Review Reports - A Comparison of Semi-Solid, Liquid, and Temporary Immersion Bioreactor Systems for Effective Plant Regeneration of <i>Gerbera jamesonii</i> “Shy Pink”

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The Authors compared 3 techniques of gerbera micropropagation. They reported interesting results but poorly described and discussed. Some important methodological information is also missing. Therefore, the manusript should be improved.

Please consider my comments and suggestions:

Title

A comparison of semi-solid, liquid and temporary immersion bioreactor systems for effective plant regeneration of Gerbera jamesonii cv. 3 ‘Shy Pink’ > A comparison of semi-solid, liquid and temporary immersion bioreactor systems for effective plant propagation (or micropropagation) of Gerbera jamesonii ‘Shy Pink’

Abstract:

v.3, 18, 28, 80...342 Not Gerbera jamesonii cv. ‘Shy Pink’ but Gerbera jamesonii cv. Shy Pink or Gerbera jamesonii ‘Shy Pink’ . The last one is more proper.

Why do You use abbreviations for TIS and SS cultures but not for liquid ones? Maybe suggest an abbreviation, e.g. 'LQ'?

v.21 'even though the number of shoots regenerated was less in number'. > 'even though the number of shoots regenerated was less'?

v.24 "The number of stomata on the abaxial surface of leaves was 11.40 and the frequency of closed stomata was 59% 25 with plants regenerated with TIS." What about other cultures?

v.67 "Gerbera jamesonii Bolus ex Hooker f. ‘Shy Pink” …" - f.?

Introduction

Quite well-justified research purpose. However:

v.72 "...although micropropagation systems using temporary im mersion cultures have not been tested." There are several articles of Mosqueda Frómeta et al. (2017-24). The Authors even cited one in Discussion (v.291).

M&M

v.84, 88 "benzyl adenine" - not benzyladenine?

Why were the BA concentrations so different for different types of cultures (SS-BA 0.1; LQ-BA 1.0)? What were media composition and BA dose in the case of TIS cultures? There was 10 ml of medium per explant in SS and LQ cultures, while in the case of TIS - 80 ml. Why were such differences? Please explain.

v.107-9 "All cultures were maintained under white light-emitting diodes (LED, 400-700 nm, 107 PSLED-1203) with the light intensity of 40 μmol m-2 s-1 photosynthetic photon flux density 108 (PPFD) and under the 16 h photoperiod."

-any information about the spectrum composition of these LEDs (blue, red, far red)?

-strictly speaking light intensity (W/m2) is not PPFD...

> 16 h/8 h (d/n) photoperiod

v.108 Fo > F₀

v.143 "garden soil (Cocopeat 51%, Peat moss 10%,...)' It is not garden soil but substrate!

v.147 "growth chamber of 80% humidity, 300 μmol m-2 s-1 PPFD, and at a temperature of 25 ± 2 oC." What about photoperiod?

Results

Did You observe regeneration of both axillary and adventitious shoots, direct and indirect regeneration? Did you count axillary and adventitious shoots separately? Have You found any somaclonal variants?

Poor description of the results. Detailed data can be provided in additional tables rather than in the text. Much more important is the information whether there were statistically significant differences among the systems in terms of the examined traits or not. Why did the Authors use statistical analyses if they do not include them in the description of the results?

v.161-166 Basically it is a repetition of data presented in Fig. 1. No information about significant differences among treatments!

v.161 The number shoots regenerated on semisolid (SS) cultures of shoots?

v.161-4 "The number shoots regenerated on semisolid (SS) cultures 161 were 6.93, whereas 3.13 and 3.03 shoots regenerated from shoot tip explants in liquid and 162 TIS cultures respectively (Fig. 1). The fresh weight (mass) of shoots (single shoot or clumps of shoots?) was 0.93, 1.2 and 0.59 g and 163 dry weight of shoots were 931.53 error???? (- inconsistent with Fig1 - 100% of dry mass content?), 1.64 and 0.55 mg with the SS, liquid and TIS cultures respectively."

I suggest calculation the dry matter content of the shoots. It is 9,8%, 9,9% > 8,7% for SS, LQ and TIS.

v.166-7 "However, 3.33 and 4.22% of shoots demonstrated hyperhydricity regenerated in SS and liquid cultures, whereas shoots regenerated on TIS cultures were healthy without any hyperdyricity." Were the difference significant or not!? Do calculate it (test on difference between two proportions).

v.170 The number of leaves with shoots was 26.80, 18.60, and 16.33 with SS, liquid, and TIS cultures. What does it mean: "The number of leaves with shoots". The number of leaves whih developed adventitious shoots?

v.171 -2 "The length of leaves with regenerated shoots was 11.33, 19.32, and 13.60 mm, whereas the width of the leaves was 6.33, 1.36 error????, and 9.44 mm respectively with SS, liquid, 172 and TIS cultures".

Lack information about significant differences among treatments!

Consider calculation of leaf width/length ratio (and area of leaf blade). W/L_R are 0.559 , 0.07????, 0.69 for SS, LQ and TIS respectively. May be there is an error for LQ leaf length?

v.188-3 "The growth characteristics such as total fresh weight (0.87 g), dry weight (75.80 mg), plantlet height (88.87 mm), shoot height (74.88 mm), number of roots (7.85 per shoot), root length (18.80 mm), petiole length (42.85 mm) leaf length (22.78 mm), leaf width (14.21 mm), leaf area (206. 94 mm2) and leaf index (1.62) were all optimum? with plantlets grown under TIS cultures when compared to SS and liquid cultures." Description too sparse. In some cases there were no proved differences among treatments. Moreover, this is not a description of the results but an assessment of them. Therefore it fits into Discussion not Results.

v. 218-220 "The highest value was recorded with the plants regenerated using the TIS system and the values Fo, F₀ Fm, Fv, and Fv/Fm were 8627.80, 48778.63, 40150.88, and 0.82 respectively." Not necessarily the highest (significantly highest), because the mens were marked with the same letters, e.g. for Fv/Fm - no proven differences, the same letters for LQ and TIS in the case of Fm and Fv.

v.242 garden soil??? It was not soil but substratum!

Discussion

The first part of the Discussion is more suited to Introduction.

v.269-70 "these systems enhance the gaseous environment and offer the most natural setting for the in vitro culture of plant regeneration at both shoot proliferation and rooting of shoots." > more natural... than… ?

v.278 "number of shoots, fresh and dry weight, and length of shoots were higher in SS and liquid cultures when compared to the TIS system." Be more precise in description of significant differences.

v.279-281 "However, the 3.33% and 4.22% shoots regenerated in SS and liquid cultures showed hyperhydricity. An optimum of 3.03 shoots was regenerated from the shoot tip explants G. jemesonii cv. ‘Shy Pink’ in TIS and they were healthy and did not show any hyperhydricity. What was medium composition and BA dose in the case of TIS cultures? Was 3,33% difference between SS and TIS cultures statistically significant? The shoot proliferation ratio of SS cultures was higher than TIS, even after taking into account 3% of vitrified shoots (6,93-3,33%>3,03...).

v.287 "In addition, exposure of explants to proper aeration during growth and differentiation in vitro will help in the improvement of the morphology and physiology of shoots [5, 10]. Were the SS shoots incorrectly formed? Did you find any morphological or physiological disorders?

v.311-313 "However, the levels of carotenoid, chlorophyll a, and chlorophyll b in the leaves of G. jemesonii cv. "Shy Pink"??? plants were at their peak when the plants were regenerated using the TIS method. This could be because the TIS culture conditions enhanced the biochemical capacity of the regenerated plants." Lack of proved significant differences!

v.322-4 "which results in a low transpiration rate [11,28]. In contrast to plants regenerated with SS and liquid cultures, the G. jemesonii cv. "Shy Pink" plants that were regenerated in the TIS in this study favor a low stomatal index and a high percentage of closed stomata." Hm, and lower dry matter content in TIS shoots than in other cultures (8,7% <9,8%). Effect or cause? By the way, what is stomatal index and percentage of closed stomata in the case of conventionally propagated gerbera plants?

v.307 "Plants! that can produce larger amounts of these pigments in micropropagated plants! would fare! better when transplanted into an ex-vitro environment." faulty style

v.330 "Comparing plants regenerated with SS and liquid cultures to those regenerated with the TIS technique, the latter showed a hundred percent survival rate four weeks following transplantation to garden soil?." What were survival rates for SS and LQ plantlets? Were the differences significant?

Hmm, without specifying the composition of the medium used in TIS cultures, it is difficult to assess whether it is a impact of the system or the influence of a different medium... It is worth to compare the cost-effectiveness of all systems. For example, the shoot proliferation ratio for SS was over 2 times higher (6.7>3) and the medium consumption was 8 fold lower while compared to TIS (except for agar).

Without cost/economics analysis, and taking into account the results of statistical analyses, the final conclusion is not sufficiently justified.

Best wishes!

Author Response

Response to reveiwer comments

Manuscript ID: horticulturae-3127702
Type of manuscript: Article
Title: A comparison of semi-solid, liquid and temporary immersion bioreactor systems for effective plant regeneration of Gerbera jamesonii cv. ‘Shy Pink’

With reference to the above, authors are thankful to anonymous reviewers for their valuable comments on the manuscript. We have revised the manuscript in the light of comments of the reviewers and incorporated all the suggestions given by them. All the changes made in the revised manuscript are presented in the tenure track format. Following are the specific responses for the quires raised by the reviewers.

Reviewer #1

Query #1. Title: A comparison……

Answer: The title has been changed as ‘A comparison of semi-solid, liquid and temporary immersion bioreactor systems for effect micropropagation of Gerbera jamesonii ‘Shy Pink’ as suggested by reviewer.

Query #2. Abstract: v.3., 18, 28, 80…..

Answer: Gerbera jamesonii ‘Shy Pink’ has been used consistently throughout the manuscript as per the suggestion.

Query #3. Why to do you abbreviations for TIS and SS cultures but not for liquid ones?...

Answer: The abbreviation ‘LQ’ was used consistently throughout the manuscript as per the suggestion.

Query #4. v.21 ‘even though the number of ….

Answer: As suggested the sentence has been modified as ‘even though the number of shoots regenerated was less’.

Query #5. v.24 “The number of stomata ….

Answer: The number of stomata with the plants regenerated with SS, LQ and TIS cultures were 21.40, 14.50 and 11.40 respectively.

The frequency of closed stomata with plants regenerated with SS, LQ and TIS cultures were 13%, 15% and 59% respectively.

Query #6. v.67. ‘Gerbera jamesonii Bolus ex Hooker f. ‘Shy Pink’..

Answer: In botanical names the word ‘ex’ is mainly used in circumstances in which there is an author (or authors) who only contributes the name of a taxon, while it is a different author (or authors) who actually validly publishes that name. Whereas ‘f’ refers to f. (after a personal noun) = fìlius: son. See Scientific Names.

Query #7. Introduction. v.72 ‘although micopropagiaon systems using temporary immersion cultures have not been tested …..

Answer: Yes, we agree few studies have been caried out on micropropagation of gerbera using liquid cultures. In the revised manuscript the sentence has been changed as ‘A method of micropropagation using various explants in several cultivars of G. jemesonii has been established [2], although micropropagation systems using temporary immersion cultures have tested in few cultivars. However, comparison of SS, LQ and TIS cultures have not been tested’. Because of this, the main goal of the current study was to micropropagation G. jemesonii ‘Shy Pink’ in temporary immersion bioreactors using the SETIS® system. Following that, the results of this approach are compared with those of conventional methods that entail LQ and SS cultures for plantlet regeneration.

Query #8. M&M. v. 84, 88 “benzyl adenine’ – not benzyladnine?

Answer: The word is presented as benzyladenine as suggested.

Query #9. Why were the BA concentration so different for different types of cultures (SS-BA 0.1; LQ-BA 1.0?....

Answer: It was typographical error and we have used in all the three types of cultures i.e., SS, LQ and TIS cultures MS medium supplemented with 0.1 mg L^-1 benzyladenine. We have carried out correction in this regard in the revised manuscript.

The utilization of medium depends upon the culture vessel – in SS and LQ cultures 5 shoots were cultured in 50 mL of medium, whereas in TIS cultures 50 shoots were cultured using 500 mL of medium.

Query #10. v. 108-9 ‘All cultures were maintained under white light-emitting diodes (LED, 400-700 nm,….

Any information about spectral composition of these LEDs..

Answer: White LED lights often mean ‘full spectrum’ or ‘broad spectrum’ lights that similar to sunlight or fluorescent light. Hence, the wavelength of the white light is 400 – 700 nm.

Strictly speaking light intensity (W/m²) is not PPFD.

Answer: We agree with this point.

16h/8h (d/n) photoperiod

Answer: We incorporated this correction.

108 Fo > F_o

Answer: We incorporated this correction.

v.143 “garden soil (Cocopaet 51%, peat mass……

Answer: We changed the word garden soil to potting medium or substrate.

v.147 “growth chamber of 80% humidity, …….

Answer: Photoperiod was ‘16h/8h (d/n) photoperiod’ and this correction is incorporated.

Query #11. Results

Did you observe regeneration of both axillary and adventitious shoots,…..

Answer: We observed only direct regeneration of axillary shoots, we have not observed the regeneration adventitious shoots. We have not carried out experiments on somacloanl variation among the regenerants.

Query #12. Poor description of the results.

161-166 Basically it is a repetition of data present in Fig. 1.

Answer: We carried out Dununs multiple comparison test for the data and the data which were presented with the figures and in the text are statistically significant. We improved the data presentation in the revised manuscript.

Query #13. The number of shoots regenerated on semisolid (SS) cultures of shoots?

Answer: We carried out corrections in the revised text.

Query #14. v. 161-4.’The number of shoots regenerated on semisolid (SS)…….

Answer: There was error in the presentation of dry weight data. The fresh weight of shoots was 0.93, 1.52 and 0.59 g and dry weight of shoots were 131.53, 1.64 and 0.55 mg with the SS, LQ and TIS cultures respectively. Correct data has been presented now in the revised text.

Query #15. v.166-7 ‘However, 3.33 and 4.22% of shoots……

Answer: The data on hyperhydricity were statistically significant according DMRT.

Query #16. v.171. The number of leaves with shoots was 26.80, 18.60……….

Answer: The number of leaves measured were with axillary shoots developed from the explants.

Query #17. ‘The length of leaves with regenerated…

Answer: There was error in the presentation of width of the leaves. The with the of the leaves with regenerated plants was 6.33, 13.6, 9.44 mm respectively with the shoots regenerated with SS, LQ and TIS. This is presented correctly in the revised text.

Query #18. v. 188-3. ‘The growth characteristics such as total fresh weight…..

Answer: The total fresh weight, plantlet height, shoot height, root length, petiole length, leaf with and leaf area and leaf index were statistically significant according DMRT (Table 1) and this data is presented in the results section.

Query #19. 216-220 ‘The highest value was recorded…….

Answer: The greatest value was found in the plants that were regenerated utilizing the TIS technique; the corresponding values for F_o, Fm, and Fv were 8627.80, 48778.63, 40150.88, in that order, in that order.

Query #20. v.242 garden soil???

Answer: The terminology potting medium/substrate has been used instead of garden soil.

Query 21. v. 269-70. ‘these systems enhance….

TIS systems enhance the gaseous environment and offer the most natural setting for the in vitro plant regeneration at both shoot proliferation and rooting of shoots compared to SS and liquid cultures.

Query #22. v.278 ‘number of shoots, fresh and dry weight…

Answer: The number of shoots regenerated, fresh and dry weight were significantly higher with the shoots regenerated with SS and LQ cultures as compared to TIS cultures.

Query #23. v. 279-281. “However, the 3.33%....

Answer: The medium composition was similar with all the cultures (SS, LQ and TIS) viz. MS medium supplemented with 0.1 mg L^-1 BA. We observed hyperhydricity of regenerated shoots with SS (3.33%) and LQ (4.22%) and this data was statistically significant according DMRT.

Query #24. v.287 In addition, exposure of explants to proper aeration …..

Answer: Shoots regenerated in SS and LQ cultures showed morphological disorders such as glassy appearance. Their stems and leaves are often thick, rigid and easily breakable.

Query #25. v.311-313. ‘However, the levels of carotenoid, ….

Answer: The leaves of plants regenerated with TIS cultures depicted higher levels of carotenoid, chlorophyll a, chlorophyl b and this might be because of enhanced biochemical capacity of regenerated plants. However, these values were not statistically significant.

Query #26. v.322-4. ‘which results…..

Answer: The number of stomata or stomatal frequency with the plants regenerated with SS, LQ and TIS cultures were 21.40, 14.50 and 11.40 respectively.

The frequency of closed stomata with plants regenerated with SS, LQ and TIS cultures were 13%, 15% and 59% respectively.

However, we have not counted the stomatal frequency and number of closed stomata in the case of conventionally propagated gerbera plants.

Query #27. v.307 ‘Plant that can produce larger…

Answer: The statement has been removed as per the suggestion.

Query #28. V.330. ‘Comparing plant regenerated …

Answer: The survival rate of shoots regenerated in SS and LQ were 85% and 80%, whereas plants regenerated with TIS showed cent percent survival and these values were statistically significant.

Query #29. Hmm, without specifying the composition of the medium…

Answer: The composition of the medium was similar in all the three types of cultures (SS, LQ and TIS) vis. MS medium supplemented with 0.1 mg L^-1 BA. We did not compare the cost analysis of regenerated plants.

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

Dear Authors, please find in the attached file my suggestions and questions for the manuscript entitled A comparison of semi-solid, liquid, and temporary immersion bioreactor systems for effective plant regeneration of Gerbera jamesonii cv. ‘Shy Pink’.

Comments for author File: Comments.pdf

Author Response

Response to reveiwer comments

Reviewer #2

Query #1. Lines 19-21. The number of shoots…

Answer: The number of shoots regenerated in each culture system is presented in the revised manuscript as per suggestion.

Query #2. Lines 23-24. Please rephrase ….

Answer: The sentence has been rephrased as per suggestion.

Query #3. In line 41-42…

Answer: More number of references have been included to substantiate the statement as per suggestion.

Query #4. Lines 50-54

Answer: The references have been added as per suggestion.

Query #5. The authors ….

Answer: The photo of Gerbera jamesonii ‘Shy Pink’ is incorporated as Figure 1 as per the suggestion. The remaining figures are renumbered accordingly.

Query #6. Lines 173-198. Figure 2 ….. and Query #7. Line 235 Fig. 6. …

Answer: The figures are formatted as per suggestion and resolution has been fixed asp MDPI author’s guide.

Query #8. Lines 260-261 please defend this claim….

Answer: Suggestion is incorporated.

Query #9. Lines 284-285. Please explain…

Answer: Suggestion is incorporated.

Query #10. This section …….

Answer: The novelty of research is substantiated as per suggestion.

Query #11. The references list….

Answer: The reference list in enriched with recent articles.

Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

The Authors improved the introductory parts of the work (Introduction, M&M) well enough. Of course, there are a few minor mistakes to correct. However, they did not act on my comments regarding Results. Therefore, I still cannot accept the manuscript in such state. :/

Please consider my comments and suggestions:

Keywords: remove: micropropagation (the same in the Title);

v.46 "the cost of the gelling agent is another element contributing to the increase in production costs" > important element ?

You don't have to automatically change "liquid" into "LQ" everywhere, for example: v.52 "continuously submerged in LQ cultures"; v.57 "tissue to be immersed in a LQ medium", v.95; v.97; v.101, ... > in liquid?

v. 71 "‘Gerbera jamesonii Bolus ex Hooker f. ‘Shy Pink’" move the full name to M&M

v.77 "However, comparison of SS, LQ and TIS cultures have not been tested." > carried out/performed?

v.104-6 "the explants were immersed in the liquidMS LQ medium containing 0.1 mg L-1 benzyladenine for 10 min for every 3 h. Fifty shoot tip explants were used for shoot multiplication in TIS and 500 mL MS medium containing 0.1 mg L-1 benzyladenine was used." repetition of information - remove one.

v.122 "were measured at the shoot multiplication stage." > were measured at the end of shoot multiplication stage.?

v.126 "were measured at the rooting of shoots stage." > were measured at the end of rooting of shoots stage.?

v.30 "The ground state fluorescence (Fo)" - not 'o' but '0'(zero)!

v.152 "potting medium/substrate" - do decide: potting medium or potting substrate

Results

"Answer: We observed only direct regeneration of axillary shoots, we have not observed the regeneration adventitious shoots. We have not carried out experiments on somacloanl variation among the regenerants." Do include this information at the beginning of Results.

Still poor description of the results! It was not corrected! The Authors still provide the same information in the text and figures. There is no point in entering, for example, the fresh weight (mass?) [mg] to the second decimal place. Why provide exact values when you can read them from a chart!? However, there is no information whether these differences were statistically significant or not! E.g. SS cultures produced twice as many shoots compared to LQ and TIS cultures. Proliferation of shoots in SS cultures was then significantly better than other cultures. Fig2. Both fresh and dry mass of TIS shoots were significantly the smallest, whereas those of LQ were the largest. The difference between SS and TIS cultures in the number from vitrified shoots were not statistically proven (the same letter b)! The same lack of statistically proven differences for petiole and shoot length! [Fig.2].

"Query #16. v.171. Answer: "The number of leaves measured were with axillary shoots developed from the explants."!? - still unclear, please simplify; numer of leaves per culture?

"Query #18. v. 188-3. Answer: The total fresh weight, plantlet height, shoot height, root length, petiole length, leaf with and leaf area and leaf index were statistically significant according DMRT (Table 1) and this data is presented in the results section." Please put this information into the text! However, the description of the results of statistical analyzes is too general. Please provide a more detailed description.

"Query #19. 216-220 Answer: The greatest value was found in the plants that were regenerated utilizing the TIS technique; faulty style :/

"Answer: We carried out Dununs multiple comparison test for the data and the data which were presented with the figures and in the text are statistically significant. We improved the data presentation in the revised manuscript. " No, they are not always statistically significant! You must indicate exactly which differences are significant and which are not.

"Query #23. v. 279-281. “However, the 3.33%.... Answer: The medium composition was similar with all the cultures (SS, LQ and TIS) viz. MS medium supplemented with 0.1 mg L-1 BA. We observed hyperhydricity of regenerated shoots with SS (3.33%) and LQ (4.22%) and this data was statistically significant according DMRT." - No, there was not proven difference between TIS (0 b) and SS (3,3% ab) - the same letter 'b' !!!!. There was proven difference beween LQ (4,2% a) and TIS (0 b).

v.340 "The two main pigments ..." > The two main groups of pigments?

v.344 "between the plants that were regenerated" > among?

v79-81 remove: Micropropagation of horticultural plants using a TIS overcomes the limitations of the SS system by reducing production costs and making scaleup and full automation possible for mass plant production [34, 35]."

v.178 hyperdyricity?

"Query #24. v.287 .. Answer: Shoots regenerated in SS and LQ cultures showed morphological disorders such as glassy appearance. Their stems and leaves are often thick, rigid and easily breakable." Put this statement in the text. Would Youlike to include any photographs?

v.210-215. Exactly the same information in the text and Table 1. Why repeat them? Wouldn't it be better to write that plantlets rooted in TIS cultures were generally the largest and those in SS cultures were the smallest? [Table 1]? The only no significant differences were found among cultures in the case of leaf index

v.228-234. Information about the lack of proven differences among plants in terms of the content of assimilation pigments should be provided in the text!

v.243 "with different culture systems were measured and data has been presented in Table 2. The highest greatest value was recorded with found in the plants that were regenerated usingutilizing the TIS system andtechnique; the corresponding values for Fo, Fm, and Fv, and Fv/Fm." No , F0, Fm, and Fv measured for TIS plantlets were generally significantly higher than those for SS plantlets! The values marked for LQ plantlets were mostly intermediate. BTW - a higher level of fluorescence indicates greater stress, so it is not clearly beneficial! I Wonder what about Fv/F0? Contrary to the Authors' statement, the differences in the Fv/Fm parameter were not statistically significant!!! This must be stated in the text! And again, there is no point in giving averages with an accuracy of two decimal places.

The same comments for analysis of stomatal index. There is no statement in the text whether the differences between plantlets were statistically significant, or not.

v.272 still 'garden soil' :/

Discussion and Conclusions

Without taking into account the results of the statistical analyses, the Discussion and Conclusions will be flawed!

v.374-7 "The results of current experiments have demonstrated that TIS is an efficient system for mass propagation of G. jemesonii ‘Shy Pink’ plantlets as compared to the conventional semisolid and liquidLQ cultures. Regeneration, growth, and accumulation of biomass by the plantlets were optimal in TIS." No there were not optimal in proliferation stage ! The better results were obtained for SS cultures in that stage - 2x greater efficiency (shoot proliferation) with a negligible increase in vitrification.

v.378. "The plantlets obtained from TIS successfully adapted and had the highest survival rate during the acclimatization stage." Where are such results presented? Was survival rate of TIS plantlets SIGNIFICANTLY higher than LQ and SS plantlets?

Or maybe it is better to recommend SS cultures in the proliferation stage and TIS cultures in the rooting stage?

Still a lot to improve... Good luck!

Author Response

Response to reviewer comments

Manuscript ID: horticulturae-3127702-R1
Type of manuscript: Article
Title: A comparison of semi-solid, liquid and temporary immersion bioreactor systems for effective plant regeneration of Gerbera jamesonii cv. ‘Shy Pink’

Query #1. Keywords: remove micropropagation (the same in the Title);

Answer: As per suggestion micropropagation has been replaced by plant regeneration in the title, keywords and other parts of the text.

Query #2. v.46 ‘the cost of the gelling agent is another element contributing to the increase in production of costs > important element?

Answer: The sentence has been rephrased as per suggestion.

Query #3. You don’t have to automatically change ‘liquid’ into ‘LQ’ everywhere, for example v.52 ‘continuously submerging LQ cultures; v.57 ‘tissue to be immersed in a LQ medium, v.95; v97; v.101 …. > in liquid?

Answer: Changes have been made as per suggestion.

Query #4. v.71 ‘Gerbera jamesonii Bolus ex Hooker f. ‘Shy Pink’ moved the full name to M&M

Answer: Changes have been made as per suggestion.

Query #5. v.104-6 ‘the explants were….

Answer: Changes have been made as per suggestion.

Query #6. v.122 ‘were measured at the root of shoot stage. > were measured a the end of rooting of shoots stage?

Answer: Yes, the changes have been made as per suggestion.

Query #7. v. 126. ‘were measured at the rooting of shoots stage. . > were measured a the end of rooting of shoots stage?

Answeer: Yes, the changes have been made as per suggestion.

Query #8. v.130 ‘The ground sate florescence (F_O) – not ‘o’ but ‘0’ (zero)

Answer: Yes, the changes have been made as per suggestion.

Query #9. v.152. ‘potting medium/substrate’ – do decide: potting medium or potting substrate

Answer: Potting medium is used wherever the work has occurred.

Query #10. Results. ‘Answer: We observed only direct regeneration of axillay shoots …

Answer: This information is included in the revised text.

Query #11. Still poor description of the results….

Answer: The results section is revised suitably and statistically significant results are presented in the light of results obtained.

Query #12. ‘Query #16 (on earlier manuscript) v. 171. Answer: the number of leaves measured with ….

Answer: The number of leaves per culture which were measured were with the newly regenerated shoots from the shoot tip explants and this information is rewritten in the revised manuscript.

Query #13. ‘Query #18. v.188-3 (on earlier manuscript). Answer: …….

Answer: The suggested information has been presented in the results section as suggested.

Query #14. Query #19. 216-220 (on earlier manuscript). Answer: The greatest value was …..

Answer: The content mentioned has been rephrased suitably in the revised text.

Query #15. Answer: We carried out Duncan’s multiple range test for the data….

Answer: The data has been represented as per the significant results.

Query #16. Query#23 (on earlier manuscript). v. 279-282…

Answer: The data has been represented in the light statistical analysis.

Query #17. V. 340. ‘The two main pigments .. > The two main groups of pigments?

Answer: Yes, two main groups of pigments. This information is incorporated in the revised text.

Query #18. 344 ‘between the plants that were regenerated’ > among?

Answer: Suggestion is incorporated in the revised text.

Query #19. v.79-81 remove: Microporpagation….

Answer: Suggestion is incorporated in the revised text.

Query #20. v. 178 hyperdyriciy?

Answer: It has been corrected in the revised manuscript.

Query #21. Query #24. v.287 (on earlier manuscript)….

Answer: The information has been incorporated in the revised text.

Query #22. v. 210-215. Exactly the same information in the text and Table 1…..

Answer: The data has been represented in the revised text.

Query #23. v.228-234. Information about the lack of proven differences among plant in ….

Answer: The data has been represented in the revised text.

Query #24. v.243. ‘with different culture systems…

Answer: The information has been represented in the revised manuscript.

Query #25. The same comments for analysis of stomatal index……

Answer: This section has been presented in the light of statistical data.

Query #26. Still ‘garden soil’

Answer: The words are replaced by ‘potting medium’.

Query #27. Discussion and conclusion. Without taking into account the results of the statistical analyses, ….

Answer: We improved the discussion and conclusion section in the light of statistical data.

Query #28. V.374-7. The results of current …..

Answer: The suggestions are implemented in the revised manuscript.

Query #29. v.378. The plantlets ……

Answer: This section has been revised as per suggestion.

Author Response File: Author Response.pdf