Novel R2R3-MYB Transcription Factor LiMYB75 Enhances Leaf Callus Regeneration Efficiency in Lagerstroemia indica
Round 1
Reviewer 1 Report
Detailed comments are presented below:
- In the Introduction, first paragraph, please provide the common name and source (Lagerstroemia indica L. (crape myrtle).
- In the Introduction, reference (Corredoira, E.; Ballester, A.; Ibarra, M.; Vieitez, A.M. Induction of somatic embryogenesis in explants of shoot cultures established from adult Eucalyptus globulus and E. saligna× E. maidenii trees. Tree Physiol. 2015, 35, 678-690) is incorrectly cited (Castanea sativa (6)).
- In the Introduction, second paragraph, in the sentence "the petioles and leaves of poplar triploid (Populus alba × P. glandulosa) × P. tomentosa was a better explant than roots [12]". "was" should be "were", also change "poplar triploid" into "triploid poplar".
- The manuscript should be checked in terms of typo mistakes. For example, in the Materials and Methods section, subsection 2.7., accession number PRJINA883013 should be PRJNA883013 (Please delete I); NL201702 should be NL210702 (both numbers are available in NCBI, which one is correct?), etc.
- I suggest the authors to use the term whole-leaf explants, because whole leaves were used for callus induction (the leaves were not cut into segments?).
- Subsection 2.7, please add the information that Lagerstroemia Jinhuang genome was used as reference genome? Please add that the online tool FGENESH was used to identify the genes (black boxes) close to the insertion sites (10 kb).
- NGS analysis should be described in more details.
- Please provide a subsection on statistical analysis in the Materials and Methods.
- Subsection 3.1., second paragraph, first sentence, please change the sentence or delete "to investigate the efficiency of the callus induction and regeneration system".
- Figure 4 - Use the genus and the species name of the plant when it is used for the first time. In other cases, including Figure and Table captions, please use the whole species name and only the first letter of the genus name. (All species are already listed in Subsection 2.8.)
- Legend of Figure 6 should be shortened.
- English improvement is required.
- Attached file has some comments.
Comments for author File: 
Comments.pdf
Author Response
Dear reviewer,
Thank you so much for your valuable comments and constructive suggestions to improve our manuscript entitled “Novel R2R3-MYB transcription factor LiMYB75 enhances leaf callus regeneration efficiency in Lagerstroemia indica” (Manuscript ID: forests-2173243). According to your suggestions, we have tried our best to revise relevant parts of the manuscript. We hope that you find this revised manuscript satisfactory for publication in Forests.
Author Response File: 
Author Response.docx
Reviewer 2 Report
The manuscript entitled "Novel R2R3-MYB transcription factor LiMYB75 enhances leaf callus regeneration efficiency in Lagerstroemia indica " reported the story about calli induction and regeneration system in crape myrtle. This MS is of interest. The study is well conducted, However, there are a few points that I hope useful for the authors to further improve the paper as below:
1．Please clearly describe the NGS analysis, and show the insertion site by IGV.
2．Did there have any other transformation methods used in Lagerstroemia indica? What’s the advantage of this method?
3．There hadn’t any description of statistical method in any figure legends or method section.
4．Since the author declared that LiMYB75 has no effect in callus induction stage, why did you examine the eight genes expression in the tissues “48 h after Agrobacterium infection”.
5．Many sentences troubled readers, such as “Surprisingly, the frequency of callus regeneration increased from 23.33% (OD600 = 0.6) to 56.67% (OD600 = 0.8) (Fig. 6d-f).” who increased? “Moreover, the expression levels of two ARABIDOPSIS RE-SPONSE REGULATOR (ARR) genes, ARR1 and ARR12, did not alter significantly between wild type and LiMYB75-overexpression leaf explants (Fig 6h).” this indicated what? “LiMYB75 might represent a novel regulatory mechanism” please describe the specific novelty regulatory mechanism. “These plants include Wolffia arrhizal [28], Deschampsia antarctica [29], and Eucalyptus globulus [6].” What’s the meaning? “This was demonstrated in L. indica in our findings.” your findings demonstrated what? please clearly showing. “The main benefit of GFP as a transgenic plant is” “[37] used GFP as a reporter gene to detect foreign genes in transgenic tobacco.”
6．The expression levels of LiMYB75 in leaves and petal have no difference, why did you allege that “leaf was the highest among different tissues.” in figure 5.
7．There have many no sense sentences in the discussion section which is not related to the main topic.
8．The MYB TFs in Arabidopsis directly suppressing LATERAL ORGAN BOUNDARIES DO-MAIN 29 expression resulted with the reduction of callus formation, and LiMYB75 also inhibited the expression levels of LiLBD18, why did the former suppress the callus formation whereas the latter enhance it?
Author Response
Dear reviewer,
Thank you so much for your valuable comments and constructive suggestions to improve our manuscript entitled “Novel R2R3-MYB transcription factor LiMYB75 enhances leaf callus regeneration efficiency in Lagerstroemia indica” (Manuscript ID: forests-2173243). According to your suggestions, we have tried our best to revise relevant parts of the manuscript. We hope that you find this revised manuscript satisfactory for publication in Forests.
The point-by-point responses to the comments and suggestions are listed below.
- Q: Please clearly describe the NGS analysis, and show the insertion site by IGV.
R: Thank you very much for the constructive suggestion. We have described in details of NGS analysis in Subsection 2.7.
- Q: Did there have any other transformation methods used in Lagerstroemia indica? What’s the advantage of this method?
R: Thank you for your kind suggestion. In our previous study, we have used axillary buds of L. indica as recipient material of Agrobacterium tumefaciens infection. The highest transformation efficiency was only 25.93%, and the obtained transgenic plants contain chimeras. the reference “Chen, M.L.; Wang, P.; Lv, F.N.; Li, Y.; Huo, Y.; Wang, Q. Construction of high-efficiency genetic transformation system of Lagerstroemia indica based on axillary buds. Mol. Plant Breed. 2022, 20, 1191-1197. in Chinese.
However, in this study, we have improved the transformation efficiency to 37.71% and positive frequency to 90%. Besides, we have simplified the genetic transformation procedure.
- Q: There hadn’t any description of statistical method in any figure legends or method section.
R: Thank you for your kind suggestion. We have added “Subsection 2.12. Statistical Analysis” in the Materials and Methods.
- Q: Since the author declared that LiMYB75 has no effect in callus induction stage, why did you examine the eight genes expression in the tissues “48 h after Agrobacterium infection”.
R: Thank you very much for your valuable comments. The time point was at callus activation stage and also the co-culture time. Callus activation stage was the original stage of callus induction and regeneration. Therefore, we selected 48 h after Agrobacterium infection
- Q: Many sentences troubled readers, such as “Surprisingly, the frequency of callus regeneration increased from 23.33% (OD600 = 0.6) to 56.67% (OD600 = 0.8) (Fig. 6d-f).” who increased? “Moreover, the expression levels of two ARABIDOPSIS RE-SPONSE REGULATOR (ARR) genes, ARR1 and ARR12, did not alter significantly between wild type and LiMYB75-overexpression leaf explants (Fig 6h).” this indicated what? “LiMYB75 might represent a novel regulatory mechanism” please describe the specific novelty regulatory mechanism. “These plants include Wolffia arrhizal [28], Deschampsia antarctica [29], and Eucalyptus globulus [6].” What’s the meaning? “This was demonstrated in L. indica in our findings.” your findings demonstrated what? please clearly showing. “The main benefit of GFP as a transgenic plant is” “[37] used GFP as a reporter gene to detect foreign genes in transgenic tobacco.”
R: Thank you. We responded to the comments one-by-one as below.
5.1. Q: “Surprisingly, the frequency of callus regeneration increased from 23.33% (OD600 = 0.6) to 56.67% (OD600 = 0.8) (Fig. 6d-f).” who increased?
R: Thank you. We have revised into “Surprisingly, the frequency of callus regeneration of LiMYB75-overexpression whole-leaf explants increased from 23.33% (OD600 = 0.6) to 56.67% (OD600 = 0.8) (Fig. 6d-f).”
5.2.Q: “Moreover, the expression levels of two ARABIDOPSIS RE-SPONSE REGULATOR (ARR) genes, ARR1 and ARR12, did not alter significantly between wild type and LiMYB75-overexpression leaf explants (Fig 6h).” this indicated what?
R: Thank you. We have revised into “ARR1 and ARR12, did not alter significantly between wild type and LiMYB75- overexpressing whole-leaf explants, indicating that these ARR genes might not play a key role in LiMYB75-induced callus regeneration (Fig 6h).”
5.3.Q: “LiMYB75 might represent a novel regulatory mechanism” please describe the specific novelty regulatory mechanism.
R: Thank you. We deleted the sentence.
5.4.Q: “These plants include Wolffia arrhizal [28], Deschampsia antarctica [29], and Eucalyptus globulus [6].” What’s the meaning?
R: Thank you. This sentence has revised into “The most often utilized auxins and cytokinins to induce calli are 2,4-D, NAA, BA, and kinetin, which are artificial plant growth regulators [6, 29-30].” in section 4.1.
5.5. Q:“This was demonstrated in L. indica in our findings.” your findings demonstrated what?
R: Thank you. This sentence has been delated into to make it clearer. Our study's findings demonstrated that 6-BA and NAA combined in various ratios can maximize Lagerstroemia indica ' callus induction rate and callus regeneration rate.
5.6.Q: “The main benefit of GFP as a transgenic plant is” “[37] used GFP as a reporter gene to detect foreign genes in transgenic tobacco.”
R: Thanks for your correction. We regret this typographical error. It has been updated in the text: “Yang et al. [38] used GFP as a reporter gene to detect foreign genes in transgenic tobacco.”
- Q: The expression levels of LiMYB75 in leaves and petal have no difference, why did you allege that “leaf was the highest among different tissues.” in figure 5.
R: Thank you. I appreciate you fixing this mistake. We apologize for our sloppy writing, which we have removed and revised manuscript.
- Q: There have many no sense sentences in the discussion section which is not related to the main topic.
R: Thank you very much for the kind suggestion. We have deleted these sentences throughout the manuscript.
- Q: The MYB TFs in Arabidopsis directly suppressing LATERAL ORGAN BOUNDARIES DO-MAIN 29 expression resulted with the reduction of callus formation, and LiMYB75 also inhibited the expression levels of LiLBD18, why did the former suppress the callus formation whereas the latter enhance it
R: Thank you very much for your comments. This might be because crape myrtle and Arabidopsis are different species, with the former suppressing callus production and the latter promoting it.
Author Response File: Author Response.docx
Round 2
Reviewer 2 Report
The authors have addressed my comments thoroughly.
