under each figure  (the value P, probability for each effect obtained from ANOVA).

Comment 13

Line 260: Add information on callus mass on which somatic embryos were counted.

Response to the comment 13

We add “somatic embryo number per 100 mg callus” in the text, line 292.

Comment 14 and 15

In line 276 it is written:˝ At higher concentrations, gibberellin significantly decreased shoot growth compared to other PGR combinations that (Table 1, 2).˝ Check this sentence. It seems it is not finished. Moreover,  from Table 1, it can be concluded that there was not significant difference in total number of embryos between lower and higher concentrations of gibberellin. When you conduct two way ANOVA, effect of each subcultivation can be presented and discussed.

In line 270 it is written:˝ On the other hand, the best quality embryos with shoots longer than 10 mm … ˝ however this is in collision with the Table 2 in which are presented data on ˝the effect of PGRs on the production of somatic embryos longer than 10 mm…˝

Response to the comment 14 and 15

Based on two-way ANOVA we have corrected these statements, lines 297- 311

Round 2

Reviewer 3 Report

The manuscript has been significantly improved and the explanations are much clearer.

However, I suggest to the authors to add ANOVA table (it can be added as Supplementary table) for Figure 2. You have written that there is significant difference for all of the factors (cultivar, auxin and TDZ concentration) and their interactions (line 226). However, it is impossible to show all of these data on the same chart. I suppose that data on Figure 2 is actually interaction of three factors (cultivar, auxin and TDZ concentration). In my opinion it is necessary to show ANOVA table for each studied factor and interaction. Moreover, whenever you have significant difference, it would be very interesting for readers if you show all the data (especially mean effect of cultivars on callus induction)

The same applies to Figure 3.

Author Response

Responses to the reviewers’ comments

Thank you very much for your critical reviewing and the valuable comments. According to the comments, we have modified and corrected our manuscript.

Comment 1: However, I suggest to the authors to add ANOVA table (it can be added as Supplementary table) for Figure 2. You have written that there is significant difference for all of the factors (cultivar, auxin and TDZ concentration) and their interactions (line 226). However, it is impossible to show all of these data on the same chart. I suppose that data on Figure 2 is actually interaction of three factors (cultivar, auxin and TDZ concentration). In my opinion it is necessary to show ANOVA table for each studied factor and interaction. Moreover, whenever you have significant difference, it would be very interesting for readers if you show all the data (especially mean effect of cultivars on callus induction). The same applies to Figure 3.

Response to the comment 1:

Table S1.  has been added showing Probability value (P) of F test from three-way analysis of variance (ANOVA) of callus and somatic embryo induction on tulip initial explants depending on auxin type, TDZ concentration and cultivar.

In Table S2. Means of the callus growth and somatic embryo number regenerated on initial expalnts for tulip cultivars, auxin types and TDZ concentrations; three-way ANOVA have been shown.

Author Response File: Author Response.docx