Cytokinin Potentials on In Vitro Shoot Proliferation and Subsequent Rooting of Agave sisalana Perr. Syn
Round 1
Reviewer 1 Report
Comments and Suggestions for AuthorsDear authors,
Agave sisalana holds significant application value in fields such as fiber extraction, pharmaceuticals, and bioenergy. However, traditional propagation methods are time-consuming and inefficient. Large-scale clonal propagation of Agave sisalana through tissue culture technology is of great importance for ecological conservation and sustainable resource utilization. Regarding the article, I have raised the following questions.
- The first occurrence of "PGRs" in the text should include its full name.
- Please provide the calculation formulas of shoots/explant，leaves/shoot and roots/shoot.
- Does the statement in line 270 "the higher concentrations of TDZ (1.5-2.5 mg L⁻¹) gave better multiplied and elongated shoots" - contradict the data presented in Table 2?
- The images/figures in the article are not sufficiently clear.
- Please expand the Discussion section to include a mechanistic analysis of how KIN, BA, and TDZ function at the physiological/molecular level.
- Please clearly state your recommended cytokinin (CTK) type and optimal concentration in the Conclusion section.
Thank you for considering these suggestions.
Best regards,
Author Response
Response_to Reviewer #1
Comments and Suggestions for Authors
Top of Form
Dear authors,
Agave sisalana holds significant application value in fields such as fiber extraction, pharmaceuticals, and bioenergy. However, traditional propagation methods are time-consuming and inefficient. Large-scale clonal propagation of Agave sisalana through tissue culture technology is of great importance for ecological conservation and sustainable resource utilization. Regarding the article, I have raised the following questions.
Response: Many thanks for your valuable comments which aimed to improve the MS. All changes or corrections you asked us to do are highlighted in yellow. Please note that there is a shift in lines number due to the corrections.
- The first occurrence of "PGRs" in the text should include its full name.
Response: Ok, done the first mention of PGRs was in the abstract and we wrote their full names then the abbreviation between two brackets, after that the abbreviation only in the rest of MS
- Please provide the calculation formula of shoots/explant leaves/shoot and roots/shoot.
Response: Ok, done I added it in the MS
- Does the statement in line 270 "the higher concentrations of TDZ (1.5-2.5 mg L⁻¹) gave better multiplied and elongated shoots" - contradict the data presented in Table 2?
Response: I corrected it to be agree with the results in the table 2
- The images/figures in the article are not sufficiently clear.
Response: Ok, done. We separated the figure into three big figures to be more clear and the numbers of photo are changed in the text accordingly.
- Please expand the Discussion section to include a mechanistic analysis of how KIN, BA, and TDZ function at the physiological/molecular level.
Response: Ok, done.
- Please clearly state your recommended cytokinin (CTK) type and optimal concentration in the Conclusion section.
Response: Ok, done.
Author Response File: 
Author Response.pdf
Reviewer 2 Report
Comments and Suggestions for Authorsplease see the attachment
Comments for author File: Comments.pdf
Author Response
Response to Reviewer #2
The following comments were made thoroughly for the manuscript improvement and revision. Many thanks for your time and efforts to improve our MS. All changes or corrections you asked us to do are highlighted in yellow.
➢ The manuscript in general is verbose, and has a high number of grammatical and awkward expressions, which profoundly weakens readability and is in need for professional language editing.
Response: We tried as we can to improve the language, we thank Dr. Judit Dobránszki who made corrections, editing, and revising the MS.
➢ The title itself is very informative; however, it might be shortened (e.g., “among three different cytokinins”), unadorned language may be used for simplicity with no loss of clarity. Authority citation should be mentioned after the scientific name in title.
Response: I changed the title to be “Cytokinin Potentials on In Vitro Shoot Proliferation, Subsequent Rooting of Agave sisalana Perr. Syn” , authority citation is included in the new title. I hope it is accepted
➢ The abstract is informative, but overly long with too many numbers that make it hard to grasp fast; it should be rewritten with a summary-style that clearly states aims, main results, and conclusions without repeating methods briefly.
Response: I rewrote the abstract so that the main important findings, the aim could be found, and I deleted some numbers to make the reading easy for fast understand
➢ Many sentences from the abstract and introduction are duplicated, including when describing the rooting behavior of the BA and TDZ treatments, with slight variation, in multiple sections.
Response: I deleted the repeated sentences as I can
➢ The introduction has a long section on Agave sisalana with some repetition of the importance, cultivation challenges and uses that should be simplified to speed up the reading.
Response: I shortened the introduction to be included information about Agaves and A. sisalana; important applications, propagation, the obstacles that face its traditional propagation so that in vitro propagation is urgently required, applications of micropropagation of Agave species then the aim of the study
➢ While the introduction is effective in laying out the necessity of micropropagation, it develops as quite long-winded and side-tracked regarding issues such as ecological destruction, nutraceutical values etc., that don’t always directly tie in to the aim of study.
Response: I am not agree with you in this point of view, because when we present the dangers that face the biodiversity of Agaves, destruction of the natural habitat and loss the biological resources of Agave, and on the other hand when we show the economic potentials and the growing demand of these species, and the various applications the depend on them, in addition to the insufficient conventional propagation even sexually or asexually, and the problems associated to its traditional propagation. All of these issues make the need to the in vitro propagation is very critical for plant resources conservation, biodiversity preservation, and large scale production, so all of these are linked directly or have strong relation to the aim of study and could answer the question why Agaves should be propagated in vitro?
➢ There is discrepancy in the explanation of cytokinin effect, especially BA, which is sometimes inhibitory and in some other way stimulating to root induction, and this requires a clarification.
Response: in the case of the BA, its accumulation at the base of the shoots in general hinders the rooting. However, if the literature describe that BA was beneficial for rooting, that can be because of two possible reasons 1. The sensitivity and need for BA may be different between plant species; 2. Moreover the applied concentration of BA also can cause different results (stimulating or hindering the subsequent rooting).
➢ The method is described in a readable way – suitable for reproducing, only that repeated description of pH-adjustment-and-autoclaving procedure for every session is not needed and it is the reason for a bulky text.
Response: I deleted the repeated part, and wrote it once in the text
➢ Mercuric chloride being technologically outdated and environmentally hazardous for sterilization, explain and/or provide justification for the application or suggest the use of safer alternatives, such as sodium hypochlorite or ethanol-based sterilizing agents.
Response: I am totally agree with you regarding this point, however we have already examined sodium hypochlorite for surface sterilization of the bulbils explants, and the contamination percentage was more than 80%, so we used mercuric chloride because it was more effective than sodium hypochlorite, and it is still used in many laboratories taking in consideration the cautions needed to get rid of its disposal or residues by a safe way
➢ The materials section is not well-organized, some sub-titles or steps for each period (e.g. culture initiation, proliferation, rooting, acclimatization) would greatly contribute to the readability.
Response: I am really sure that the material section is organized according to in vitro propagation protocols by this sequence of steps, if I could understand your comment
➢ Single images (Figure 1 in particular) are at low resolution and contain a large number of small images that are barely visible and do not support the experimental procedure properly; there is a need for better quality images with clearer identification.
Response: You are right, we separated this photo into three photos that could be visible as we can, we identified each photo clearly, we will provide these photos in Tiff extension to be in a high quality as supplementary files
➢ Findings are comprehensive however narratives are sometimes mixed with interpretative commentary, detracting from the lucidity of scientific reporting and should be rigorously distinguished.
Response: I revised the results to be more clear
➢ Data in the tables is extensive, statistical and not entirely referred to, nor discussed in the text with the result that there is a disconnect in the text narrating findings.
Response: The most important issue for any in vitro propagation protocol is the multiplication rate as well as the rooting capacity of the regenerated shoots, so I mainly focus on them but I refer to all the data
➢ Most of the text in the discussion section reproduces the same concepts and facts already described in the introduction, such as the importance and current uses of the Agave plants, which should be discussed and interpreted in a more profound way.
Response: I deleted the repeated facts and information which are mentioned in the introduction
➢ The discussion is mostly outdated in some references and some references are repeated multiple times, by incorporating new studies and preventing over-citation of the same application would increase a scholar approach.
Response: most of the outdated references are on Agave sisalana, I had to count them to present the fact that the literatures on it are limited and not recent which are belong to Debnath et al. 2010, Carneiro-dos Santos et al. 2014, Hazra etal. 2002, Nikam et al. 2003)
Moreover, I deleted the sentences that not related to the topic of MS as “cytokinins and stress” then I deleted the corresponding references, also I deleted the repeated ref. to reduce over-citation of the same publication as I can. I added new studies in the discussion
(Tahir et al. 2024; Bidabadi et al. 2020; Martins et al. 2022; van Voorthuizen et al. 2021; Wang et al. 2025; Yang et al. 2021, Ali et al. 2022), other more studies that related to the topic and the obtained results are included
➢ No comparison on the differential influence of the employed cytokinins on shoot multiplication and rooting at either a physiological or molecular level is provided as it would add a scientific base to the discussion.
Response: I tried as I can to add some information on the molecular mechanism of CKs,
Sorry, the entire authors are not specific in this point, so we couldn’t discuss it deeply.
We will take this point in consideration in the future
➢ Although the conclusion recapitulates the results, it generalizes the view of cytokinin application in plant tissue culture and does not stress sufficiently the new insights provided or practical use.
Response: I added sentences on the optimal CK and its concentration would be recommended for a successful in vitro propagation protocol of Agave sisalana, the future studies that should be investigated as well the further prospective
➢ Summary would benefit from some specific data or results (eg “TDZ at 1.0mgL-1 gave highest shoot number but failed to support rooting”) to support the recommendation of BA at 4.0mgL-1.
Response: ok, done
➢ The statistical design is proper and replication seems to be enough, however, an experimental design should be more organized, especially for the definition of treatment groups and sub cultivations times.
Response: We refered to number of replicates and number of explants per replicate in each experiment, number of subcultures in Materials and Methods section, if I could understand your comment
➢ In the ex-vitro acclimatization part although a number of treatments gives 100% survival, no clear indication is given about what point the acclimatization is successful, and no visual or physiological statements are done.
Response: the new roots that are induced and developed on the plants as well as new leaves that are emerged were the visual or physiological signs that indicated the successful of acclimatization, and all plants were survived (no plant die) in the treatments that gave 100% survival, knowing that the plants were followed up until they became one year old
➢ A number of scientific redundancies (repeated effects of TDZ on root inhibition; and the rationale of bulbil propagation) have diminished the concise concord and scientific integrity of this manuscript.
Response: I deleted as I can the repeated words regarding the inhibitory behavior of TDZ on rooting
➢ Overall, the manuscript reports on a well-designed and appropriate study for micropropagation of A. sisalana, however, it needs careful language edition, elimination of redundancy, improvement of figure quality, and clarification on rooted behavior and cytokinin interpretation to be considered for publication.
Response: We tried to do the best, we hope the corrections made be accepted, thanks for your valuable guidance
Author Response File: Author Response.pdf
Reviewer 3 Report
Comments and Suggestions for AuthorsThe authors Seliem et al. in their manuscript “In Vitro Comparative Study among Three Different Cytokinins: Shoot Proliferation, Subsequent Rooting and Acclimatization of Agave sisalana” underlined the potential of some cytokinins to improve the growth of sisal species in vitro culture and later in the acclimatization phase. This seems of great importance since the experiment is well-conducted and the manuscript is written following a logical order, but many changes, clarifications, and English revisions are required before publishing
Abstract
The importance of research and the main results are reported.
Line 19-22: Please change the phrase to “In vitro micropropagation of A. sisalana via axillary shoot proliferation from bulbil explants was attained using Murashige and Skoog medium (MS) supplemented with cytokinins 6-benzyladenine (BA), kinetin (KIN), or thidiazuron (TDZ).
Introduction:
In this section, the authors explained how their work is related to other studies in the published literature. I recommend shortening the introduction, as some paragraphs are quite long. Please review this section and provide a concise background that is relevant to the study.
Line 46: Change “peoples” to “people”
Lines 60+Line 62 and Line 64+Line 78+Line 82: Please, write “agave” as “Agave”
Line 111: Please, add the full name of BA “6-benzyladenine” for the first time, and then use the abbreviation BA in the rest of the manuscript.
Line 117: The full names should be added for these abbreviations (i.e. BA, KIN and TDZ)…..
Materials and Methods
Line 134: Change “by dipping” to “immersed”
Line 206+ Line 208: Please, write the probability level in lowercase as “p≤ 0.05”
Results
Table 1: replace the phrase “MS without cytokinin” with “Control” as the authors already defined the MS without cytokinin as control in lines 159-160, writing “MS basal medium without PGRs served as control”
Line 249+Line 275+Line 306: Add the full name of BA as 6-benzyladenine
Line 250+Line 276+Line 307: Add the word “analysis” after “ANOVA”
Line 255: Please, check that the statement “12 months” is correct in the sentence” After three subcultures, four weeks each (12 months) “?
Lines 265-266: Please, edit this statement as “while only the first two treatments of TDZ (0.5 and 1.0 mg L-1) produced roots on the shoots (Figure 1J and K).”
Line 305: I suggest merging Table 3 with Table 2, adding one column titled “Acclimatization percentage after 40 days" to Table 3.
Line 317: I suggest separating this Figure into three different figures to increase the quality of the images, since the current photos are small and blurry.
Discussion
This section needs to be shortened, and the authors should focus on highlighting their results, supported by adequate citations and discussion from the literature review.
Lines 327-376: The authors added more and longer information already mentioned in the introduction regarding the importance of Agave species, overexploitation, conventional propagation, etc.
Please, revise the paragraphs included in the indicated lines and summarize the useful information to start the discussion of your results from “Earlier attempts were made to improve the in vitro multiplication rate of A. sisalana via direct shoot organogenesis…….
Author Response
Response to Reviewer #3
Comments and Suggestions for Authors
The authors Seliem et al. in their manuscript “In Vitro Comparative Study among Three Different Cytokinins: Shoot Proliferation, Subsequent Rooting and Acclimatization of Agave sisalana” underlined the potential of some cytokinins to improve the growth of sisal species in vitro culture and later in the acclimatization phase. This seems of great importance since the experiment is well-conducted and the manuscript is written following a logical order, but many changes, clarifications, and English revisions are required before publishing
Response: Many thanks for your valuable comments which aimed to improve the MS. All changes or corrections you asked us to do are highlighted in yellow. Please note that there is a shift in lines number due to the corrections.
Abstract
The importance of research and the main results are reported.
Response: Many thanks for your encouragement
Line 19-22: Please change the phrase to “In vitro micropropagation of A. sisalana via axillary shoot proliferation from bulbil explants was attained using Murashige and Skoog medium (MS) supplemented with cytokinins 6-benzyladenine (BA), kinetin (KIN), or thidiazuron (TDZ).
Response: Ok, done.
Introduction:
In this section, the authors explained how their work is related to other studies in the published literature. I recommend shortening the introduction, as some paragraphs are quite long. Please review this section and provide a concise background that is relevant to the study.
Response: Ok, done.
Line 46: Change “peoples” to “people”
Response: Ok, done.
Lines 60+Line 62 and Line 64+Line 78+Line 82: Please, write “agave” as “Agave”
Response: Ok, done.
Line 111: Please, add the full name of BA “6-benzyladenine” for the first time, and then use the abbreviation BA in the rest of the manuscript.
Response: it is mentioned for the first time in the abstract where we wrote its full name then the abbreviation between two brackets, so I corrected Kinetin to KIN
Line 117: The full names should be added for these abbreviations (i.e. BA, KIN and TDZ).
Response: in the abstract, we wrote the full name of cytokinins under study then the abbreviation in two brackets, so after that should be the abbreviations only.
Materials and Methods
Line 134: Change “by dipping” to “immersed”
Response: Ok, done.
Line 206+ Line 208: Please, write the probability level in lowercase as “p≤ 0.05”
Response: Ok, done.
Results
Table 1: replace the phrase “MS without cytokinin” with “Control” as the authors already defined the MS without cytokinin as control in lines 159-160, writing “MS basal medium without PGRs served as control”
Response: Ok, done.
Line 249+Line 275+Line 306: Add the full name of BA as 6-benzyladenine
Response: Ok, done.
Line 250+Line 276+Line 307: Add the word “analysis” after “ANOVA”
Response: the word “ANOVA” is the abbreviation of Analysis of Variance, we used to write it as “ANOVA” in our published papers and it is accepted what do you think?
Line 255: Please, check that the statement “12 months” is correct in the sentence” After three subcultures, four weeks each (12 months) “?
Response: you are right, I mean 12 weeks, I corrected it
Lines 265-266: Please, edit this statement as “while only the first two treatments of TDZ (0.5 and 1.0 mg L-1) produced roots on the shoots (Figure 1J and K).”
Response: Ok, done.
Line 305: I suggest merging Table 3 with Table 2, adding one column titled “Acclimatization percentage after 40 days" to Table 3.
Response: I cant merge table 2 with table 3, because table 2 is applying MS free for the shoots produced only on BA and TDZ for root induction. While table 3 is the acclimatization for all rooted plantlets in the study (which produced directly on MS free and KIN supplemented media, and those shoots that produced on BA and TDZ then they needed to transfer on MS free for root induction).
Line 317: I suggest separating this Figure into three different figures to increase the quality of the images, since the current photos are small and blurry.
Response: Ok, done. We separated the figure into three big figures to be more clear and the numbers of photo are changed in the text accordingly.
Discussion
This section needs to be shortened, and the authors should focus on highlighting their results, supported by adequate citations and discussion from the literature review.
Response: Ok, done.
Lines 327-376: The authors added more and longer information already mentioned in the introduction regarding the importance of Agave species, overexploitation, conventional propagation, etc.
Please, revise the paragraphs included in the indicated lines and summarize the useful information to start the discussion of your results from “Earlier attempts were made to improve the in vitro multiplication rate of A. sisalana via direct shoot organogenesis
Response: Ok, done.
Author Response File: Author Response.pdf
Round 2
Reviewer 3 Report
Comments and Suggestions for AuthorsThe authors of "Cytokinin Potentials on In Vitro Shoot Proliferation and Subsequent Rooting of Agave sisalana Perr. Syn" have addressed the reviewer's comments and made the necessary revisions to their manuscript. I recommend accepting the manuscript in its current form.
