Review Reports

Round  1

Reviewer 1 Report

It is a well conducted study but will need a good proof reading.

Few corrections:

Page 2, line 88- replace 1s wiith 1 a

Page 2, line 91- clarify new explants?

Page 3, line 102- Reniform?

Clarify if light or dark assisted with the embryo formation/maturation- provide data

Did the histological analysis provide source of embryo formation?

Page 3- line 123- typo

It would be nice to explain why DKW performed better than MS and B5

Page 4, line 138- arrange figures in order 1b, 1d - where is 1c?

Page 6, line 172- 2,4D has to be removed from the medium - so its obvious embryo maturation failed

strengthen the discussion further by adding more details on response to the medium and better clarity on histological analysis.

Author Response

Point 1: Page 2, line 88- replace 1s with 1a.
Response 1: Thanks for your suggestions. There must be a misunderstanding here. The initial materials used for the study were immature seeds (Fig. 1a), while the callus induction materials were the stems of well-developed seedings (Fig. 1o). Thus, we delete the word “initial” to avoid misunderstanding. (P2, line 86).
Point 2: Page 2, line 91- clarify new explants?
Response 2: Thanks for your suggestions. According to your suggestions, we insert the word “stem” in order to make the readers understand better (P2, line 89-90).
Point 3: Page 3, line 102- Reniform?
Response 3: Thanks for your suggestions. According to your comments, we re-write the sentence (P3, line 99-101).
Point 4: Clarify if light or dark assisted with the embryo formation/maturation- provide data.
Response 4:  Thanks for your suggestions. According to your comments, we insert a new table (Table 4), and make a detail illustration about the effect of light on the somatic embryos maturation in the results (P6, line 183-192).
Point 5: Did the histological analysis provide source of embryo formation?
Response 5:  Thanks for your suggestions. We think the reviewers maybe want to ask us where the somatic embryo originated in the explant. In our study, the explant produced calluses firstly, and then procambia was produced from the calluses, which was shown in Fig. 3a. And it obviously belongs to indirect somatic embryogenesis. And we will make a detailed observation about the origin of the procambium in our subsequent experiments.
Point 6: Page 3- line 123- typo
Response 6: According to your comments, we revised the mistake. (P3, line 122-123).
Point 7: It would be nice to explain why DKW performed better than MS and B5
Response 7: Thanks for your suggestions. According to the results of our pre-experiment, we found that the calluses induction rate was highest in DKW medium, and embryogenic calluses were only obtained on DKW medium. So we speculated that DKW medium is more suitable for callus induction in K. paniculata. However, we ignored the role of PGRs. Thus we revise the conclusion of this part to make our study more precise (P4, line 138-140, 142-143). And in order to provide better reference for readers, we also make a careful analysis about the difference among the three basic media in our discussion (P12, line 311-321).
Point 8: Page 4, line 138- arrange figures in order 1b, 1d - where is 1c?
Response 8: Thanks for your suggestions. According to your comments, we all think that some pictures in Fig. 1 are repetitive and have no value to show, so we adjusted Fig. 1 and figure legend (P5, line 167-173).
Point 9: Page 6, line 172: 2,4-D has to be removed from the medium - so its obvious embryo maturation failed.
Response 9: Thanks for your suggestions. We think the reviewer maybe want to ask why we don't use 2,4-D during somatic embryos maturation. 2,4-D is an important plant growth regulator during somatic embryogenesis. And many studies have shown that it plays a great role in embryogenic calluses induction and somatic embryos development (Ahn et al. 2017; Nunes et al. 2018; Prakash and Gurumurthi 2009), which also was shown in the article (P11, line 270-280). And the development and maturation of somatic embryos are two independent processes. As far as we know, there is no relevant literature to show that 2,4-D has an effect on the maturation of somatic embryos in woody plants (Corredoira et al. 2013; El-Mahrouk et al. 2010; García-Mendiguren et al. 2016; Jayanthi et al. 2015; Martínez et al. 2017; Nunes et al. 2018; Prakash and Gurumurthi 2009). Thus, we haven't designed experiments to prove its role in embryo maturation. And according to your comments, we will carry out the related research about the effect of 2,4-D on somatic embryos maturation in our subsequent experiment.
Ahn C-H, Tull AR, Montello PM, Merkle SA (2017) A clonal propagation system for Atlantic white cedar (Chamaecyparis thyoides) via somatic embryogenesis without the use of plant growth regulators. Plant Cell, Tissue and Organ Culture (PCTOC) 130:91-101. http://doi:10.1007/s11240-017-1206-7
Nunes S et al. (2018) Somatic embryogenesis of hybrid Pinus elliottii var. elliottii × P. caribaea var. hondurensis and ploidy assessment of somatic plants. Plant Cell, Tissue and Organ Culture (PCTOC) 132:71-84. http://doi:10.1007/s11240-017-1311-7
Prakash MG, Gurumurthi K (2009) Effects of type of explant and age, plant growth regulators and medium strength on somatic embryogenesis and plant regeneration in Eucalyptus camaldulensis. Plant Cell, Tissue and Organ Culture (PCTOC) 100:13. http://doi:10.1007/s11240-009-9611-1
Corredoira E, Valladares S, Martínez MT, Vieitez AM, San José MC (2013) Somatic embryogenesis in Alnus glutinosa (L.) Gaertn. Trees 27:1597-1608. http://doi:10.1007/s00468-013-0907-8
El-Mahrouk M, Dewir YH, Omar AMK (2010) In vitro propagation of adult strawberry tree (Arbutus unedo L.) through adventitious shoots and somatic embryogenesis. Propagation of Ornamental Plants 10:93-98
García-Mendiguren O, Montalbán IA, Goicoa T, Ugarte MD, Moncaleán P (2016) Environmental conditions at the initial stages of Pinus radiata somatic embryogenesis affect the production of somatic embryos. Trees 30:949-958. http://doi:10.1007/s00468-015-1336-7
Jayanthi M, Susanthi B, Mohan NM, Mandal PK (2015) In vitro somatic embryogenesis and plantlet regeneration from immature male inflorescence of adult dura and tenera palms of Elaeis guineensis (Jacq.). SpringerPlus 4:256
Martínez MT, San José MC, Vieitez AM, Cernadas MJ, Ballester A, Corredoira E (2017) Propagation of mature Quercus ilex L. (holm oak) trees by somatic embryogenesis. Plant Cell, Tissue and Organ Culture (PCTOC) 131:321-333. http://doi:10.1007/s11240-017-1286-4
Point 10: Strengthen the discussion further by adding more details on response to the medium and better clarity on histological analysis.
Response 10: Thanks for your suggestions. According to your comments, we modify our discussion part (P11, line 263-266, 267-269, 281-286; P12, line 311-321), and discussed the results of histological analysis in details (P13, line 363-375).

Note: all of the changes (text or location) in our manuscript have been highlighted with yellow.

Author Response File: Author Response.docx

Reviewer 2 Report

Manuscript ID: forests-396664

Somatic embryogenesis: an important pathway in Koelreuteria paniculata Laxm. Xiong Yang et al.

K. paniculata has a difficulty in large-scale propagation because of hard seed shells and long dormancy period. In this manuscript, authors investigate the effects of basal medium and plant growth regulator on callus induction and plantlet regeneration for the efficient propagation by tissue culture technique. Although authors improved the efficiency of somatic embryo production, methods were similar to former reports. However it should be considered that this is a first report for establishment of plant regeneration via somatic embryogenesis in K. paniculata. Nevertheless it is difficult to understand author’s suggestions from their data and additional data is required for their conclusions. Following points should be addressed:

1. Title

Title should be specific, concise and informative. But this title does not indicate the subject matter. Title should be changed.

2. In Abstract, Materials and Methods part is too long. Materials and Methods should be compactly summarized in Abstract.

3. Abstract

Authors describe that roots were sub-cultured on 1/2DKW medium to get complete plantlets. Did authors sub-culture roots? Is it for rhizogenesis? Furthermore, authors describe “Rhizogenesis was induced by clonal culture (on 1/2DKW medium?)” in Materials and Methods, and “For clonal culture, the highest regeneration rate (rhizogenesis?) was observed on 1/4DKW” in Results. Readers will get confused. Abstract should be changed to more accessible.

4. P2L68-70:

Are plant growth regulators different from hormone? If these indicate same mean, term should be unified.

5. P2L77:

The sentence should be changed to “embryo development was completed”.

6. P3L99:

What is a development medium? Is this medium used for regeneration? If development medium is used for regeneration, “regeneration medium” is easy to understand.

7. How authors calculated the rate of callus induction, percentage of somatic embryogenesis and percentage of rooted plants? Based on the number of explants? Calculation methods should be included in Materials and Methods.

8. Table 1

Authors suggest that callus is efficiently induced by DKW basal medium. However effects of basal media on callus induction could not be correctly evaluated, because concentrations of BA, NAA and 2,4-D were different among basal media. There are no basal media with same concentrations of PGRs. For evaluating the effects of basal media, basal media with same conditions have to be compared. Additional data should be required.

9. Table 2

Embryogenic callus was efficiently induced by the culture on IEM1 and IEM2, however, IEM3 and IEM4 had negative effects on callus culture, such as production of aerial roots and slow growth. Authors suggest that the higher 2,4-D concentration affected efficient callus induction in IEM1 and IEM2. However, the concentrations of NAA are also different among these media. Efficient induction might be caused by lower concentration of NAA or combination of higher 2,4-D and lower NAA. For elucidating effects of 2,4-D on callus induction, additional data is required.

10. Fig. 1-3

These figures include redundant information. For example, I could get same information for the morphological characteristics of calli from Fig. 1 f-j. Is it necessary? These figures should be constructed by photographs with the minimum necessary information.

Figure parts should be denoted by clear larger letters. Black and white arrows are unclear in Fig. 3. Then it is difficult to understand author’s suggestions.

11. Table 3

Why the concentrations of 2,4-D and NAA are different in this experiment? Effects of NAA and 2,4-D on SE frequency have to be compared with same concentration. Why the optimal concentration of NAA is 10 times lower than that of 2,4-D? Is this difference caused by the efficiency of uptake or responsibility?

12. Although authors suggest that the light condition affects growth of somatic embryos, results are only represented in several photographs (Fig. 1 l, o, p, q). Light condition has strong effects on several tissue culture traits in many plant species. Data for light effects on SE frequency and callus induction should be included in manuscript.

13. Order of Table 4 and Fig. 4 is backward.

Are average rooting rate (Fig.4) and rhizogenesis frequency (Table 4) different or same? There are several discrepancies in this manuscript. Should be checked and corrected. Again, calculation method of these values should be included in manuscript.

14. P10L242

The sentence “Tissue culture technology through SE is an important method of plant regeneration” is difficult to follow and unclear meaning. Please re-write this sentence for clarify.

15. SE is abbreviation for somatic embryogenesis. Nevertheless authors used SE (P10L242…) and somatic embryogenesis (P10L256, L261…). Should be unified.

16. Which compositions are different between DKW and MS medium? Authors described DKW medium contain heavy metals, such as nickel. Is high performance of DKW medium associated with characteristics of K. paniculata as a heavy metal accumulator?

17. In this study, seedlings derived from immature seeds were used as explant. How about the potential of seedlings derived from mature seeds or other tissues as explant sources? Physiological status of explant affects the tissue culture traits, such as callus induction and shoot regeneration. Authors should discuss the effectiveness of their improved method (explant, basal medium and PGRs) in comparison with previous reports.

18. P11L324: Quiroz-Figuerosa et al. (2006)

This citation in text is different from other citations. Please check instruction manual.

19. In clonal culture, authors discuss the effects of basal media (DKW, 1/2DKW, 1/4DKW) on rooting frequency. However, there are no data for this experiments. Data should be included in manuscript.

20. P12L342

1/4KDW? Is it 1/4DKW? Should be corrected.

Author Response

Point 1: Title should be specific, concise and informative. But this title does not indicate the subject matter. Title should be changed.

Response 1: Thanks for your suggestions. According to your comments, we changed the title. (P1, line 2-3)

Point 2: In Abstract, Materials and Methods part is too long. Materials and Methods should be compactly summarized in Abstract. Authors describe that roots were sub-cultured on 1/2DKW medium to get complete plantlets. Did authors sub-culture roots? Is it for rhizogenesis? Furthermore, authors describe “Rhizogenesis was induced by clonal culture (on 1/2DKW medium?)” in Materials and Methods, and “For clonal culture, the highest regeneration rate (rhizogenesis?) was observed on 1/4DKW” in Results. Readers will get confused. Abstract should be changed to more accessible.

Response 2: Thanks for your suggestions. According to your comments, we condense the materials and methods in the abstract. And we are sorry that we make a misunderstanding in the abstract. 1/2 DKW medium was used for somatic embryos germination, and 1/4 DKW medium was used for clonal culture. And we re-write these sentences about somatic embryos maturation and clonal culture in the abstract (P1, line 21-23).

Point 3: P2L68-70: Are plant growth regulators different from hormone? If these indicate same mean, term should be unified.

Response 3: Thanks for your suggestions. According to your comments, we revise the term (P2, line 68).

Point 4: P2L77: The sentence should be changed to “embryo development was completed”.

Response 4: Thanks for your suggestions. According to your comments, we revised the sentence (P2, line 75).

Point 5: P3L99: What is a development medium? Is this medium used for regeneration? If development medium is used for regeneration, “regeneration medium” is easy to understand.

Response 5: Thanks for your suggestions. Development medium was used for somatic embryos differentiation, which was similar with regeneration medium. According to your comments, we replace “development medium” with “regeneration medium” (P3, line 97, 102; P6, line 180; P12, line 349).

Point 6: How authors calculated the rate of callus induction, percentage of somatic embryogenesis and percentage of rooted plants? Based on the number of explants? Calculation methods should be included in Materials and Methods.

Response 6: Thanks for your suggestions. According to your comments, we add detailed formulas in our materials and methods. (P3, line 131-134).

Point 7: Table 1: Authors suggest that callus is efficiently induced by DKW basal medium. However, effects of basal media on callus induction could not be correctly evaluated, because concentrations of BA, NAA and 2,4-D were different among basal media. There are no basal media with same concentrations of PGRs. For evaluating the effects of basal media, basal media with same conditions have to be compared. Additional data should be required.

Response 7: Thanks for your suggestions. We all agree with you. According to the results of our pre-experiment, the callus induction rate was the highest in DKW medium, but the role of PGRs on calluses induction could not be ignored. According to your comments, we revise our conclusions about this part (P4, line 138-140, 142-143). And we will determine the effect of basic medium on SE in our subsequent experiments.

Point 8: Table 2: Embryogenic callus was efficiently induced by the culture on IEM1 and IEM2, however, IEM3 and IEM4 had negative effects on callus culture, such as production of aerial roots and slow growth. Authors suggest that the higher 2,4-D concentration affected efficient callus induction in IEM1 and IEM2. However, the concentrations of NAA are also different among these media. Efficient induction might be caused by lower concentration of NAA or combination of higher 2,4-D and lower NAA. For elucidating effects of 2,4-D on callus induction, additional data is required.

Response 8: Thanks for your suggestions. We all agree with you. The concentration of 2,4-D and NAA all play a role in calluses proliferation. Thus, we revise the result in this part. (P4, line 152-153; P11, line 267-269).

Point 9: Fig. 1-3: These figures include redundant information. For example, I could get same information for the morphological characteristics of calli from Fig. 1 f-j. Is it necessary? These figures should be constructed by photographs with the minimum necessary information. Figure parts should be denoted by clear larger letters. Black and white arrows are unclear in Fig. 3. Then it is difficult to understand author’s suggestions.

Response 9: Thanks for your suggestions. According to your comments, we revise these figures (Fig. 1, Fig. 2 and Fig.3) and the figure legends (P5, line 167-171; P8, line 225-229; P9, line 232-240).

Point 10: Table 3: Why the concentrations of 2,4-D and NAA are different in this experiment? Effects of NAA and 2,4-D on SE frequency have to be compared with same concentration. Why the optimal concentration of NAA is 10 times lower than that of 2,4-D? Is this difference caused by the efficiency of uptake or responsibility?

Response 10: Thanks for your suggestions. The concentration of 2,4-D and NAA was confirmed based on previous research about litchi (Raharjo and Litz 2007; Yu et al. 2000), which also belongs to Sapidaceae. And we all agree with your opinion. Effects of NAA and 2,4-D on SE frequency have to be compared with same concentration. The data in our study can't be used to compare the effects of the two PGRs, so we revise our results and discussion (P6, line 178-179; P11, line 281-286). Comparisons of the two PGRs at the same concentration will be carried out in our subsequent studies.

Raharjo SHT, Litz RE (2007) Somatic embryogenesis and plant regeneration of litchi (Litchi chinensis Sonn.) from leaves of mature phase trees. Plant Cell, Tissue and Organ Culture 89:113-119. http://doi:10.1007/s11240-007-9219-2

Yu C, Chen Z, Lu L, Lin J (2000) Somatic embryogenesis and plant regeneration from litchi protoplasts isolated from embryogenic suspensions. Plant Cell, Tissue and Organ Culture 61:51-58. http://doi:10.1023/a:1006446506041

Point 11: Although authors suggest that the light condition affects growth of somatic embryos, results are only represented in several photographs (Fig. 1 l, o, p, q). Light condition has strong effects on several tissue culture traits in many plant species. Data for light effects on SE frequency and callus induction should be included in manuscript.

Response 11: Thanks for your suggestions. According to your comments, we add the effects of light on the growth of cotyledon embryos (Table 4; P6, line 184-192), and hope to provide readers with a better understanding of the effects of light on somatic embryos maturation. And we all agree with you. Light is an important factor in plant development. However, previous studies have not shown that light is beneficial to callus induction and somatic embryogenesis. In the research about strawberry tree (El-Mahrouk et al. 2010) and Larix kaempferi (Kim 2015), the researchers all pointed out that light inhibited the callus induction and SE. So, we have not conducted experiments on the two stages. However, the effect of light on callus induction and SE in K. paniculate has not been confirmed. And we all think this suggestion can be used for our future research.

El-Mahrouk M, Dewir YH, Omar AMK (2010) In vitro propagation of adult strawberry tree (Arbutus unedo L.) through adventitious shoots and somatic embryogenesis. Propagation of Ornamental Plants 10:93-98

Kim Y-W (2015) Initiation of embryogenic callus from mature zygotic embryos in Japanese larch (Larix kaempferi). Journal of Plant Biotechnology 42:223-227. http://doi:JPB-42-233

Point 12: Order of Table 4 and Fig. 4 is backward. Are average rooting rate (Fig.4) and rhizogenesis frequency (Table 4) different or same? There are several discrepancies in this manuscript. Should be checked and corrected. Again, calculation method of these values should be included in manuscript.

Response 12: Thanks for your suggestions. According to your comments, the order of Table 4 (now Table 5) and Fig. 4 was adjusted. The average rooting rate in Fig. 4 and rhizogenesis frequency in Table 4 (now Table 5) are same. According to your comments, we revise the discrepancies in our manuscript (P10, line 252).

Point 13: P10L242: The sentence “Tissue culture technology through SE is an important method of plant regeneration” is difficult to follow and unclear meaning. Please re-write this sentence for clarify.

Response 13: Thanks for your suggestions. According to your comments, we revise the sentence (P11, line 258-259).

Point 14: SE is abbreviation for somatic embryogenesis. Nevertheless authors used SE (P10L242…) and somatic embryogenesis (P10L256, L261…). Should be unified.

Response 14: Thanks for your suggestions. According to your comments, we revise these terms (P11, line 275, 280).

Point 15: Which compositions are different between DKW and MS medium? Authors described DKW medium contain heavy metals, such as nickel. Is high performance of DKW medium associated with characteristics of K. paniculata as a heavy metal accumulator?

Response 15: Thanks for your suggestions. According to your comments, we make a detailed comparison of the three basic media in the discussion part (P12, line 311-321), and found that we made an imprecise conclusion. Although the DKW medium contains nickel, it lacks cobalt. So, whether heavy metals play a role in this process is not certain, because there are differences in other components among three basic media. And we will make more detailed analysis about the effects of basic medium in our future study, and hope we can find something more meaningful.

Point 16: In this study, seedlings derived from immature seeds were used as explant. How about the potential of seedlings derived from mature seeds or other tissues as explant sources? Physiological status of explant affects the tissue culture traits, such as callus induction and shoot regeneration. Authors should discuss the effectiveness of their improved method (explant, basal medium and PGRs) in comparison with previous reports.

Response 16: Thanks for your suggestions. We all agree with you. The physiological status also has an effect on SE. In the study, immature seeds were selected because the shell of mature seed is hard, which is not good for the experiment. The leaves and stems of adult trees contain more secondary metabolites and brown easily. The leaves of the seedlings had the same results, which brown easily. And this is the first study on somatic embryogenesis in K. paniculate, so there are no previous studies to make a comparison. But we inserted the results of other woody plants in the discussion section for reference (P11, line 263-266, 281-286).

Point 17: P11L324: Quiroz-Figuerosa et al. (2006). This citation in text is different from other citations. Please check instruction manual.

Response 17: Thanks for your suggestions. According to your comments, we revise the citation (P12, line 357).

Point 18: In clonal culture, authors discuss the effects of basal media (DKW, 1/2DKW, 1/4DKW) on rooting frequency. However, there are no data for this experiment. Data should be included in manuscript. P12L342: 1/4KDW? Is it 1/4DKW? Should be corrected

Response 18: Thanks for your suggestions. In our pre-experiment, the 1/4DKW medium is much better than the other two media, so we only provide the results about the 1/4DKW medium. According to your comments, we provide the data as a supplementary table (Table S1). As a reference, we also list the results in response letter.

Author Response File: Author Response.docx

Round  2

Reviewer 2 Report

I re-reviewed a revised manuscript (forests-396664). Manuscript has been carefully revised with incorporating my comments.