Somatic Embryogenesis and Plant Regeneration from Stem Explants of Pomegranate

Round 1

Reviewer 1 Report

Dear authors

The following changes are required::

Abstract

ü  In general, this section is poorly written. It is written simply. This section should include the most important findings from this study. As a result, this section should be improved.

ü  The authors should follow the title in the introduction section. This content of this section should follow the sequence of writing in the materials and method and results

ü  The experimental design and the number of factors should be provided

ü  The aim is not correctly written

ü  The authors should provide the name of methods used in this study

ü  The authors should provide some scored values such as root length, callus induction, embryogenic callus induction, and somatic embryo induction

ü  The authors should present a decisive conclusion that is derived from the research in the final line of the abstract and provide a single line of future prospects.

Keywords

ü  The title words should not be used as keywords. As a result, the keyword structure needs to be changed.

Introduction

ü  Some information about the effects of concentration of different hormones on the callus induction should be added

ü  Some information about the types of organogenesis should be included

ü  Along with the hypothesis statement, the authors should write a few sentences about the knowledge gap that their research has filled.

ü  The authors should also include a statement of novelty at the conclusion. What new research-related activities or correlations have authors made in this study versus earlier ones?

ü  The broad and detailed objectives ought to be included.

Materials and Methods

ü  All abbreviations in Materials and methods should be given in their full name for the first time.

ü  All parts of MM should be supported by the references

ü  The chemical structure of compounds mentioned in this study should be corrected especially in the subscription of number

ü  A section about the statistical data analysis should be added

ü  It is necessary to include the name of the experimental design (One way or Two way ANOVA) and the number of investigated factors.

ü  It is necessary to add the method's name for the means comparison.

Results

ü  The method of the means comparison like LSD, Duncan should be added in the captions of all tables

ü  The signs + and – should be defined as the concentration in the Table 1

ü  It is necessary to include the status of the significant level between various treatments of all parameter studies.

ü  All table captions need to be improved and expanded upon.

Discussion

ü  This section is generally poorly written. Some sentences are not related to the interpretation of results and are written as a review.

ü  The results are reiterated in this section. The significance of each parameter used in this study needs to be explained by the authors.

Conclusion

ü  The authors should summarize the most significant findings because this section has been written simply.

ü  Furthermore, additional works about this research should be added as the future works.

Extensive correction is needed

Author Response

Response to Reviewer 1 Comments

Dear authors

The following changes are required:

Abstract

• In general, this section is poorly written. It is written simply. This section should include the most important findings from this study. As a result, this section should be improved.

Reply - We thank the reviewer for the comment. According to the suggestion, we have rewritten the Abstract section to highlight the improvement of our system compared to the existing protocols and our contribution to the area of pomegranate tissue culture and plant regeneration. The revised Abstract is copied here:

“Abstract: Plant regeneration through somatic embryogenesis provides a solution for maintaining and genetically improving crop or fruit varieties with desirable agronomic traits. For the fruit tree pomegranate (Punica granatum L.), despite of some successful applications, the existing somatic embryogenesis protocols are limited by low availability of explants and susceptibility to browning. To address these problems, in this study, we developed a effective system for induction of high vigor pomegranate somatic embryos derived from stem explant. The usage of stem ex-plant break through the difficulty in material obtaining, thus make our system suitable for wide-spread commercial production. To enhance the performance of our system, we screened the optimal culture condition for each step. The results showed that inoculating stem explants onto a Murashige and Skoog (MS) medium supplemented with 1.0 mg/L 6-benzylaminopurine (6-BA) and 1.0 mg/L 1-naphthaleneacetic acid (NAA) achieved the best induction rate and growth status of pomegranate calli (induction rate = ~72%), and MS medium containing 0.5 mg/L 6-BA and 1.0 mg/L NAA was the optimal condition for the induction of embryogenic calli and somatic embryos (induction rate = ~74% and 79%, respectively). The optimal subculture period for embryo-genic calli was found to be 30-35 days. Strong roots were then induced in the developed somatic embryo seedlings survived and grew at a good condition after transplantation to natural environment, indicating the good vitality of the induced pomegranate somatic embryos. Together, our system provides a solution to mass somatic embryo induction and plant regeneration of pomegranate and lays a foundation for future genetic transformation and bioengineering improvement of pomegranate with favorable agronomic traits.”

• The authors should follow the title in the introduction section. This content of this section should follow the sequence of writing in the materials and method and results

Reply - We thank the reviewer for the comment. According to the suggestion, we have reorganized the Abstract to make sure the logic is more proper and all necessary parts have been included.

• The experimental design and the number of factors should be provided

Reply - We thank the reviewer for the suggestion. In the revised Abstract, we have listed the factors we tested in this study. The relevant sentence is copied here:

“To enhance the performance of our system, we screened the optimal explants, subculture cycles and combination of basal media and plant growth regulators for each step.”

• The aim is not correctly written

Reply - We thank the reviewer for the suggestion. We have rewritten the aim of this study in Abstract. The revised sentence is copied here:

“For the fruit tree pomegranate (Punica granatum L.), despite of some successful applications, the existing somatic embryogenesis protocols are limited by low availability of explants and susceptibility to browning. To address these problems, in this study, we developed an effective somatic embryogenesis system for pomegranate using stem segments as explant.”

• The authors should provide the name of methods used in this study

Reply - We thank the reviewer for the suggestion. Actually, there is no official name for the somatic embryogenesis system. And due to the limited space in Abstract, we listed the details of methods in Materials and Methods section.

• The authors should provide some scored values such as root length, callus induction, embryogenic callus induction, and somatic embryo induction

Reply - We thank the reviewer for the suggestion. We have added these details in the revised Abstract. The relevant sentences are copied here:

“The results showed that inoculating stem explants onto a Murashige and Skoog (MS) medium supplemented with 1.0 mg/L 6-benzylaminopurine (6-BA) and 1.0 mg/L 1-naphthaleneacetic acid (NAA) achieved the best induction rate and growth status of pomegranate calli (induction rate = ~72%), and MS medium containing 0.5 mg/L 6-BA and 1.0 mg/L NAA was the optimal condition for the induction of embryogenic calli and somatic embryos (induction rate = ~74% and 79%, respectively).”

• The authors should present a decisive conclusion that is derived from the research in the final line of the abstract and provide a single line of future prospects.

Reply - We thank the reviewer for the suggestion.

Keywords

• The title words should not be used as keywords. As a result, the keyword structure needs to be changed.

Reply - We thank the reviewer for the comment. We have changed the key words into “in vitro culture; fruit tree; callus induction; plant growth regulators; optimal culture condition” according to the suggestion.

Introduction

• Some information about the effects of concentration of different hormones on the callus induction should be added

Reply - We thank the reviewer for the suggestion. We have expanded our background introductions about the effects of concentration of different hormones on the callus induction in pomegranate. The relevant sentences are copied here:

“Plant growth regulators play important roles in regulating cell growth, division, and differentiation during in vitro plant tissue culture. For example, auxin is usually used for eliciting plant rooting and vegetative propagation from stem and leaf cuttings, and cytokinin is necessary for promoting somatic embryogenesis [18-21]. However, since plant tissues cannot produce auxin and cytokinin under in vitro culture conditions, exogenous supplementation of auxin and cytokinin is critically required for ensuring plant embryogenic callus induction and somatic embryogenesis [22-24].”

• Some information about the types of organogenesis should be included

Reply - We thank the reviewer for the comment. However, in this study, the system we established is for somatic embryogenesis for pomegranate rather than organogenesis. We suppose the wrong manuscript title makes the misleading. In the revised manuscript, we have corrected the title into “Somatic embryogenesis and plant regeneration from stem explants of pomegranate”.

• Along with the hypothesis statement, the authors should write a few sentences about the knowledge gap that their research has filled.

Reply - We thank the reviewer for the suggestion. In the revised manuscript, we have re-written the sentences at end of Introduction to highlight the hypothesis statement and the break though of our study. The relevant sentences are copied here:

“In pomegranate, despite of some successful applications of somatic embryogenesis, its recalcitrant nature post challenges to the somatic embryo induction due to a major reason: the existing protocols mainly employ cotyledonary or leaf explants, however, the low availability of cotyledonary tissues limits the widespread commercial application, while the leaf-derived materials derived from leaves are prone to browning, leading to tissue culture failure [25]. Thus, a stable and effective somatic embryogenesis system is urgently required for pomegranate.

To address the above-mentioned problem, in this study, we aim to establish an effective somatic embryogenesis system for pomegranate using easily acquired explants. And we further explored the optimal culture conditions for the induction and development of somatic embryos, to improve the efficiency of somatic embryogenesis in pomegranate. Our system offers a solution to bridge over the difficulties of mass somatic embryo in-duction and plant regeneration in pomegranate, and also provides a foundation for the future genetic transformation and bioengineering improvement of pomegranate with desirable traits.”

• The authors should also include a statement of novelty at the conclusion. What new research-related activities or correlations have authors made in this study versus earlier ones?

Reply - We thank the reviewer for the comment and suggestion. Accordingly, we have added statements of novelty of our studies in both the last paragraph of Introduction and Conclusion.

• The broad and detailed objectives ought to be included.

Reply - We thank the reviewer for the suggestion. We have revised our wording to make our statement on the detailed and broad objectives clearer. The relevant sentences are copied here:

“To address the above-mentioned problem, in this study, we aim to establish an effective somatic embryogenesis system for pomegranate using easily acquired explants. And we further explored the optimal culture conditions for the induction and development of somatic embryos, to improve the efficiency of somatic embryogenesis in pomegranate. Our system offers a solution to bridge over the difficulties of mass somatic embryo in-duction and plant regeneration in pomegranate, and also provides a foundation for the future genetic transformation and bioengineering improvement of pomegranate with desirable traits.”

Materials and Methods

• All abbreviations in Materials and methods should be given in their full name for the first time.

Reply - We thank the reviewer for pointing this out. We have carefully checked the abbreviation usage throughout the entire manuscript and all the full names have been given when the abbreviations first appear.

• All parts of MM should be supported by the references

Reply - We thank the reviewer for the comment. Necessary citations have been added to the Materials and Methods section.

• The chemical structure of compounds mentioned in this study should be corrected especially in the subscription of number. A section about the statistical data analysis should be added

Reply - We thank the reviewer for the comments. We have added the chemical formula of three plant growth regulators in Methods and Materials section 2.2, when they first appear in the main text. And we also added the details of statistical tests for each comparison in the corresponding section (Method section 2.2, 2.3, 2.4 and 2.6), included the statistic method applied for the test and the statistic program. The relevant sentences are copied here:

“Factorial Analysis of Variance (ANOVA) was performed to assess the statistical significance level among 6 different combinations of basal medium and plant growth regulators (see Table 1 for details) in IBM SPSS Statistics (IBM Corp. New York, USA). Duncan’s Multiple Range test (DMRT) was then conducted to measure the specific difference be-tween each pair of culture conditions.”

“Statistical differences among 9 combinations of different 6-BA and NAA concentrations (detailed in Table 2) were measured by two-way ANOVA using SPSS software. Multiple comparison procedure was performed by the post hoc DMRT.”

“The difference in induction of embryogenic calli between the two types of explants was evaluated by one-way ANOVA in SPSS software.”

“Two-way ANOVA was implemented to quantify the statistically significant level among the 9 combinations of different 6-BA and NAA concentrations (detailed in Table 3), and DMRT was employed to measure the pairwise differences between culture conditions.”

• It is necessary to include the name of the experimental design (One way or Two way ANOVA) and the number of investigated factors.

Reply - We thank the reviewer for the suggestion. We have added the details of statistical tests for each comparison in the corresponding section (Method section 2.2, 2.3, 2.4 and 2.6), included the statistic method applied for the test and the statistic program. The relevant sentences are copied here:

“Factorial Analysis of Variance (ANOVA) was performed to assess the statistical significance level among 6 different combinations of basal medium and plant growth regulators (see Table 1 for details) in IBM SPSS Statistics (IBM Corp. New York, USA). Duncan’s Multiple Range test (DMRT) was then conducted to measure the specific difference be-tween each pair of culture conditions.”

“Statistical differences among 9 combinations of different 6-BA and NAA concentrations (detailed in Table 2) were measured by two-way ANOVA using SPSS software. Multiple comparison procedure was performed by the post hoc DMRT.”

“The difference in induction of embryogenic calli between the two types of explants was evaluated by one-way ANOVA in SPSS software.”

“Two-way ANOVA was implemented to quantify the statistically significant level among the 9 combinations of different 6-BA and NAA concentrations (detailed in Table 3), and DMRT was employed to measure the pairwise differences between culture conditions.”

It is necessary to add the method's name for the means comparison.

Reply - We thank the reviewer for the suggestion. Duncan’s Multiple Range test (DMRT) was then conducted to measure the specific difference be-tween each pair of culture conditions. In revised manuscript, we have added this detail in the corresponding section (Method section 2.2, 2.3, 2.4 and 2.6).

Results

• The method of the means comparison like LSD, Duncan should be added in the captions of all tables

Reply - We thank the reviewer for the suggestion. In the study, Duncan’s Multiple Range test was used to measure the mean difference between pairs of culture conditions, and we have added this detail in the captions of each table, following the suggestion.

The signs + and – should be defined as the concentration in the Table 1

Reply - We thank the reviewer for the suggestion, and we have revised Table 1 accordingly.

• It is necessary to include the status of the significant level between various treatments of all parameter studies.

Reply - We thank the reviewer for the comment. We have added the information of significant levels at the right place of the main text. The relevant sentences were copied here:

“Among the 9 combinations, the medium supplemented with 0.5 mg/L 6-BA and 1 mg/L NAA (B2) was most suitable for the induction of pomegranate embryogenic calli, where the induction rate (74.44 ± 5.09%) was significantly higher than all the other culture conditions (p < 0.05)”

“In contrast, the induction using stem segments performed better than that using hypocotyls, with a significantly higher induction rate of embryogenic calli (80.00 ± 4.95%; p < 0.05)”

• All table captions need to be improved and expanded upon.

Reply - We thank the reviewer for the comment and suggestion. We have expanded table captions to include the details of statistical test.

Discussion

• This section is generally poorly written. Some sentences are not related to the interpretation of results and are written as a review.

Reply - We thank the reviewer for the comment and suggestion. We have re-written our discussion to remove the sentences not closely relevant to result interpretation, and expanded our discussion by comparing our results to those of previous studies. Now the Discussion section is better organized and the logic is clearer.

• The results are reiterated in this section. The significance of each parameter used in this study needs to be explained by the authors.

Reply - We thank the reviewer for the comment and suggestion. We have removed the redundant description of results in Discussion section and added brief explanation to the impact of each parameter, such as concentration of plant growth regulators and explants, on the somatic embryogenesis in pomegranate. The relevant sentences are copied here:

“In this study, we assessed the performance of the combination of different types of plant growth regulators, 6-BA, NAA and IBA, in pomegranate somatic embryogenesis, and showed that supplementing the MS medium with 6-BA and NAA achieved the best balance between callus induction rate and growth status (Table 1). NAA is a synthetic auxin that is usually used for eliciting plant rooting and vegetative propagation from stem and leaf cuttings [18,19], and 6-BA is plant growth regulator of cytokinin family that can stimulate plant growth and development. However, for both types of plant growth regulators, high concentrations in the medium were found to have adverse impacts on the callus induction and growth. In our system, the appropriate 6-BA concentration for pomegranate somatic embryogenesis is 0.5 mg/L and the optimum NAA concentration is 1.0 mg/L, where the induction rates of calli, embryogenic calli and somatic embryos were ~72%, 74% and 79%, respectively (Tables 1 and 2). It is because that auxins at high concentrations are toxic to plants, while a high cytokinin concentration inhibits auxin polar transport and thus inhibits somatic embryogenesis [21,33,34]”

“In general, all types of plant tissues with strong meristematic ability can be used as explants for tissue culture, but different explants plant have different abilities in forming calli [37]. Jaidka and Mehra [38] demonstrated the success of using pomegranate stem explant in inducing calli when cultured on MS medium containing 4.0 mg/L NAA, 2.0 mg/L Kin and 15% coconut water, indicating the feasibility of using stem explants for pomegranate somatic embryogenesis. In addition, Yang and Ludders [39] reported organogenesis in pomegranate initiated from stem explant. In the current study, we first induced pomegranate somatic embryogenesis using stem segments as explant and showcased a better performance of using stem explant in producing pomegranate calli, in terms of both induction rate and the growth status of embryogenic calli, than using hypocotyls. Although pomegranate calli and somatic embryos can also be generated using other types of explants, such as leaf, shoot tip, nodal segment and cotyledonary tissue, these explants exhibit their own shortcomings: shoot tips are not able to provide enough materials, and the materials from leaves and leafstalk are prone to browning, which leads to tissue culture failure [25]. Comparatively, stem segment is more easily assessable, and the embryogenic calli formed by stem segments did not show obvious browning and grew well. These advantages make stem explants more suitable for initial culture, especially for industrial and commercial production.”

Conclusion

• The authors should summarize the most significant findings because this section has been written simply.

Reply - We thank the reviewer for the comment. Following the suggestion, we re-wrote our Conclusion section to highlight our break throughs in the area of pomegranate somatic embryogenesis. The relevant paragraph is copied here:

“In this study, we established an effective system for somatic embryogenesis and plant regeneration in pomegranate using stem segments as explants. In our system, the MS medium supplemented with 0.5 mg/L 6-BA and 1.0 mg/L NAA achieved the best performance in pomegranate somatic embryogenesis, where the induction rates of calli, embryogenic calli and somatic embryos were ~72%, 74% and 79%, respectively. The good somatic embryo induction indicates the effectiveness and stability of our system. In addition, compared to the existing systems, the stem explant employed in our protocol is more easily acquired and suitable for widespread commercial production. Taken together, our system overcomes the recalcitrant nature of pomegranate and provide a solution to mass somatic embryo induction and plant regeneration of pomegranate.”

• Furthermore, additional works about this research should be added as the future works.

Reply - We thank the reviewer for the suggestion. Accordingly, we added a sentence about our future work in pomegranate area. The relevant sentence is copied here:

“With the help of this somatic embryogenesis protocol, we aim to build a genetic transformation system for future bioengineering improvement of pomegranate with favorable agronomic traits.”

Comments on the Quality of English Language

Extensive correction is needed

Reply - We thank the reviewer for the comment. We have sent our manuscript to Nature Research Editing Service for editing for proper English language, grammar, punctuation, spelling, and overall style by one or more of the highly qualified native English speaking editors (the verification code 304F-6FF1-7EF9-B692-0B23). The quality of English language should match the publication requirement.

Please see the attachment.

Author Response File: Author Response.pdf

Reviewer 2 Report

The MS entitled “Somatic embryogenesis and organogenesis from stem explants of pomegranate” with authors Jingting Wang, Xinhui Xia, Jingwen Tang, Jun Wang, Wenhao Zhu, Ming Qian, Jiyu Li, Guangrong Cui, Yuchen Yang, Gaihua Qin and Jingjing Qian presents new data and is interesting but needs minor improvement to be published.

The pomegranate (Punica granatum L.) belongs to the family Punicaceae, which includes only one genus and two species. Punica is classified as the only genus in its family due to the unique structure of the ovary and fruit. The pomegranate is widely cultivated in India, Southeast Asia, Malaysia, East, tropical Africa, and the United States. Its fruits have high nutritional and medicinal value, and the tree is also used as an ornamental. The fruit is rich in minerals, vitamins, antioxidant polyphenols and tannins. Traditionally, the pomegranate is propagated vegetatively through cuttings, but it takes a long time for these cuttings to be cultivated in the field. Seed propagation is not the preferred propagation method. These difficulties can be avoided by applying biotechnological techniques and developing protocols for rapid and efficient regeneration to cultivate elite genotypes.

The article follows the order required by the journal: Introduction, Materials and Methods, Results, Discussion, Conclusions. Abstract is informative; The introduction is good, but it would be good if the authors added information on tissue cultures of the species; the purpose of the study is not clearly stated and some of the text should be moved to a more appropriate place (e.g. in results). Over 70% of the cited literature is from the last 5 years. However, the quality of the manuscript will improve if the authors use clearer and more precise sentences. It is essential to minor improve Introduction, Purpose, Material and Methods.

I have some comments on the text:

L123 - In my opinion, it is more correct to use the term “Plant growth regulators” or “Phytohormones” instead of hormone

L132 – L135 - The sentence should be moved to Discussion or Results if it is not part of the methods used by the authors

L184 – In Table 1 the concentrations of the phytoregulators used must be indicated.

L218 - The table is redundant, these results are explained in the text

L262 - Is adaptation of seedling not necessary?

L323 - Тhe number of the cited author is missing

The authors should carefully and critically review publications on the subject and highlight more clear their novel contribution.

Author Response

Please see the attachment.

Author Response File: Author Response.docx

Reviewer 3 Report

Lines 34-35: 'Our system provides a valuable model that can facilitate future molecular breeding and genetic improvement of pomegranate with favorable agronomic traits', this is a strong statement for the abstract, considering that you did not conduct such research. 

LInes 83-88: Please state the clear objectives of the study, not the achieved goals. 

Figures are very beneficial. 

Line 323: Please rewrite this sentence: Ashwinikumar et al. revealed that stem segments can be used as explants to induce calli of Rosa rugosa on the medium containing 0.5 mg/L IBA and 0.5 mg/L NAA, indicating the feasibility of using stem explants for pomegranate somatic embryogenesis. Results for Rosa can not directly indicate the same for pomegranate. Instead of pomegranate please consider writing 'other shrub and/or woody species'.

Lines 325 - 334 please add appropriate references. 

Please supplement the conclusion section with exact data and concentrations of the hormones that provided certain percentages of successfully established, differentiated, rooted plantlets...

Author Response

Please see the attachment.

Author Response File: Author Response.docx

Reviewer 4 Report

The manuscript "Somatic embryogenesis and organogenesis from stem explants of pomegranate" deals with the topic of in vtro conservation in culture and microclonal propagation is one of the oldest known edible fruit tree species - Punica granatum L.. In addition, well-developed somatic embryo production technology can be used also for genetic transformation, e.g. through CRISPR-mediated genome editing, which promotes genetic improvement of favourable phenotypes. In this regard, the chosen topic of the manuscript may be of interest to both breeders and geneticists working with this crop.

The manuscript is very well illustrated and contains a sufficiently large number of experiments to optimise the individual steps of somatic embryoid production and to increase organogenesis from callus when introduced into in vitro culture of stem explants of pomegranate. Nevertheless, the manuscript requires improvement:

1. First of all, it should be noted that the research topic has been studied by various researchers for quite some time, therefore it is imperative to definitely expand the discussion section and compare your experiments with similar experiments already conducted and published by other scientists. Here I think the following review will be very useful:  Jaime A. Teixeira da Silva, Tikam Singh Rana, Diganta Narzary, Nidhi Verma, Deodas Tarachand Meshram, Shirish A. Ranade, Pomegranate biology and biotechnology: A review, Scientia Horticulturae, Volume 160, 2013, Pages 85-107, ISSN 0304-4238, https://doi.org/10.1016/j.scienta.2013.05.017

Special attention should be focused on the plant growth regulators used in nutrient media and their ratio (auxin/cytokinin) for callus induction and somatic embryogenesis in pomegranate. It is desirable for the authors to expand this part of the paper a little.

2. It is necessary to give an extended description of the statistical processing of the data. In what programme it was carried out, what test was used to assess the level of significance. This information should also be included in the notes of Tables 1-4.

3. in Table 4 it is better to present not "Somatic embryo induction percentage(%)", but to indicate the number of somatic embryoids formed from one callus explant.

4 Figure 2, 5, 6 - the scale bar should be placed in the lower right corner

5. L - 59     … CRISPR-mediated genome editing… - the full name of the method should be given

6. L - 100       -  IBA – decipher the first time the abbreviation is mentioned

7. L 112- 113 - (0.5, 1.0 and 1.5 mg/L 6-benzylaminopurine (6-BA), 1-naphthaleneacetic acid 112 (NAA) and indole-3-butyric acid (IBA)) - the transcript is indicated the first time it is mentioned, and it is not necessary to do so thereafter

8. L 115  -….in a 16/8 h light/dark 115 light cycle….. – the intensity of the lighting and which light sources were used should be specified

9. in vivo and in vitro in the text of the manuscript should be written in italics

Author Response

Response to Reviewer 4 Comments

The manuscript "Somatic embryogenesis and organogenesis from stem explants of pomegranate" deals with the topic of in vtro conservation in culture and microclonal propagation is one of the oldest known edible fruit tree species - Punica granatum L. In addition, well-developed somatic embryo production technology can be used also for genetic transformation, e.g. through CRISPR-mediated genome editing, which promotes genetic improvement of favourable phenotypes. In this regard, the chosen topic of the manuscript may be of interest to both breeders and geneticists working with this crop.

The manuscript is very well illustrated and contains a sufficiently large number of experiments to optimise the individual steps of somatic embryoid production and to increase organogenesis from callus when introduced into in vitro culture of stem explants of pomegranate. Nevertheless, the manuscript requires improvement:

Reply - We thank the reviewer for the positive comments and appreciation of our research.

1. First of all, it should be noted that the research topic has been studied by various researchers for quite some time, therefore it is imperative to definitely expand the discussion section and compare your experiments with similar experiments already conducted and published by other scientists. Here I think the following review will be very useful: Jaime A. Teixeira da Silva, Tikam Singh Rana, Diganta Narzary, Nidhi Verma, Deodas Tarachand Meshram, Shirish A. Ranade, Pomegranate biology and biotechnology: A review, Scientia Horticulturae, Volume 160, 2013, Pages 85-107, ISSN 0304-4238, https://doi.org/10.1016/j.scienta.2013.05.017

Reply - We thank the reviewer for the thoughtful suggestion. We have expanded our discussions by clarifying 1) impacts of the types and concentrations of plant growth regulators on pomegranate somatic embryogenesis based on both our own results and previous studies. and 2) the advantages of the explants we used for pomegranate somatic embryogenesis in this study over those used in previous studies. The review suggested by the reviewer is also discussed and cited in Discussion section. The relevant sentences are copied here:

“However, different combinations of plant growth regulators are required for in vitro propagation pomegranate when using different explants [25]. For instance, Kantharajah et al. [36] showed that MS medium supplemented with 1 mg/L 6-BA and 0.4 mg/L NAA gave rise to the best callus initiation and growth from nodal explants, while the medium containing 1 mg/L 6-BA archived the best performance for leaf explant-derived callus culture. These results indicate that the choice of plant growth regulators used in nutrient medium and their ratio (auxin/cytokinin) are largely depending on the morphogenic potentials of the pomegranate explants employed for in vitro culture.”

“In general, all types of plant tissues with strong meristematic ability can be used as explants for tissue culture, but different explants plant have different abilities in forming calli [37]. Jaidka and Mehra [38] demonstrated the success of using pomegranate stem explant in inducing calli when cultured on MS medium containing 4.0 mg/L NAA, 2.0 mg/L Kin and 15% coconut water, indicating the feasibility of using stem explants for pomegranate somatic embryogenesis. In addition, Yang and Ludders [39] reported organo-genesis in pomegranate initiated from stem explant. In the current study, we first induced pomegranate somatic embryogenesis using stem segments as explant and showcased a better performance of using stem explant in producing pomegranate calli, in terms of both induction rate and the growth status of embryogenic calli, than using hypocotyls. Although pomegranate calli and somatic embryos can also be generated using other types of explants, such as leaf, shoot tip, nodal segment and cotyledonary tissue, these explants exhibit their own shortcomings: shoot tips are not able to provide enough materials, and the materials from leaves and leafstalk are prone to browning, which leads to tissue culture failure [25]. Comparatively, stem segment is more easily assessable, and the embryogenic calli formed by stem segments did not show obvious browning and grew well. These advantages make stem explants more suitable for initial culture, especially for industrial and commercial production.”

2. Special attention should be focused on the plant growth regulators used in nutrient media and their ratio (auxin/cytokinin) for callus induction and somatic embryogenesis in pomegranate. It is desirable for the authors to expand this part of the paper a little.

Reply - We thank the reviewer for the thoughtful suggestion. We have expanded our discussion in the revised manuscript to compare the different effects of plant growth regulators on in vitro culture of pomegranate, especially using different explants. The relevant sentences are copied here:

“However, different combinations of plant growth regulators are required for in vitro propagation pomegranate when using different explants [25]. For instance, Kantharajah et al. [36] showed that MS medium supplemented with 1 mg/L 6-BA and 0.4 mg/L NAA gave rise to the best callus initiation and growth from nodal explants, while the medium containing 1 mg/L 6-BA archived the best performance for leaf explant-derived callus culture. These results indicate that the choice of plant growth regulators used in nutrient medium and their ratio (auxin/cytokinin) are largely depending on the morphogenic potentials of the pomegranate explants employed for in vitro culture.”

3. It is necessary to give an extended description of the statistical processing of the data. In what programme it was carried out, what test was used to assess the level of significance.

Reply - We thank the reviewer for the comment. We have added the details of statistical tests for each comparison in the corresponding section (Method section 2.2, 2.3, 2.4 and 2.6), included the statistic method applied for the test and the statistic program. The p-value level for the definition of significant level has been added in the notes of the relevant tables in the revised manuscript. The revised sentences in the main text are copied here:

“Factorial Analysis of Variance (ANOVA) was performed to assess the statistical significance level among 6 different combinations of basal medium and plant growth regulators (see Table 1 for details) in IBM SPSS Statistics (IBM Corp. New York, USA). Duncan’s Multiple Range test (DMRT) was then conducted to measure the specific difference be-tween each pair of culture conditions.”

“Statistical differences among 9 combinations of different 6-BA and NAA concentrations (detailed in Table 2) were measured by two-way ANOVA using SPSS software. Multiple comparison procedure was performed by the post hoc DMRT.”

“The difference in induction of embryogenic calli between the two types of explants was evaluated by one-way ANOVA in SPSS software.”

“Two-way ANOVA was implemented to quantify the statistically significant level among the 9 combinations of different 6-BA and NAA concentrations (detailed in Table 3), and DMRT was employed to measure the pairwise differences between culture conditions.”

4. in Table 4 it is better to present not "Somatic embryo induction percentage(%)", but to indicate the number of somatic embryoids formed from one callus explant.

Reply - We thank the reviewer for the suggestion. We have added one more column in Table4 to present the number of somatic embryoids formed from one callus explant. The result also revealed that the culture condition supplemented with 0.5 mg/L 6-BA and 0.5 mg/L NAA gave rise to the higher number of somatic embryoids from one single callus explant.

5. Figure 2, 5, 6 - the scale bar should be placed in the lower right corner

Reply - We thank the reviewer for pointing this out and sorry for the mistake. We have modified the Figure 2, 5, 6 in the revised manuscript.

6. L - 59… CRISPR-mediated genome editing… - the full name of the method should be given. For example, via Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-mediated genome editing, which facilitates the genetic improvement of favorable phenotypes.

Reply - We thank the reviewer for pointing this out. We have revised accordingly.

7. L - 100....- IBA – decipher the first time the abbreviation is mentioned.

Reply - We thank the reviewer for pointing this mistake out and sorry for our carelessness. We have added the full name “indole-3-butyric acid” before the abbreviation.

8. L 112- 113 - (0.5, 1.0 and 1.5 mg/L 6-benzylaminopurine (6-BA), 1-naphthaleneacetic acid 112 (NAA) and indole-3-butyric acid (IBA)) - the transcript is indicated the first time it is mentioned, and it is not necessary to do so thereafter.

Reply - We thank the reviewer for pointing this out. In the revised manuscript, only the abbreviations are used there.

9. L 115...-….in a 16/8 h light/dark 115 light cycle….. – the intensity of the lighting and which light sources were used should be specified.

Reply - We thank the reviewer for the suggestion. We used LED lights and the lighting intensity was 1600 lx. These details have been specified in the revised manuscript. The relevant sentence is copied here:

“The culture was maintained at a constant temperature of 22±2 °C and illumination in-tensity of 1600 xl by LED lights at a 16/8 h light/dark cycle”

10. in vivo and in vitro in the text of the manuscript should be written in italics.

Reply - We thank the reviewer for the careful check and comment. We have corrected the format of ‘in vivo and in vitro’ in the revised manuscript.

Please see the attachment.

Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

The authors have been addressed all comments

moderate correction is needed

Reviewer 4 Report

The authors have taken all my comments into account and improved the manuscript