The Prospect of Physiological Events Associated with the Micropropagation of Eucalyptus sp.

Round 1

Reviewer 1 Report

Comments for the manuscript: The Prospect of Physiological Events Associated with the Micropropagation of Eucalyptus sp.

Dear authors: Please read the following review article which you failed to cite.

Muhammad Nakhooda and Shri Mohan Jain (2016) A REVIEW OF EUCALYPTUS PROPAGATION AND CONSERVATION Propagation of Ornamental Plants 16(4): 101-119

There are several references unrelated to the statement.

Please rewrite the manuscript in the following order: the establishment of plantlets (phase I), multiplication and differentiation (phase II), rooting (phase III), and acclimatisation (phase IV) for better understanding.

Micropropagation: direct and indirect adventitious shoot regeneration, axillary shoot multiplication, shoot elongation, rooting, somatic embryo induction, development and conversion, finally acclimatization. In each aspect, discus how various factors affecting the process at the physiological and molecular levels.

L53-55: Please rewrite this sentence “Vegetative progeny is achieved by applying multiple techniques of forest tree propagation, such as grafting, layering, root cuttings and rooting of the shoot depends on the physiological structure, shape, performance and age of the donor tree (Monteuuis, 2017)”.

L56-58: “Over the past few decades, conventional breeding tools implemented in forestry improvement program have become the main strategy to multiply Eucalyptus and obtain resistant genotypes (Aggarwal, Kumar, Sharma, & Reddy, 2012).” Cite related articles. Aggarwal et al. (2012) investigated several factors influencing micropropagation of a selected elite clone of Eucalyptus tereticornis Sm.

L58: obtain resistant genotypes? Indicate the biotic or abiotic factor.

L63: Figure 1 is not related to this statement.

L72: Strangely, you have cited this review. There are publications for Eucalyptus and its very basic information. Please delete it “Mazri & Meziani, 2015”

L91: Eucalyptus regeneration (Chieng, Chen, Sim, & Goh, 2014) again strange citation.

L95-96: Please rewrite this sentence “Organogenesis is the vegetative propagation of plant organs like flower buds, shoots, and roots from the cells under in vitro conditions.”

L102: cell suspension that is produced from callus “RamIrez-Mosqueda & Iglesias-Andreu, 2015”. The authors regenerated vanilla through callus culture, not from the cell suspension. Please cite publications related to Eucalyptus.

L103: “genetically” modified plants

L106-108: In Eucalyptus, the composition of different factors such as carbon, various vitamins, amino acids, gelling agent, macro- and micronutrients and other additives have affected the efficacy of both types of organogenesis (R Abiri et al., 2017). Again, strange citation R Abiri et al. (2017) reported rice somatic embryogenesis!

L110: expand the abbreviation “2,4-D”

L115: direct regeneration? Shoot/root/plant?

L189: Da Costa et al., 2013? Again, strange citation! Please cite the original article not this review paper. This review provides information about mineral nutrition, sugar (carbohydrate), light and auxin on rooting of E. globulus and E. saligna.

L195-197: “Adventitious root (AR) development in Eucalyptus is a complex and multifactorial procedure influenced by the plant age, stress conditions, environmental factors, genetic traits, mineral nutrition and phytohormones (Da Costa et al., 2013)” cite the original article.

L255: in Eucalyptus (Grzyb & Mikuła, 2019). Again, wrong citation

L358-361: Not relevant to this topic: Role of Auxins on Root Structure and Formation “Induction of embryogenic callus is a key to form adventitious bud and to achieve this target, different molecular pathways of hormonal levels and genes have contribut to the active mechanism of callus induction (Craig, 2012; Ho et al., 1998; Z.-C. 360 Huang, Zeng, & Lu, 2010; Z. Huang & Li, 2020)”.

L363-370: Not relevant to this topic: Role of Auxins on Root Structure and Formation “It is noted that low level of NAA (0.5 mg L-1) could elongate shoot length and increase shoot numbers in Eucalyptus dunnii. Nevertheless, the higher level of NAA (> 0.5 364 mg L-1) enhanced hyperhydric structures and thus caused damage in the shoot elongation (Navroski, Reiniger, & Pereira, 2015). The combination of polyvinylpyrrolidone (PVPP; 250 mg L-1) and 6-benzylaminopurine (2.2 μM) did not have impact on microshoot hyperhydration and increased the rate of multiplication rate in E. globulus under in vitro conditions (George et al., 2008b; Gonzalez, Rios, Aviles, & Sa¡nchez-Olate, 2011). Interestingly, hyperhydration without influencing new shoots formation can occurr due to the decrease of sucrose concentration (Gonzalez et al., 2011)”.

L375: (PIN) proteins and auxin influx AUXIN1/LIKEAUX1 (AUX/LAX) (Benra et al., 2019). Again, wrong citation. Please explain why this strange citation? The article is not providing any rooting information!

L378: Jin et al., 2016 is not listed in the reference

L404-405: “Rout, Mahato, & Senapati, 2008; Vengadesan & Pijut, 2009”. Again, wrong citation. Please focus on the effect of cytokinins on Eucalyptus

L405: The inclusion of NAA – why here? “Role of cytokinins in micropropagation of Eucalyptus”

L 405-408: “The inclusion of NAA into the media helps in shoot induction, multiplication, and elongation in some plant species, This could be due to removal of phenolic compounds by competing for the active forms of auxin oxidase (IAA-O) enzyme, associated in the oxidation of phenols, thereby assisting BA in the shoot multiplication (Sugimura et al., 2005)”. Not clear please rewrite

L527-529: “Primary somatic embryogenesis and regeneration from seedling derived callus were documented for the first time in E. × Liechow” (Ochatt & Abirachede•Darmency, 2019; Overvoorde et al., 2010)”. Again, wrong citation

L554: qas found (typo)

Table 1: Watt, 199?

Reference (Incomplete)

77. Huang Z, Li H (2020) Control of oxidative stress by a combination of PBU, BAP and DMTU enhances 1019 adventitious shoot formation in Eucalyptus urophylla. Plant Cell Tissue and Organ Culture
78. Monteuuis O (2017). Vegetatively propagating forest trees.
79. Moura LCd, Xavier A, Cruz ACFd, Gallo R, Gatti KC, Miranda NA, et al. (2017) Effects of explant type, culture media and picloram and dicamba growth regulators on induction and proliferation of somatic embryos in Eucalyptus grandis x E. urophylla1
80. Revista Õrvore 41. 1101
81. Moura LCd, Xavier A, Cruz ACuFd, Gallo R, Miranda NA, Otoni WC (2019) Auxin pulse in the induction of somatic embryos of Eucalyptus 1103
82. Revista Õrvore 43.

 

Author Response

Dear Reviewer,

 

We have edited the manuscript based on your valuable advice. We strongly believe that the current version is improved accordingly. We hope this will be to your satisfaction.

 

Stay safe,

Rambod

Reviewer 1

Comments for the manuscript: The Prospect of Physiological Events Associated with the Micropropagation of Eucalyptus sp.

Dear authors: Please read the following review article which you failed to cite.

 

Comment 1

Muhammad Nakhooda and Shri Mohan Jain (2016) A REVIEW OF EUCALYPTUS PROPAGATION AND CONSERVATION Propagation of Ornamental Plants 16(4): 101-119

Answer

Thank you for the comment. The mentioned reference has been added as follows (Line 147):

 

Implementing the outcomes of analysis on micropropagation of Eucalyptus, like other plants, demonstrated that post-acclimatisation and graviperception architecture are controlled by the exogenous auxin analogue, added in the pre-rooting culture media ( Nakhooda et al., 2014; Nakhooda & Jain, 2016).

 

This reference has also been added to the Reference List (line 827-829)

 

Comment 2

There are several references unrelated to the statement.

 

Answer

Thank you for the comment. We have replaced the references. All the edited, deleted, added and modified references have been stated in the answer to the reviewers file.

 

Comment 3

Please rewrite the manuscript in the following order: the establishment of plantlets (phase I), multiplication and differentiation (phase II), rooting (phase III), and acclimatisation (phase IV) for better understanding.

Micropropagation: direct and indirect adventitious shoot regeneration, axillary shoot multiplication, shoot elongation, rooting, somatic embryo induction, development and conversion, finally acclimatization. In each aspect, discus how various factors affecting the process at the physiological and molecular levels.

 

Answer

Thank you for the suggestion. We agree that another strategy to write the paper is the mentioned suggestion, however we would like to mention that our study has been designed to focus on the effect of different factors on tissue culture of Eucalyptus. To achieve the mentioned goal, we started off the manuscript by introducing organogenesis and somatic embryogenesis. We then continued with the importance of root and the root and shoot interactions as these are the most important phase and difficulties in Eucalyptus tissue culture. Our literature results showed that the suggested style is broad and to cover the available data we need to re-write and change the whole structure of the manuscript. Therefore, based on the deadline of the special issue, we think, we could not re-structure the draft based on the suggestion on time.  However, your suggestion is greatly appreciated and we think it will add on to the current knowledge, therefore we are working on the suggestion for our future publication. We have to inform that, our team is working on the mentioned suggestion and plan to write a book accordingly.  We have written the manuscript as follows and, with all due respect to the valuable idea of reviewer, we hope the viewer accepts the current format and style.

 

1. Introduction
2. General Features of Micropropagation and their Applications in Eucalyptus

2.1. Organogenesis

2.2. Somatic Embryogenesis

3. Importance of Eucalyptus Root Architecture and Behaviour
4. The Relationship Between Root structure and Shoot System in Eucalyptus
5. Role of Plant Growth Regulators (PGRs) in Micropropagation of Eucalyptus

5.1. Role of Auxins on Root Structure and Formation

5.2. Effect of Ethylene on Root Architecture

5.3. Role of cytokinins in micropropagation of Eucalyptus

5.4. Role of Auxins in Somatic Embryogenesis

6. Influence of Environmental and External Factors on Micropropagation of Eucalyptus

6.1. Effect of Media

6.2. Importance of Nitrogen, Calcium, Boron, and Colchicine on Micropropagation of Eucalyptus

6.3. Effect of Carbohydrate

6.4. Light and Radiation Effect

7. Regeneration and Acclimatisation of Eucalyptus
8. Conclusion

 

Comment 4

L53-55: Please rewrite this sentence “Vegetative progeny is achieved by applying multiple techniques of forest tree propagation, such as grafting, layering, root cuttings and rooting of the shoot depends on the physiological structure, shape, performance and age of the donor tree (Monteuuis, 2017)”.

 

Answer

Thank you. The mentioned sentences have been re-written as follows (Line 53-56):

Vegetative progeny methods are broadly being used for asexual propagation of forest trees. The main vegetative propagation techniques are grafting, layering, root cuttings and rooting of the shoot. Generally speaking, the effectiveness of the methods depends on the physiological structure, shape, performance and age of the donor tree (Monteuuis, 2017).

 

Comment 5

L56-58: “Over the past few decades, conventional breeding tools implemented in forestry improvement program have become the main strategy to multiply Eucalyptus and obtain resistant genotypes (Aggarwal, Kumar, Sharma, & Reddy, 2012).” Cite related articles. Aggarwal et al. (2012) investigated several factors influencing micropropagation of a selected elite clone of Eucalyptus tereticornis Sm.

 

Answer

Thank you for the comment. We have replaced the reference as follows (Line 767):

 

Naidoo, S., Slippers, B., Plett, J. M., Coles, D., & Oates, C. N. (2019). The road to resistance in forest trees. Frontiers in Plant Science, 10, 273.

 

Comment 6

L58: obtain resistant genotypes? Indicate the biotic or abiotic factor.

 

Answer

Thank you addressing this. We have edited the sentence and now reads as (Line 56-58):

 

“Over the past few decades, conventional breeding tools which have been implemented in forestry improvement program become the main strategy to multiply Eucalyptus forest trees and obtain resistant genotypes”

 

Comment 7

L63: Figure 1 is not related to this statement.

 

Answer

The followed statement has been added, and the figure is replaced after it:

Line 71-72 “The advantages and disadvantages of micropropagation method in Eucalyptus are presented in Figure 1 (Hussain et al., 2012).”

 

Comment 8

L72: Strangely, you have cited this review. There are publications for Eucalyptus and its very basic information. Please delete it “Mazri & Meziani, 2015”

 

Answer

Many thanks for the comment. The mentioned reference has been deleted.

 

Comment 9

L91: Eucalyptus regeneration (Chieng, Chen, Sim, & Goh, 2014) again strange citation.

 

Answer

Thank you for the comment. We have replaced the reference with relevant reference (Line 87).

Line 777-779: Girijashankar, V., Sharma, K. K., Balakrishna, P., & Seetharama, N. (2007). Direct somatic embryogenesis and organogenesis pathway of plant regeneration can seldom occur simultaneously within the same explant of sorghum. Journal of SAT Agricultural Research, 3(1), 1-3.

 

Comment 10

L95-96: Please rewrite this sentence “Organogenesis is the vegetative propagation of plant organs like flower buds, shoots, and roots from the cells under in vitro conditions.”

 

Answer

Thank you for the comment. We have rewritten it as follows (Line 92-95):

Organogenesis is the vegetative propagation by which plant organs like flower buds, shoots, and roots are produced from the cells and tissues (the unusual points of origin) under in vitro conditions.

 

Comment 11

L102: cell suspension that is produced from callus “RamIrez-Mosqueda & Iglesias-Andreu, 2015”. The authors regenerated vanilla through callus culture, not from the cell suspension. Please cite publications related to Eucalyptus.

 

Answer

Thank you for the comment. The reference was replaced as follows (Line 782):

18. Johns, A.E. Lessons for Plant Micropropagation. Educreation Publishing, 2019; p. 85.

 

Comment 12

L103: “genetically” modified plants

 

Answer

Thank you for the comment. As it was suggested, the term “genetically” was added (Line 98).

 

Comment 13

L106-108: In Eucalyptus, the composition of different factors such as carbon, various vitamins, amino acids, gelling agent, macro- and micronutrients and other additives have affected the efficacy of both types of organogenesis (R Abiri et al., 2017). Again, strange citation R Abiri et al. (2017) reported rice somatic embryogenesis!

 

Answer

Thank you for the comment. The mentioned reverence was replaced with the relevant reverence as follows (Line 784-785):

Kendurkar, S. V., & Rangaswamy, M. (2018). In Vitro Approaches for the Improvement of Eucalyptus. In Biotechnologies of Crop Improvement, Volume 1 (pp. 159-214). Springer, Cham.

 

Comment 14

L110: expand the abbreviation “2,4-D”

 

Answer

Thank you for the comment. The following full name has been added. Th sentence now reads as (Lines 102-105):

For instance, indirect organogenesis of Eucalyptus camaldulensis hybrids, the combination of naphthyl acetic acid (NAA) and 6-benzylaminopurine (BAP) shows a better efficacy as compared to application of zeatin, kinetin, casein hydrolysate, and 2,4-Dichlorophenoxyacetic acid (2,4-D) [21].

 

Comment 15

L115: direct regeneration? Shoot/root/plant?

 

Answer

Thank you for the comment. The mentioned sentence has been deleted.

 

Comment 16

L189: Da Costa et al., 2013? Again, strange citation! Please cite the original article not this review paper. This review provides information about mineral nutrition, sugar (carbohydrate), light and auxin on rooting of E. globulus and E. saligna.

 

Answer

Thank you for the comment. The mentioned reference was replaced with the correct reference as follows (Line 831-832):

Geiss, G., Gutierrez, L., & Bellini, C. (2018). Adventitious root formation: new insights and perspectives. Annual Plant Reviews online, 127-156.

 

Comment 17

L195-197: “Adventitious root (AR) development in Eucalyptus is a complex and multifactorial procedure influenced by the plant age, stress conditions, environmental factors, genetic traits, mineral nutrition and phytohormones (Da Costa et al., 2013)” cite the original article.

 

Answer

Thank you for the comment. As we found that the mentioned reference is crossed reference, therefore we just cited the original reference which was Da Costa et al., 2013. However, we have added both original and crossed references now (Line 185). The following statement is the cited in Vilasboa et al., (2019)

 

 “Adventitious root development is the result of an intricate interaction of endogenous and environmental factors, including phytohormones, light, temperature and mineral nutrition (Da Costa et al., 2013).”

 

The new citation has also been added in the reference list as follows (Line 836-837): Vilasboa, J., Da Costa, C. T., & Fett-Neto, A. G. (2019). Rooting of eucalypt cuttings as a problem-solving oriented model in plant biology. Progress in Biophysics and Molecular Biology, 146, 85-97.

 

Comment 18

L255: in Eucalyptus (Grzyb & Mikuła, 2019). Again, wrong citation

 

Answer

Thank you for the comment. The mentioned line and references has been deleted.

 

Comment 19

L358-361: Not relevant to this topic: Role of Auxins on Root Structure and Formation “Induction of embryogenic callus is a key to form adventitious bud and to achieve this target, different molecular pathways of hormonal levels and genes have contribut to the active mechanism of callus induction (Craig, 2012; Ho et al., 1998; Z.-C. 360 Huang, Zeng, & Lu, 2010; Z. Huang & Li, 2020)”.

 

Answer

Thank you for the comment. The mentioned paragraph is deleted.

 

Comment 20

L363-370: Not relevant to this topic: Role of Auxins on Root Structure and Formation “It is noted that low level of NAA (0.5 mg L-1) could elongate shoot length and increase shoot numbers in Eucalyptus dunnii. Nevertheless, the higher level of NAA (> 0.5 364 mg L-1) enhanced hyperhydric structures and thus caused damage in the shoot elongation (Navroski, Reiniger, & Pereira, 2015). The combination of polyvinylpyrrolidone (PVPP; 250 mg L-1) and 6-benzylaminopurine (2.2 μM) did not have impact on microshoot hyperhydration and increased the rate of multiplication rate in E. globulus under in vitro conditions (George et al., 2008b; Gonzalez, Rios, Aviles, & Sa¡nchez-Olate, 2011). Interestingly, hyperhydration without influencing new shoots formation can occurr due to the decrease of sucrose concentration (Gonzalez et al., 2011)”.

 

Answer

Thank you for the comment. The mentioned paragraph is deleted.

 

Comment 21

L375: (PIN) proteins and auxin influx AUXIN1/LIKEAUX1 (AUX/LAX) (Benra et al., 2019). Again, wrong citation. Please explain why this strange citation? The article is not providing any rooting information!

 

Answer

Thank you for the comment. The correct reference is added (Line 331). We have to inform that, in the first version of the draft we added the references manually, after that we added the references by endnote software and we guess that some of the errors happened due to this.

 

Comment 22

L378: Jin et al., 2016 is not listed in the reference.

 

Answer

Thank you for the comment. The reference has been added (Line 917-918).

 

Comment 23

L404-405: “Rout, Mahato, & Senapati, 2008; Vengadesan & Pijut, 2009”. Again, wrong citation. Please focus on the effect of cytokinins on Eucalyptus

 

Answer

Many thanks for the comment. The mentioned references have been replaced with the related references.  Lines 357-358, references 86,87, and 88.

 

Comment 24

L405: The inclusion of NAA – why here? “Role of cytokinins in micropropagation of Eucalyptus”

 

Answer

Thank you for the comment. The mentioned sentence is deleted.

 

Comment 25

L 405-408: “The inclusion of NAA into the media helps in shoot induction, multiplication, and elongation in some plant species, This could be due to removal of phenolic compounds by competing for the active forms of auxin oxidase (IAA-O) enzyme, associated in the oxidation of phenols, thereby assisting BA in the shoot multiplication (Sugimura et al., 2005)”. Not clear please rewrite

 

Answer

Thank you for the comment. The mentioned sentence has been deleted.

 

Comment 26

L527-529: “Primary somatic embryogenesis and regeneration from seedling derived callus were documented for the first time in E. × Liechow” (Ochatt & Abirachede•Darmency, 2019; Overvoorde et al., 2010)”. Again, wrong citation

 

Answer

Thank you for the comment. After editing the section “Role of Auxins in Somatic Embryogenesis”, the mentioned lines have been deleted. 

 

Comment 27

L554: qas found (typo)

 

Answer

Thank you for the comment. The typo was corrected as follows (Line 485-487):

“Although 2,4-D is a popular phytohormone used in SE induction, but picloram was found to be more effective and stronger auxin in SE in case of Eucalyptus species”

 

Comment28

Table 1: Watt, 199?

 

Answer

Thank you for the comment. Since we have changed the references to the number the mentioned reference is also edited. However, in the bibliography section, we have edited the reference as well. The reference is edited, Watt 1999.

 

Comment 29

Reference (Incomplete)

1. Huang Z, Li H (2020) Control of oxidative stress by a combination of PBU, BAP and DMTU enhances 1019 adventitious shoot formation in Eucalyptus urophylla. Plant Cell Tissue and Organ Culture
2. Monteuuis O (2017). Vegetatively propagating forest trees.
3. Moura LCd, Xavier A, Cruz ACFd, Gallo R, Gatti KC, Miranda NA, et al. (2017) Effects of explant type, culture media and picloram and dicamba growth regulators on induction and proliferation of somatic embryos in Eucalyptus grandis x E. urophylla1
4. Revista Õrvore 41. 1101
5. Moura LCd, Xavier A, Cruz ACuFd, Gallo R, Miranda NA, Otoni WC (2019) Auxin pulse in the induction of somatic embryos of Eucalyptus 1103
6. Revista Õrvore 43.

 

Answer

Thank you for the comment. The mentioned references have been edited as follow:

 

Line 956, reference 95 (Huang 2020) has been completed.

Line 764, reference 10 (Monteuuis 2017) has been completed.

Line 812, reference 30 (Moura et al., 2017) has been completed.

Line 862, reference 53 (Moura et al., 2019) has been completed.

 

 

Reviewer 2 Report

Review of the Forests-948534 manuscript:

Dear Editor and Authors,

The manuscript has been clearly and legibly written.

It reviews the tissue culture of Eucalyptus, a woody evergreen plant species recalcitrant to in vitro regeneration. The authors investigated the influence of plant growth regulators (PGRs), nutrients and of some environmental features on the regenerative potential of Eucalyptus. Additionally, the analysis of the basic correlation between genetics and tissue culture has been presented.

 

Personally, I would enrich the section “Conclusion”. It is to summarize the main achievement of the Authors’ review of the subject literature. The Authors’ opinion on the prospects of the development of the regeneration protocols would undoubtedly make an added value.

 Technical remarks:

• In Forests, references in the text should be presented as numbers placed in square brackets.
• The manuscript needs a slight proofreading since I have found some minor issues regarding surface errors such as misspellings and mistakes in punctuation, e.g.: the sentence “Reactive oxygen species act in an oxidative mechanism pathway and activate the different 448 cellular” (page 11, lines 448) seems unfinished.

Best regards,

Reviewer

Author Response

Dear Reviewer,

We have edited the manuscript based on your valuable advice. We strongly believe that the current version is improved accordingly. We hope this will be to your satisfaction.

 

Stay safe,

Rambod

Reviewer 2

Dear Editor and Authors,

The manuscript has been clearly and legibly written.

It reviews the tissue culture of Eucalyptus, a woody evergreen plant species recalcitrant to in vitro regeneration. The authors investigated the influence of plant growth regulators (PGRs), nutrients and of some environmental features on the regenerative potential of Eucalyptus. Additionally, the analysis of the basic correlation between genetics and tissue culture has been presented.

 

 Comment 1

Personally, I would enrich the section “Conclusion”. It is to summarize the main achievement of the Authors’ review of the subject literature. The Authors’ opinion on the prospects of the development of the regeneration protocols would undoubtedly make an added value.

 

Answer

Thank you for the comment. We have edited the section as follows (Line 712-738):

The micropropagation technology has matured to become popular in the biotechnology filed where many researchers are looking to establish “recipes” to use in vitro culture. So far, numbers of recipes have been established successfully for several plant species, without special training or adaptation. Nonetheless, there are plenty of plant species where little micropropagation investigation has been performed on them and appropriate protocols may or may not be fully developed. In summary, the current review paper focused on the most relevant physiological advances in the micropropagation of Eucalyptus from the cell to acclimatisation. In this project, we highlighted the inner scenario of the interaction between the internal and external factors which play a role in the tissue culture of Eucalyptus. We also demonstrated that the ability of selected PGRs to form organogenesis and somatic embreyogenesis remains the most significant and yet often the most problematic aspect of Eucalyptus propagation. Additionally, our review emphasised on the relationship between the key plant growth regulators (auxins, cytokinins, ethylene) and the formation of roots and shoots in correlation with molecular and biochemical events.

On the other hand, we showed that other external factors such as nutrients, nitrogen, calcium, boron, colchicine, carbohydrate, light and radiation have the regulatory function on the physiological aspects of Eucalyptus plant in vitro conditions. As mentioned, regeneration protocols through organogenesis or somatic embryogenesis need to be optimized depending on specific genotypic requirements. For the majority of woody plants especially Eucalyptus, the application of tissue culture technology presents yet many bottlenecks particularly due to the induction conditions, the genotype, the number of shoot and root, the percentages of shoot and root elongation, the number of embryos produced, the maturation, the regeneration and embeling and acclimatization rate and performance. The advanced information of the current paper will help researchers to focus on the optimum conditions of Eucalyptus tissue culture based on physiological phenomenon. Notwithstanding the large number of researches which have been conducted on the micropropagation of Eucalyptus over the decades, additional investigations are also required to identify, discover, and ultimately overcome some of the current barriers, and develop a strategic approach for the industrial purposes.

 

Technical remarks:

 Comment 2

• In Forests, references in the text should be presented as numbers placed in square brackets.

Answer

Thank you for the comment. All the references have been corrected based on journal format.

 

Comment 3

• The manuscript needs a slight proofreading since I have found some minor issues regarding surface errors such as misspellings and mistakes in punctuation, e.g.: the sentence “Reactive oxygen species act in an oxidative mechanism pathway and activate the different 448 cellular” (page 11, lines 448) seems unfinished.

Answer

Thanks for the comment. The mentioned sentence is corrected and we have double checked the draft and edited, accordingly.

 

Best regards,

Reviewer

 

Reviewer 3 Report

The manuscript provides updated information on the physiological, biochemical and molecular aspects related to the micropropagation of economically important Eucalyptus species. The analysis concerns the two most reliable methods used under the in vitro propagation of these plants: organogenesis and somatic embryogenesis. The authors broadly discuss the morphological and physiological aspects of root regeneration and the relationship between the root structure and the shoot system in plants obtained via in vitro culture. The article presents in detail also the relationship between the key plant growth regulators (auxins, cytokinins, ethylene) and the formation of roots and shoots in correlation with molecular and biochemical events. Additionally the relationship between auxins and initiation of somatic embryogenesis is discussed. Much of the manuscript text is devoted to discussing the regulatory impact of the in vitro conditions (nutrient, sources of nitrogen, calcium, boron, colchicine, carbon, and light quality) on the physiological aspects of Eucalyptus micropropagation.

               In my opinion, the reviewed article will be a valuable source of information for researches working on optimizing protocols of micropropagation of Eucaliptus sp. and related species. The authors point to hirherto unexplored research fields that would allow for a deeper understanding of the processes taking place during the microporpagation of these woody plants. Generally, the manuscript text is very well written, with numerous references to the latest literature, and clearly designed Figures. However, I believe that in chapter 7 it would be necessary to complete the information concerning the genetic fidelity of seedlings (as indicated by the subtitle) because in the text little is written about it. Apart this, a few following corrections should be made in the text before its publication:

 

5. Role of Plant Growth Regulators (PGRs) in Micropropagation of Eucalyptus

 

5.1. Role of Auxins on Root Structure and Formation

Page 9, line 378

“Jin et al., 2016”   This citation is not included in the reference list.

 

5.4. Role of Auxins in Somatic Embryogenesis

Page 13, lines 548-550

1. urophylla x Eucalyptus grandis or E. grandis x E. urophylla.....Which of these two names is correct?

 

 

 

6. Influence of Environmental and External Factors on Micropropagation of Eucalyptus

 

6.2. Importance of Nitrogen, Calcium, Boron, and Colchicine on Micropropagation of Eucalyptus

 

Pages 663-668, lines 548-550

The information in these two sentences is doubled.

 

7. Regeneration, Acclimatisation and Genetic Fidelity of the Emblings

Table 1

Check, please this citation: “Pinto et al., 2008b”. There is only one item by this author on the Reference list.

“Muralidharan and Mascarenhas 1995”  This citation is not included in the reference list.

 

Page 19, line 814

“4-CCPU”    Include, please full name of this compound into the text.

References

Please, complete the missing bibliographic data within the citations. Names of the species should be written in italic in the whole manuscript text.

 

More detailed comments can be found in the text of revised manuscript.

 

Comments for author File: Comments.pdf

Author Response

Dear Reviewer,

We have edited the manuscript based on your valuable advice. We strongly believe that the current version is improved accordingly. We hope this will be to your satisfaction.

 

Stay safe,

Rambod

The manuscript provides updated information on the physiological, biochemical and molecular aspects related to the micropropagation of economically important Eucalyptus species. The analysis concerns the two most reliable methods used under the in vitro propagation of these plants: organogenesis and somatic embryogenesis. The authors broadly discuss the morphological and physiological aspects of root regeneration and the relationship between the root structure and the shoot system in plants obtained via in vitro culture. The article presents in detail also the relationship between the key plant growth regulators (auxins, cytokinins, ethylene) and the formation of roots and shoots in correlation with molecular and biochemical events. Additionally the relationship between auxins and initiation of somatic embryogenesis is discussed. Much of the manuscript text is devoted to discussing the regulatory impact of the in vitro conditions (nutrient, sources of nitrogen, calcium, boron, colchicine, carbon, and light quality) on the physiological aspects of Eucalyptus micropropagation.

               In my opinion, the reviewed article will be a valuable source of information for researches working on optimizing protocols of micropropagation of Eucaliptus sp. and related species. The authors point to higher to unexplored research fields that would allow for a deeper understanding of the processes taking place during the microporpagation of these woody plants. Generally, the manuscript text is very well written, with numerous references to the latest literature, and clearly designed Figures.

 

Comment 1

However, I believe that in chapter 7 it would be necessary to complete the information concerning the genetic fidelity of seedlings (as indicated by the subtitle) because in the text little is written about it. Apart this, a few following corrections should be made in the text before its publication:

 

Answer

Many thanks for the comment. As the reviewer mentioned correctly, we didn’t elaborate the genetic fidelity of seedling and as this is a broad topic, we decided to remove it from the draft.

 

 Comment 2

1. Role of Plant Growth Regulators (PGRs) in Micropropagation of Eucalyptus

 5.1. Role of Auxins on Root Structure and Formation

Page 9, line 378

“Jin et al., 2016”   This citation is not included in the reference list.

 

Answer

Thanks for the comment. We have added the mentioned reference in the bibliography list.

 

Comment 3

5.4. Role of Auxins in Somatic Embryogenesis

Page 13, lines 548-550

1. urophylla x Eucalyptus grandis or E. grandis x E. urophylla.....Which of these two names is correct?

 

Answer

Thanks for the comment. We have corrected the name of plants as follow:

1. urophylla x E. grandis

 

Comment 4

1. Influence of Environmental and External Factors on Micropropagation of Eucalyptus

 6.2. Importance of Nitrogen, Calcium, Boron, and Colchicine on Micropropagation of Eucalyptus

 Pages 663-668, lines 548-550

The information in these two sentences is doubled.

 

 Answer

Thanks for the comment. Lines 549-551 has been deleted. Accordingly, lines 665- 668 has been deleted.

 

Comment 5

1. Regeneration, Acclimatisation and Genetic Fidelity of the Emblings

Table 1

Check, please this citation: “Pinto et al., 2008b”. There is only one item by this author on the Reference list.

 

Answer

Thank you for the comment. The correct reference is Pinto et al., 2008, and we have corrected it accordingly.

 

Comment 6

“Muralidharan and Mascarenhas 1995”  This citation is not included in the reference list.

 

Answer

The mentioned reference has been added.

 

 Comment 7

Page 19, line 814

“4-CCPU”    Include, please full name of this compound into the text.

  

Answer

Thank you for the comment. The term “N‐(2‐chloro‐4‐pyridyl)‐N'‐phenylurea” has been added.

 

Comment 8

References

Please, complete the missing bibliographic data within the citations. Names of the species should be written in italic in the whole manuscript text.

 

Answer

We have corrected all. 

 

Comment 9

More detailed comments can be found in the text of revised manuscript.

Page 2- Figure

 

Answer

Many thanks for the comment. We have edited the figure accordingly.

 

Comment 10

Page 3- Line 108

 

Answer

Thanks for the comment. The reference has been changed to the relevant reference as follow:

Kendurkar, S. V., & Rangaswamy, M. (2018). In Vitro Approaches for the Improvement of Eucalyptus. In Biotechnologies of Crop Improvement, Volume 1 (pp. 159-214). Springer, Cham.

 

Comment 11

Page 4, lines 125 and 126.

 

Answer

Thanks for the comment. Phase is changed to type. Trough is changed to through.

 

Comment 12

Page 6 line 217.

 

Answer

Thanks for the comment. Cauased is changed to caused

 

Comment 13

Page 9 line 378.

 

Answer

Thanks for the comment. The mentioned reference is added.

 

Comment 14

Page 12 line 524.

 

Answer

Thanks for the comment. The parenthesis is removed.

 

Comment 15

Page 13 line 549-551.

 

Answer

Thanks for the comment We couldn’t open the comments of reviewer on the pdf draft, however we guess it’s about repeating the content which reviewer 1 also mentioned about it. We have deleted the mentioned sentence.

 

Comment 16

Page 13 line 555.

 

Answer

Thanks for the comment. We have included a space.

 

Comment 17

Page 15 line 663-657.

 

Answer

Thanks for the comment. The repeated sentence is deleted.

 

Comment 18

Page 17 line 740.

 

Answer

Thanks for the comment. RAP is changed to PAR

 

Comment 19

Page 17 line 770.

 

Answer

Thanks for the comment. Exlant has been changed to explants.

 

Comment 20

Page 18, reference

 

Answer

Thanks for the comment. The reference has been corrected.

 

Comment 21

(Muralidharan and Mascarenhas 1995) and Watt, 199.

 

Answer

Thanks for the comment.  Muralidharan and Mascarenhas 1995 has been added to the bibliography and Watt, 199 has been corrected to Watt, 1999.

 

Comment 22

Question marks in the table by reviewer.

 

Answer

Thanks for the comment. The mentioned rows were empty in the reference and we have added “-“ in the table.

 

Comment 23

Page 18 line 794.

 

Answer

Thanks for the comment. The second “however” has changed to “On the other hand”.

 

Comment 24

Page 19 line 814.

 

Answer

Thanks for the comment. Full name (N‐(2‐chloro‐4‐pyridyl)‐N'‐phenylurea) has been added.

 

Comment 25

Page 19 line 828.

 

Answer

Thanks for the comment. Collectively has been changed to summarising.

 

Comment 26

Complete the bibliography.

 

Answer

Thanks you for the comment. All the references have been checked again.

 

Round 2

Reviewer 1 Report

The authors revised the manuscript according to the reviewer's comments. 

Please address the following comments:

L327: effect instead of impact

L348: ARR1, IAA12, and TPL genes should be italicized (please do it for all genes e.g. L354-355)

L386: Please rewrite

L404: ascorbate peroxidase (Non-italic)

L544: Please rewrite

L621: 6.4. Light and Radiation Effect

Please indicate the optimal light intensities for in vitro propagation of Eucalyptus sp.

Author Response

Dear reviewer,

Many thanks for your kind attention to improving the quality of the paper. We have applied all the changes based on the comments as follow, and we hope to have your valuable feedback on the paper. 

 

Thank you again for spending your valuable time, and supporting our paper.

 

Best wishes,

Rambod

 

Please address the following comments:

Comment 1: L327: effect instead of impact

Answer: Thank you. The word “impact” has been replaced with “effect” in the mentioned line.

Comment 2: L348: ARR1, IAA12, and TPL genes should be italicized (please do it for all genes e.g. L354-355).

Answer: Thank you. All the names have been changed to italic style.

Comment 3: L386: Please rewrite

Answer: Thank you. The sentence has been re-written as follow:

Despite the initial and secondary somatic embryos formation, these developments did not eventuate any seedling or/and germination induction.

Comment 4: L404: ascorbate peroxidase (Non-italic).

Thank you. The term “ascorbate peroxidase” has been changes to non-italic style.

Comment 5: L544: Please rewrite

Thank you. The three sweetness have been slightly modified as follow:

Similarly, the dry and fresh weight of Eucalyptus were increased when the plant was cultivated on MS media enriched with the higher concentrations of Ca and B [51, 114, 115]. Boron deficiency, under ex vitro condition, caused seedling inhibition of E. grandis. Mainly, this inhibition occurred in the meristematic shoots regions, thus helped to impair the lateral and apical buds’ initiation [106].

L621: 6.4. Light and Radiation Effect

Please indicate the optimal light intensities for in vitro propagation of Eucalyptus sp.

Comment 4:

Answer: We have added the following paragraph.

 

Light intensity (quantity) is associated with photosynthetic activity and changes in vegetative growth, dry matter accumulation, and carbon fixation [181]. Generally, plants under high light intensity produce or develop thicker leaves, which can increase photosynthetic capacity per unit of area, improving the plant's ability to utilize light for carbon fixation [182]. It is shown that sudden changes in light intensity stimulated rooting [183]. Study on micropropagation of Eucalyptus ficifolia demonstrated that a light intensity of 10 µmol m^-2 s^-1 developed fibrous root system. Furthermore, incubation of E. ficifolia on media supplemented with riboflavin under a light intensity of 10 µmol m^-2 s^-1 developed the subsurface roots with either short or no laterals [184]. Reportedly, a higher rate of shoot formation and organ regeneration was obtained in Eucalyptus plants under reduced (50% reduced) or indirect light compared to normal light [185]. In addition, an increase in some growth parameters of E. urograndis tissues was reported in response to high light intensity (250 µmol m^-2 s^-1) [186]. On the contrary, Miranda et al [187] reported that the light quality was more important than the light intensity in vitro development of E. urophylla. It is believed that the variations of Eucalyptus responses to light quantity in vitro are largely dependent on the genotype x light x time or genotype x time interactions [188].