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Temporary Immersion Bioreactor for In Vitro Multiplication of Raspberry (Rubus idaeus L.)

Horticulturae 2025, 11(7), 842; https://doi.org/10.3390/horticulturae11070842

by Bruno Reyes-Beristain¹, Eucario Mancilla-Álvarez¹, José Abel López-Buenfil² and Jericó Jabín Bello-Bello^3,*

Reviewer 1:

Valbona Sota

Reviewer 2: Anonymous

Reviewer 3:

Branka Vinterhalter

Horticulturae 2025, 11(7), 842; https://doi.org/10.3390/horticulturae11070842

Submission received: 20 June 2025 / Revised: 9 July 2025 / Accepted: 16 July 2025 / Published: 17 July 2025

(This article belongs to the Special Issue Tissue Culture and Micropropagation Techniques of Horticultural Crops)

Round 1

Reviewer 1 Report (Previous Reviewer 1)

Comments and Suggestions for Authors

Dear authors,

You tried to optimize the manuscript, and you did well in this regard.

Some elements for which I do not agree with the experimental design clearly can't be changed anymore. It was designed this way from the beginning. In some aspects, it does not seem cost-effective.

However, the paper is now more comprehensive, and the discussion section is well-supported by the results you obtained.

Kind regards,

Comments for author File: Comments.pdf

Author Response

July 04, 2025

Dear Reviewer

Thanks for your comments on our manuscript ID: horticulturae- 3740157, titled: “Temporary immersion bioreactor for in vitro multiplication of raspberry (Rubus idaeus L.)”. The reviewers’ suggestions and recommendations have been addressed point-by-point. We have made major changes. The new changes were colored in blue during the manuscript. Besides, all suggestions have improved our manuscript substantially. Reviewers' suggestions and recommendations are appended below (C= Comment and R=Response):

C1: Micropropagation itself does not produce disease-free plants. You have to perform meristem culture first, or any therapy (thermotherapy, cryotherapy, cryotherapy or electrotherapy) and after verifying their phytosanitary status and confirm as disease-free, you can multiply the obtained product via micropropagation. I strongly recommend to revise this sentence.

R: We thank you for your constructive and high-quality feedback. The sentence has been revised and rewritten

C2: In my opinion, this is not cost effective. However, it’s clear you can’t do anything more in this case.

R: Thank you for your observation. I agree with you. However, due to the experimental focus on physiological responses and the need for controlled conditions, a lower explant density was necessary.

Example: This protocol can be scaled up to 4 liter bioreactors. Two liters of culture medium corresponds to 40 explants, with a total of 228 shoots per TIB.

C4: This subsection has the same caption as subsection 4.3 below

R: Thank you for your observation. These repeated subsections were revised and rewritten.

Yours sincerely,

Author Response File: Author Response.pdf

Reviewer 2 Report (Previous Reviewer 2)

Comments and Suggestions for Authors

Dear Authors,

I would guess that 7 explants were placed in 500 ml of medium. This would be the most accurate representation. However, the current rounding to 71 ml is much more accurate than the rounding to 75 ml.

Best regards,

Reviewer

Author Response

July 04, 2025

Dear Reviewer

C1: would guess that 7 explants were placed in 500 ml of medium. This would be the most accurate representation. However, the current rounding to 71 ml is much more accurate than the rounding to 75 ml.

R: Thank you for your observation. Yes, 7 explants in 500 mL was the setup used, and we agree that 71 mL per explant is a more precise representation than rounding to 75 mL.

Yours sincerely,

Author Response File: Author Response.pdf

Reviewer 3 Report (Previous Reviewer 3)

Comments and Suggestions for Authors

New version of the manuscript has been considerably improved, but some corrections like the change of chapter title were not done. I confirm here that both immersion frequency and frequency of immersion are equally valid so that this remark can be neglected. But there are also some new remarks connected with the portions of text that were changed.

In 2.1. pressure 15 psi should be expressed in bar units.
In experiment I authors claimed use of vessels with 500 ml capacity containing 250 ml medium. In experiment II size of vessels was not stated, but according to Fig. 1.they are the same vessels as those in exp I. In exp III vessels are 1000 ml with 500 ml medium. However, from Fig. 1 this seems to be the same vessels as those from exp I and II. This means that the amount of medium per explant was not 25, 50, 71 and 100 ml, but 12.5, 25, 35,7 and 50 ml. The entire text dealing with medium amount per explant needs to be checked.
In chapter 3.1. as you compare only two groups, you cannot use highest, greatest, lowestbut instead use better, longer ,shorter...
I believe that the bar length in Fig. 1. is not correct. Bar lenght would better correspond to vessels of 350 ml than 500 ml.
Presentation of results for chapter 3. 4. should be made differently. First the difference between the content of chlorophyll and carotenoids should be done having in mind differences of cultivation. Higher content of Chlorophyll (a, b and total) and carotenoid (β-carotene) in shoots culture in TIS should be stated irrespectively of immersion frequencywhich by itself has no statistical significance here. Histograms of Chl a and Chl b are not well distinguished by their color.
In chapter 3.5. first sentence is not correct, as there is no statistical significance for total Chl content among the different culture medium volumes per explants.

Comments on the Quality of English Language

Language editing is recommended.

Author Response

July 04, 2025

Dear Reviewer

C1: In 2.1. pressure 15 psi should be expressed in bar units.

R: Thank you for your observation. The words have been revised and rewritten as suggested.

C2: In experiment I authors claimed use of vessels with 500 ml capacity containing 250 ml medium. In experiment II size of vessels was not stated, but according to Fig. 1. they are the same vessels as those in exp I. In exp III vessels are 1000 ml with 500 ml medium. However, from Fig. 1 this seems to be the same vessels as those from exp I and II. This means that the amount of medium per explant was not 25, 50, 71 and 100 ml, but 12.5, 25, 35,7 and 50 ml. The entire text dealing with medium amount per explant needs to be checked.

R: Thank you for your observation. We have reviewed the fig.1 and revised the text accordingly to ensure that the medium volume per explant is accurately reported throughout the manuscript.

Note: It is important to mention that all experiments were conducted under equal conditions. That is, different containers can be used if the same conditions are met regarding the volume of culture medium and the number of explants.

C3: In chapter 3.1. as you compare only two groups, you cannot use highest, greatest, lowest but instead use better, longer, shorter...

R: Thank you for your observation. The words have been revised and rewritten as suggested.

C4: I believe that the bar length in Fig. 1. is not correct. Bar lenght would better correspond to vessels of 350 ml than 500 ml.

R: Thank you for your observation. The scale bar in Fig. 1 has been corrected to match the actual 500 mL vessel dimensions.

C5: Presentation of results for chapter 3. 4. should be made differently. First the difference between the content of chlorophyll and carotenoids should be done having in mind differences of cultivation. Higher content of Chlorophyll (a, b and total) and carotenoid (β-carotene) in shoots culture in TIS should be stated irrespectively of immersion frequency which by itself has no statistical significance here. Histograms of Chl a and Chl b are not well distinguished by their color.

R: Thank you for your observation. Chapters 3 and 4 present results from independent experiments; therefore, the data were analyzed and discussed separately. We have clarified the differences in chlorophyll and carotenoid content based on the cultivation system, and improved the histogram colors to better distinguish Chl a and Chl b.

C6: In chapter 3.5. first sentence is not correct, as there is no statistical significance for total Chl content among the different culture medium volumes per explants.

R: Thank you for your observation. The first sentence of Chapter 3.5 has already been corrected and rewritten for clarity.

Yours sincerely,

Author Response File: Author Response.pdf

Round 2

Reviewer 3 Report (Previous Reviewer 3)

Comments and Suggestions for Authors

I wish to thank Editors for the opportunity to once again inspect the corrected text of this manuscript. Unfortunately, authors did not make all of the corrections they were told to do and did not provide adequate explanations, is need they claim making all of these corrections which is not true.

Authors made corrections on remarks C1 and C 3. However, the main remarks, which are of utmost importance for this paper, (C2 and C4 related to the size of bioreactors) are still not addressed. Authors claim that they used the bioreactor vessel size of 500 mL containing 250 mL medium for experiments 1 and 2. and bioreactor vessel with total capacity of 1000 mL with 500 mL medium for experiment 3 (Chapter 2.4.). But according the bar sizes in Fig. 1, vessel seem to be of same size in all experiments. According to the size of presented bars and air filters it seems that volume of vessel is some 350 - 500 mL. If this assumption is correct authors need to adjust the text to fit Fig.1. It also means that the amount of medium per explant was not 25, 50, 71 and 100 ml, but 12.5, 25, 35.7 and 50 ml. If authors still claim that they used 1000 mL jars in experiment 3, then a new image with corrected bar size is needed.

Authors did not make correction for remarks C5 and C6. They had to improve color presentation of Chl a and Chl b in histograms. Authors also did not provide corrected text. They had to point out that there are no significant statistical difference in content of total Chl in Chapter 3.5.

In my opinion text will be acceptable for publishing only after clarification of repeated problems that I observed and described in my present and previous recommendations.

Author Response

We thank you for your constructive and high-quality feedback. The comments were insightful and greatly contributed to strengthening the manuscript. We have carefully addressed all points raised and made substantial revisions throughout. We believe the manuscript has significantly improved as a result, and we remain open to making further changes should additional concerns arise during the next stage of review. Please see the attachment.

Author Response File: Author Response.pdf

This manuscript is a resubmission of an earlier submission. The following is a list of the peer review reports and author responses from that submission.

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

Dear authors,

I have attached the document with my comments. You have worked hard and tried to build a manuscript based on literature reviews and some statistical analysis, rather than focusing on the experimental design and following an appropriate methodology. I hope my comments will be somehow useful for further studies.

Best regards,

Comments for author File: Comments.pdf

Author Response

May 15, 2025

Ms. Hailey Xia

Section Managing Editor

Dear Reviewer

Thanks for your comments on our manuscript ID: horticulturae-3629438, titled: “Temporary immersion bioreactor for in vitro multiplication of raspberry (Rubus idaeus L.)”. The reviewers’ suggestions and recommendations have been addressed point-by-point. We have made major changes. The new changes were colored in blue in the manuscript. Besides, all suggestions have improved our manuscript substantially. Reviewers' suggestions and recommendations are appended below (C= Comment and R=Response):

C1: I suggest to delete this sentence since it’s not correct. Regarding genetic stability, the conventional vegetative propagation methods have been always effectively used. The method which is discussed to cause genetic instability is exactly in vitro culture. Regarding phytosanitary status, in vitro culture does not spontaneously produce virus-free plants. For this, specific protocols should be followed, which, in your study, you didn’t do, since it was not the purpose of this study.

R: Thank you for your observation, you are right!. The paragraph has been revised and rewritten.

C2: Is better to elaborate a bit more on the advantages of PTC.

R: Thank you for your observation. The paragraph has been revised and more advantages have been added.

C3: Which type is this? All these bioreactors: SETIS, RITA, PlantForm… are TIB bioreactors (Temp. immersion bioreactors)

R: We appreciate your observation. According to Bello-Bello et al. (2024), two types of culture systems use bioreactors: (1) permanent immersion systems (PIS), where explants are continuously submerged in culture media, and (2) temporary immersion systems (TIS), where explants are only temporarily submerged in culture media. There are several TIS models available (Bravo-Ruíz et al. 2024), including the Recipient for Automated Temporary Immersion (RITA®) (Vitropic, St-Mathieu de Tréviers, France) (Alvard et al. 1993); Temporary Immersion Bioreactor (TIB) (Escalona et al. 1999); Temporary Immersion Modular Bioreactor (BioMINT®) (CICY, Yucatan, Mexico) (Robert et al. 2006); Ebb-and-Flow Bioreactor (Ducos et al. 2007); Box-in-Bag Bioreactor (Ducos et al. 2010); Mobloc Advance Temporary Immersion System (MATIS®) (CID Plastiques, Valergues, France) (Etienne et al. 2013); PlantForm™ Bioreactor (Plant Form AB, Lomma, Sweden) (Welander et al. 2014); SETIS™ Bioreactor (Vervit, Beervelde, Belgium) (Vervit 2024); GreenTray® (IRTA, Barcelona, Spain) (Cantabella et al. 2022); Temporary Immersion Modular System (SMIT®) (Invitroorquid, Mexico City, Mexico) (Invitroorquid® 2024), among other examples. TIB is a Cuban patent and consists of twin flasks.

Bello-Bello, J. J., Mancilla-Álvarez, E., and Spinoso-Castillo, J. L. (2025). Scaling-up procedures and factors for mass micropropagation using temporary immersion systems. In Vitro Cellular & Developmental Biology-Plant, 1-12. https://doi.org/10.1007/s11627-024-10480-7

Bravo-Ruíz IN, González-Arnao MT, Hernández-Ramírez F, López-Domínguez J, Cruz-Cruz CA (2024) Types of temporary immersion systems used in commercial plant micropropagation. In Micropropagation methods in temporary immersion systems 2759:9‒24. New York, NY: Springer US. https://doi.org/10.1007/978-1- 0716-3654-1_2

Alvard D, Cote F, Teisson C (1993) Comparison of methods of liquid medium culture for banana micropropagation. Plant Cell Tiss Org Cult 32:55–60. https://doi.org/10.1007/BF00040116

Escalona M, Lorenzo JC, Daquinta M, Desjardins Y, Borroto CG (1999) Pineapple (Ananas comosus L. Merr) micropropagation in temporary immersion systems. Plant Cell Rep 18:743–748. https://doi.org/10.1007/s002990050653

Robert ML, Herrera-Herrera JL, Castillo E, Ojeda G, Herrera-Alamillo MA (2006) An efficient method for the micropropagation of Agave species. Methods Mol Biol 318:165–178. https://doi.org/10.1385/1-59259-959-1:165

Ducos JP, Labbe G, Lambot C, Petiard V (2007) Pilot scale process for the production of pre-germinated somatic embryos of selected robusta (Coffea canephora) clones. In Vitro Cell Dev Biol – Plant 43:652–659. https://doi.org/10.1007/s11627-007-9075-0

Ducos JP, Terrier B, Courtois D (2010) Disposable bioreactors for plant micropropagation and mass plant cell culture. Adv Biochem Eng Biotechnol 115:89–115. https://doi.org/10.1007/10_2008_28

Etienne H, Bertrand B, Georget F, Lartaud M, Montes F, Decham E, Verdeil JL, Barry-Etienne D (2013) Development of coffee somatic and zygotic embryos to plants differs in the morphological, histochemical and hydration aspects. Tree Physiol 33:640–653. https://doi.org/10.1093/treep hys/tpt034

Welander M, Persson J, Asp H, Zhu LH (2014) Evaluation of a new vessel system based on temporary immersion system for micropropagation. Sci Hortic 179:227–232. https://doi.org/10.1016/j.scienta.2014.09.035

Vervit (2024) SETIS™ bioreactor temporary immersion systems in plant micropropagation. Available online:http:// www.setissystems.be.

Cantabella D, Mendoza CR, Teixidó N, Vilaró F, Torres R, Dolcet-Sanjuan R (2022) GreenTray® TIS bioreactor as an effective in vitro culture system for the micropropagation of Prunus spp. Rootstocks and analysis of the plant-PGPMs interactions. Sci Hortic 291:110622. https://doi.org/10.1016/j.scienta.2021.110622

Invitroorquid® (2024) Available online: https://invitroorquid.com.mx/sitio/

C4: Write as SETIS^TM

R: Thank you for your observation. The term has been revised and changed as suggested.

C5: Write as PlantForm^TM

R: Thank you for your observation. The term has been revised and changed as suggested.

C6: Are you referring to any prototype or handmade TIB??

R: Thank you for your observation. Yes, in this study, we are referring to the TIB prototype originally design by Escalona et al. [19].

C7: Is a prototype or handmade by you??

R: Thank you for your observation. Yes, in this study, the TIB was a handmade bioreactor based on the prototype from Escalona et al. [19]. We assembled this bioreactor.

C8: To avoid repetition, I suggest to use here: supplemented with ….

R: Thank you for your observation. The sentence has been revised and rewritten as suggested.

C9: What about this??

R: I appreciate your comment, you are right! We deleted this paragraph that doesn't make sense.

C10: NO pictures were provided in the results section for the in vitro cultures. I strongly recommend to provide pictures

R: Thank you for your recommendation. Pictures for the in vitro cultures have been provided as suggested

C11: NO pictures were provided in the results section for the multiplication in TIBs. I strongly recommend to provide pictures

R: Thank you for your recommendation. Pictures for the multiplication in TIBs have been provided as suggested

C12: So, you have used the Twin flask system. So, your TIB has a name… I recommend to use this name throughout the manuscript.

R: Thank you for your recommendation. In this study, we used a handmade TIB bioreactor based on the Twin flask system from Escalona et al. [19] and the name was suggested by these authors.

C13: In my opinion, immersing only 5 explants in 250 ml of medium, results in a very high cost of production.

R: Thank you for your observation. However, the mass propagation for any species it is important to adjust the volume of the culture medium to optimize the culture conditions. In addition, the evaluation of explant density per bioreactor is an important factor that is use to determine nutrient consumption per explant, available headspace, competition between explants, and light irradiance. Mass production scaling is first done in small containers. The results of this study can be scaled up to larger capacity bioreactors. For more details, see the article: Bello-Bello, J. J., Mancilla-Álvarez, E., and Spinoso-Castillo, J. L. (2025). Scaling-up procedures and factors for mass micropropagation using temporary immersion systems. In Vitro Cellular & Developmental Biology-Plant, 1-12. https://doi.org/10.1007/s11627-024-10480-7

C14: As I understand, here you have 4 treatments regarding medium volume, and 4 evaluations regarding explants’ number in each medium volume: In total 16 combination for 2 variables: medium volume and explants number/volume

R: Thank you for your observation. We did not specify this methodology well. The experiments were performed independently not in combination. In all cases, different explants per bioreactor were used (20, 10, 7 and 5 explants) in glass jars with a total capacity of 1000 mL with 500 mL of multiplication medium. The Experiments under this design correspond to 25, 50, 75 and 100 mL per explant, respectively. This sentence has been revised and changed in the corresponding section for a better understanding.

C15: These are standard formulas, so no need to put here. In this case, it’s enough to put the reference only the reference.

R: Thank you for your recommendation. All the standard formulas in this section were eliminated. In addition, the section has been revised and rewritten for a better understanding.

C16: How did you prepare the microscopic samples. How many microscopic fields did you evaluate for each treatment. I imagine you have used leaves from each treatment: in vivo plants, semisolid medium, all TIBS combinations. How many leaves did you evaluate in each case?

R: Thank you for your observation. The microscopic samples were prepared as follow: “the abaxial epidermis of the developed leaves were cleaned, and then carefully smeared with nail varnish in the mid-area between the central vein and the leaf edge. The thin films were peeled off from the leaves surface, mounted on a glass slide, covered with a cover slip, and then lightly pressured with fine point tweezers”. All samples were observed in 1 mm² using a 40X objective under a microscope. This section has been revised and rewritten for a better understanding.

C17: What magnification did you use?? NO picture was provided in the results section for the microscopic observation. I strongly recommend to put pictures.

R: Thank you for your recommendation. No picture was provided due to the low quality obtained. However, the taken pictures were used for stomata quantification. Here, we show you some examples of the pictures.

C18: In the abstract, you mention hyperhydricity. How did you evaluate it in each culture system??

R: Thank you for your observation. The evaluation of the shoots hyperhydricity have been added in Materials and Methods section: “The shoots that were fragile, translucent stems and leaves were identified as hyperhydric shoots. The hyperhydricity rate was calculated using the formula: hyperhydric shoots/normal shoots × 100”.

C19: 5 explants/TIB is a very low number even if you perform 4 replicates.

R: Thank you for your observation. As I mentioned in a previous comment, mass production scaling is first done in small containers. The results of this study can be scaled up to larger capacity bioreactors. Few explants were used due to the capacity of the bioreactor.

C20: First of all, did you easily stabilized the in vitro plantlets from the mother plants? Which was the multiplication rate after subculturing for 3 times in semisolid medium? This analysis is necessary because you did 3 subcultures, 28 days each, and after 3 months you started testing TIBs.

R: Thank you for your observation. The stabilization of the in vitro plantlets from the mother plants was not easy. However, the multiplication rate after subculturing for 3 times in semisolid medium was not evaluated due to the plant material in the first subcultures does not expresses a high shoot proliferation potential. Therefore, this analysis has not been carried out because the objective of this study was to evaluated the TIB efficiency in comparison with semisolid medium system. In addition, it is necessary to perform subcultures to have a high stock of explants to carry out the experiments.

C21: Which was the immersion time and frequency here for the TIB? As I understand you put 250 ml in TIB and 5 explants

R: Thank you for your observation. The immersion time and frequency of 2 minutes every 8 hours have been added in Table 1 and the corresponding section.

C22: As I understand, you put 250 ml in TIB and 5 explants

R: Thank you for your observation. Yes, for the evaluation of different immersion frequencies in temporary immersion a glass jars with a total capacity of 500 mL with 250 mL of culture medium was used at a rate of 50 mL of liquid multiplication medium was added per explant, placing five explants per bioreactor. This section has been revised and rewritten for a better understanding.

C23: Where are the results for the section 2.4?? As I understood, you have used 16 combinations.

R: Thank you for your observation. We did not specify this methodology well. The experiments were performed independently not in combination. In all cases, different explants per bioreactor were used (20, 10, 7 and 5 explants) in glass jars with a total capacity of 1000 mL with 500 mL of multiplication medium. The experiments under this design correspond to 25, 50, 75 and 100 mL per explant, respectively. This sentence has been revised and changed in the corresponding section for a better understanding.

C24: Where are the results for the section 2.4???

R: Thank you for your observation. The results for the section 2.4 are in Table 1, III. However, in the Results section this has been revised and rewritten.

C25: Write as in vitro multiplication

R: Thank you for your observation. The word has been revised and changed as suggested.

C26: I suggest to delete this since it lacks data starting from methodology and many more.

R: Thank you for your suggestion. The methodology was described in detail for better understanding. We request to maintain this section.

C27: The same here

R: Thank you for your suggestion. The methodology was described in detail for better understanding. We request to maintain this section.

C28: Not enough data and explanation for this stage….

R: Thank you for your observation. The data has been included; the survival rate during the acclimatization stage was included in the Table 1.

C29: However, in the results you presented for the acclimatization stage, you didn’t find differences in survival percentage regarding the culture systems or the immersion frequency!!

R: I appreciate your comment. However, the shoots without symptoms of hyperhydricity were used for acclimatization stage. This observation has been revised and rewritten for a better understanding.

C30: Plants under in vitro conditions are characterized by mixotrofic growth, a situation which is auto regulated during the acclimatization stage. Since you did not observe differences among treatments during the acclimatization stage, this long discussion here is somehow not so useful.

R: I appreciate your comment, you are right! Photomixotrophic growth is auto regulated during the acclimatization stage. However, photosynthetic pigments also promote in vitro shoots development. We consider this discussion useful and a phrase was included in this section: “Chlorophyll and carotenoid content were greater in temporary immersion compared to semisolid medium. These results suggest that the increase in photosynthetic pigments during temporary immersion could promote in vitro shoots development”. We request to maintain this section.

C31: Mixtrotrophism is something in between heterotrophic growth and autotrophic ones, meaning that some organic compounds are synthetized, and some others are absorbed by the medium (if the medium contains such)…. I suggest to deeply revise this sentence.

R: Thank you for your comment. The sentence is correct; however, we have made modifications to the sentence as you suggested: “The in vitro photomixotrophism refers to the capacity of explants to acquire energy from the culture medium and/or as a product of in vitro photosynthesis”.

C32: I strongly suggest to deeply revise this. The percentage of sucrose is the same in all media. For example, if you have a concentration of 3% of sucrose, you will add 3 gr if you will prepare 100 ml of media, or 6 gr if you have to prepare 200 ml of media. In all cases, the proportions are maintained.

R: I appreciate your comment, you are right! We deleted this sentence that doesn't make sense.

C33: Beside many functions, the stomatal density is related to the transpiration during ex vitro adaptation. Since you did not observe differences among treatments during the acclimatization stage, this long discussion here is somehow not so useful.

R: I appreciate your comment, you are right! The stomatal density is related to the transpiration during ex vitro adaptation. We do not directly assess stomatal density. However, stomatal index and percentage of closed stomata also affect the in vitro shoots development. We wanted to study the effect of stomata on the in vitro systems evaluated prior to the acclimatization stage. We consider this discussion useful. We request to maintain this discussion.

Please address all correspondence concerning this manuscript to me at: jericobello@gmail.com

Thanking your kind attention to our submission, we hope to hear from you soon.

Yours sincerely,

Dr. Jericó Jabín Bello-Bello

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

Dear Authors,

The subject of the work is important from the point of view of efficient production of elite raspberry propagation material.

However, one of the points of the methodology - 2.4 (effect of volume of culture medium) raises doubts as to whether it was presented correctly, whether the authors did not make a mistake. Therefore, it seems necessary for the work to be returned to the authors for correction. If there is an error in this part, it affects the correctness of the presented results and discussion.

I have presented my doubts in the text of the manuscript.

Best regards,

Reviewer

Comments for author File: Comments.pdf

Author Response

May 15, 2025

Ms. Hailey Xia

Section Managing Editor

Dear Reviewer

C1: Please, write what type of bioreactor it is?

Alvard D, Cote F, Teisson C (1993) Comparison of methods of liquid medium culture for banana micropropagation. Plant Cell Tiss Org Cult 32:55–60. https://doi.org/10.1007/BF00040116

Ducos JP, Terrier B, Courtois D (2010) Disposable bioreactors for plant micropropagation and mass plant cell culture. Adv Biochem Eng Biotechnol 115:89–115. https://doi.org/10.1007/10_2008_28

Vervit (2024) SETIS™ bioreactor temporary immersion systems in plant micropropagation. Available online:http:// www.setissystems.be.

Invitroorquid® (2024) Available online: https://invitroorquid.com.mx/sitio/

C2: Should be given for which specific values.

R: Thank you for your observation. The specific values were added.

C3: how many minutes

R3: The immersion time and frequency of 2 minutes every 8 hours have been added in the manuscript.

C4: Change the record to the correct one

R4: Thank you for your observation. The record has been revised and corrected as suggested.

C5: The goal is too general. Please be more specific.

R5: Thank you for your recommendation. The goal has been revised and rewritten. The objective of this study was to establish a protocol for the in vitro propagation of R. idaeus using a temporary immersion bioreactor (TIB), under intermittent immersion periods and culture medium volumes at the in vitro multiplication stage.

C6: Fragment to be removed.

R6: I appreciate your comment, you are right! We deleted this paragraph that doesn't make sense.

C7: Name this part 'experiment I' because it is called that later on, e.g. in tables.

R7: Thank you for your recommendation. The experiments names have been changed as suggested.

C8: Please add a photo of your device.

R8: Thank you for your recommendation. Photographs of the device have been provided as suggested. See TIB installation originally design by Escalona et al. [19].

C9: Please add information about the vessels the semisolid cultures were in and what volume of medium was used for them.

R9: Thank you for your observation. Additional information was included: “For semisolid cultures, glass jars with a total capacity of 500 mL with 250 mL of culture medium was used at a rate of 50 mL per explant to maintain equal culture conditions”, placing five explants per bioreactor. This section has been revised and rewritten for a better understanding.

C10: How many jars, how many explants in jars, how many bioreactors?

R10: Thank you for your observation. Additional information was included: In all cases, glass jars with a total capacity of 500 mL with 250 mL of culture medium was used at a rate of 50 mL of liquid multiplication medium was added per explant, placing five explants per bioreactor. Four jars in semisolid medium and four bioreactors were used for each culture system. This section has been revised and rewritten for a better understanding.

C11: how much medium, what is the volume of the vessel, how many explants in the vessel?

R11: Thank you for your observation. Additional information was included. In all cases, glass jars with a total capacity of 500 mL with 250 mL of culture medium was used at a rate of 50 mL of liquid multiplication medium was added per explant, placing five explants per bioreactor. Four jars in semisolid medium and four bioreactors were used for each culture system. This section has been revised and rewritten for a better understanding.

C12: There is probably some mistake here. Multiples of 75ml and 100ml are not 250ml. I was wondering if maybe the conversion is to the volume of the vessel, but 20 x 25 = 500, 10 x 50 = 500, 75 x 7 = 525, 5 x 100 = 500. Maybe the experiment was set up differently. 20, 10, 7 or 5 explants were thrown into the vessel for 250ml of medium. Then, per explant, we have 12.5, 25, 35.7 and 50ml of medium. If that was the case, the results in Table 1 are assigned incorrectly, so the conclusions will also be incorrect.

R12: Thank you for your observation. However, the experiments were performed independently not in combination. In all cases, different explants per bioreactor were used (20, 10, 7 and 5 explants) in glass jars with a total capacity of 1000 mL with 500 mL of multiplication medium. The experiments under this design correspond to 25, 50, 75 and 100 mL per explant, respectively. This sentence has been revised and changed in the corresponding section for a better understanding.

C13: Was this intended as an experiment? How many shoots were taken for each combination?

R13: Thank you for your observation. Yes, it was an experiment, with a total of 150 shoots of approximately 2-3 cm in length, without hyperhydricity symptoms, from different culture systems were used.

C14: also for acclimatization? 'Four replicates', and there were three bioreactors. What was the replication?

R14: Thank you for your observation. No, for acclimatization three replicates of fifty shoots from different culture systems were used, with a total of 150 shoots.

C15: It would be good if you could add photos from your experiments in bioreactors.

R15: Thank you for your recommendation. Photos for the in vitro cultures have been provided as suggested.

Please address all correspondence concerning this manuscript to me at: jericobello@gmail.com

Thanking your kind attention to our submission, we hope to hear from you soon.

Yours sincerely,

Dr. Jericó Jabín Bello-Bello

Author Response File: Author Response.pdf

Reviewer 3 Report

Comments and Suggestions for Authors

Article „Temporary immersion bioreactor for in vitro multiplication of raspberry (Rubus idaeus L.)" by B. Reyes-Beristain, E.Mancilla-Álvarez et al. provides a nice presentation of advantages which temporary immersion bioreactor systems have over the conventional techniques for in vitro multiplication of raspberry on semisolid medium.

Experimental treatments are well planed and presented. Authors accurately defined the optimal conditions for in vitro culture multiplication of R. idaeus in a temporary immersion bioreactor. The obtained large production of new shoots which rooted en mas with a survival rate to exvitro greenhouse conditions of 96-99%. In the introduction and discusion authors present previous studies that were done TIB cultivation of R.ideus comparing thist results with those that they obtained in the present study.

However there are some points which should be corrected which I would like to show to authors. It is mostly related to some inapropriate use of terminology.

In chapter 2.1. Plant Material and In vitro Establishment, last paragrph should be erased as it apparently remained there by mistake.
In the chapter 2.2. and elsewhere I strongly recommend that "culture system " should be replaced by "method of cultivation ".
Your Murashige and Skoog medium is of unknown composition. You can not declare just the number of grams of some probably commercial, ready made mixture and then provide reference. I suggest you to write down MS composition (look in other papers) or just state the manufacturer of the commercial MS product. What was the percentage of employed sucrose?
In the chapter 2.3. Effect of immersion frequency on shoot regeneration you need to replace "immersion frequency" with "frequency of immersion". Thus instead of "The frequencies were evaluated every 4, 8..." it should be "evaluated for immersion that was done every 4, 8 or 12 hours"

This corrections need to be incorporated in all of the results, tables, legends, discussion etc.

Chapter 2.4. Effect of medium volume per explant on shoot regeneration. Your sentence "...different volumes of culture medium (25, 50, 75 and 100 mL per explant) were evaluated with 20, 10, 7 and 5 explants per bioreactor "is not clear enough. TIB that you used had 500 ml volume. If you placed in a TIB 20 explants and then added 25 ml per explant or 10 explants with 50 ml per explants .... than TIB becomes overcrowded by the end of treatment which will certainly have some side effects.

So if you studied effect volume per explant than you had to vary only the volume of medium in the TIB, keeping the same number of explants. Look how it is described in reference 21. Anyhow make a correction by finding a better way to describe what you have done.

Change the title of chapter 3.4. from " Chlorophyll and carotenoid determination during immersion frequency" into " Chlorophyll and carotenoid content in relation to different frequency of immersions".

Legends of Table 1 need some corrections. Instead of "Effect of different immersion frequencies and culture medium volumes on in vitro multiplication of raspberry (Rubus idaeus L.) at 4 weeks of culture" it should be "Effect of different frequencies of temporary immersion and volumes of culture medium on .... ". Also you should state number of explants per treatment for every treatment type (I, II, III), number of explants per culture vessel and volume of media per explants.

In the first column of the table instead of "Frequency every.." it should be "Immersion every.."

Legends for Fig. 2. and Fig 3. correct according to instructions for Table 1.
As the paper is dealing with mass production of shoots in bioreactors it would perhaps be interesting for readers to show some photographs of your TIB instalation.
In legend of Fig. 3. it is not stated how cultures were produced, please be accurate.

Comments on the Quality of English Language

There are some remarks

Author Response

May 15, 2025

Ms. Hailey Xia

Section Managing Editor

Dear Reviewer

Article „Temporary immersion bioreactor for in vitro multiplication of raspberry (Rubus idaeus L.)" by B. Reyes-Beristain, E. Mancilla-Álvarez et al. provides a nice presentation of advantages which temporary immersion bioreactor systems have over the conventional techniques for in vitro multiplication of raspberry on semisolid medium.

Experimental treatments are well planned and presented. Authors accurately defined the optimal conditions for in vitro culture multiplication of R. idaeus in a temporary immersion bioreactor. The obtained large production of new shoots which rooted in mass with a survival rate to ex vitro greenhouse conditions of 96-99%. In the introduction and discussion authors present previous studies that were done TIB cultivation of R. ideus comparing these results with those that they obtained in the present study.

However, there are some points which should be corrected which I would like to show to authors. It is mostly related to some inappropriate use of terminology.

C1: In chapter 2.1. Plant Material and In vitro Establishment, last paragraph should be erased as it apparently remained there by mistake.

R: I appreciate your comment, you are right! We deleted last paragraph.

C2: In the chapter 2.2. and elsewhere I strongly recommend that "culture system " should be replaced by "method of cultivation ".

R2: Thank you for your observation. The term has been revised and replaced.

C3: Your Murashige and Skoog medium is of unknown composition. You cannot declare just the number of grams of some probably commercial, ready made mixture and then provide reference. I suggest you to write down MS composition (look in other papers) or just state the manufacturer of the commercial MS product. What was the percentage of employed sucrose?

R3: Thank you for your observation. The composition of the MS medium was according to the international formulation medium of Murashige and Skoog [21], which was already cited in the manuscript. This culture medium was prepared with stock solutions in the laboratory. The formulation medium includes 3% sucrose. ¨This culture medium was prepared with stock solutions¨.

C4: In the chapter 2.3. Effect of immersion frequency on shoot regeneration you need to replace "immersion frequency" with "frequency of immersion". Thus instead of "The frequencies were evaluated every 4, 8..." it should be "evaluated for immersion that was done every 4, 8 or 12 hours".

R4: Thank you for your observation. The suggested term has been revised and replaced.

C5: These corrections need to be incorporated in all of the results, tables, legends, discussion etc.

R5: I appreciate your comment. These corrections have been revised and incorporated as suggested in all sections.

C6: Chapter 2.4. Effect of medium volume per explant on shoot regeneration. Your sentence "...different volumes of culture medium (25, 50, 75 and 100 mL per explant) were evaluated with 20, 10, 7 and 5 explants per bioreactor "is not clear enough. TIB that you used had 500 ml volume. If you placed in a TIB 20 explants and then added 25 ml per explant or 10 explants with 50 ml per explants .... than TIB becomes overcrowded by the end of treatment which will certainly have some side effects.

R6: Thank you for your observation. We did not specify this methodology well. The experiments were performed independently not in combination. In all cases, different explants per bioreactor were used (20, 10, 7 and 5 explants) in glass jars with a total capacity of 1000 mL with 500 mL of multiplication medium. The Experiments under this design correspond to 25, 50, 75 and 100 mL per explant, respectively. This sentence has been revised and changed in the corresponding section for a better understanding.

C7: Change the title of chapter 3.4. from " Chlorophyll and carotenoid determination during immersion frequency" into " Chlorophyll and carotenoid content in relation to different frequency of immersions".

R7: Thank you for your observation. The title has been revised and rewritten.

C8: 1. Legends of Table 1 need some corrections. Instead of "Effect of different immersion frequencies and culture medium volumes on in vitro multiplication of raspberry (Rubus idaeus L.) at 4 weeks of culture" it should be "Effect of different frequencies of temporary immersion and volumes of culture medium on .... ". Also you should state number of explants per treatment for every treatment type (I, II, III), number of explants per culture vessel and volume of media per explants.

R8: Thank you for your observation. The legends of Table 1 have been revised and rewritten.

C9: In the first column of the table instead of "Frequency every..." it should be "Immersion every..."

R9: Thank you for your observation. The term in the first column of Table 1 have been revised and rewritten as suggested.

C10: Legends for Fig. 2. and Fig 3. correct according to instructions for Table 1.

R10: Thank you for your observation. The legends of Fig. 2. and Fig 3 have been revised and rewritten.

C11: As the paper is dealing with mass production of shoots in bioreactors it would perhaps be interesting for readers to show some photographs of your TIB instalation.

R:11: Thank you for your recommendation. Pictures for the in vitro cultures have been provided as suggested. See TIB installation originally design by Escalona et al. [19].

C12: In legend of Fig. 3. it is not stated how cultures were produced, please be accurate.

R12: Thank you for your observation. However, the produced cultures have been stated in Table 1 as number of shoots per explant. The in vitro shoots and acclimation of raspberry plantlets to ex vitro conditions are described in detail in materials and methods section.

Figure 4. In vitro shoots and acclimation of raspberry (Rubus idaeus L.) plantlets to ex vitro conditions. a) Shoots after 4 weeks of in vitro culture in different culture systems, b) acclimatized plantlets after 4 weeks in greenhouse, c) plantlets after 8 weeks of cultivation, and d) planting of R. idaeus after 12 weeks of culture.

Please address all correspondence concerning this manuscript to me at: jericobello@gmail.com

Thanking your kind attention to our submission, we hope to hear from you soon.

Yours sincerely,

Dr. Jericó Jabín Bello-Bello

Author Response File: Author Response.pdf

Reviewer 4 Report

Comments and Suggestions for Authors

The manuscript has sufficient scientific merit for publication. It is recommended to implement minor textual revisions, remove paragraphs that do not align with the Materials and Methods section, and clarify the recommendation of the most effective treatment in the conclusion.

Comments for author File: Comments.pdf

Author Response

May 15, 2025

Ms. Hailey Xia

Section Managing Editor

Dear Reviewer

Thanks for your comments on our manuscript ID: horticulturae-3629438 “Temporary immersion bioreactor for in vitro multiplication of raspberry (Rubus idaeus L.)”. The reviewers’ suggestions and recommendations have been addressed point-by-point. We have made major and major changes. The new changes were colored in blue in the manuscript. Besides, all suggestions have improved our manuscript substantially. Reviewers' suggestions and recommendations are appended below (C=Comment and R=Response):

C1: The terms in vitro and ex vitro should be italicized.

R: Thank you for your observation. All terms in vitro and ex vitro were italicized

C2: Replace “Multiplication” with another keyword that is not included in the article title. Example: Micropropagation; Physiological effects

R: Thank you for your observation. The keyword has been replaced by Micropropagation.

C3: The aim…..It is suggested to complement with '...bioreactor, under intermittent immersion periods and culture medium volumes'

R: Thank you for your observation. The paragraph has been revised and rewritten.

C4: The specific cultivar employed in the experiments must be clearly stated.

R: Thank you for your observation. The specific cultivar employed was included.

C5: This text should be comments from the article's authors or another reviewer. Consider removing it.

R: I appreciate your comment, you are right! We deleted this sentence that doesn't make sense.

C6: The water supply volume, use of micro-sprinkler technology, and flow rate parameters should be clarified for reproducibility.

R: Thank you for your observation. The paragraph has been revised and rewritten.

C7: In Conclusions… What protocol is recommended based on the data presented? What volume of culture medium? What intermittent immersion period?

R: Thank you for your observation. The paragraph has been revised and rewritten.

Please address all correspondence concerning this manuscript to me at: jericobello@gmail.com

Thanking your kind attention to our submission, we hope to hear from you soon.

Yours sincerely,

Dr. Jericó Jabín Bello-Bello

Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

Dear authors,

The manuscript is deeply improved. However, I have left many comments, some similar to my first revisions. Some paragraphs must be deleted entirely since the results do not support them. Many clarifications and optimizations are needed. Also, you have to avoid repetitions throughout the manuscript.

Kind regards,

Comments for author File: Comments.pdf

Comments on the Quality of English Language

Only some minor corrections are needed.

Author Response

May 22, 2025

Dear Reviewer

Thanks for your comments on our manuscript ID: horticulturae-3629438, titled: “Temporary immersion bioreactor for in vitro multiplication of raspberry (Rubus idaeus L.)”. The reviewers’ suggestions and recommendations have been addressed point-by-point. We have made major changes. The new changes were colored in blue during the manuscript. Besides, all suggestions have improved our manuscript substantially. Reviewers' suggestions and recommendations are appended below (C= Comment and R=Response):

C1: biotechnological and C2: independent

R: Thank you for your observation. The words have been revised and rewritten as suggested.

C3: See a comment in the result section

R: Thank you for your comment. The recommendation of the use of in vitro stabilized cultures was included. “In vitro stabilized cultures were used for the evaluation of the different method of cultivation” in the aforementioned multiplication medium.

C4: Avoid repetitions

R: Thank you for your observation. The sentence has been revised, repetitions were avoided, and rewritten for a better understanding. “This culture medium was prepared with stock solutions and was adjusted” to a pH of 5.7 ± 0.1.

C5: In the first submission, you had mentioned 3 subcultures…

R: Thank you for your observation. In section 2.2 the number of subcultures were deleted because the term in vitro stabilized cultures were included.

C6: I still insist that putting only 5 explants / bioreactor is still a very low number even for small scale plant production. It increases the costs too much. 4 bioreactors means that you have worked with 20 plantlets in total.

R: Thank you for your observation. In our study, the decision to work with five explants per bioreactor was aligned with our experimental objective: “The objective of this study was to establish a protocol for the micropropagation of raspberry (Rubus idaeus L.) using the temporary immersion bioreactor, under intermittent immersion periods and culture medium volumes.” The density may seem low for commercial-scale production, it's important to clarify that our work is situated within an experimental optimization protocol. For production using 4 L TIBs, with 40 explants per bioreactor, and 2 L of medium. This significantly improves medium utilization and reduces unit costs.

4 TIBs × 40 explants each = 160 explants total

C7: English revisions needed

R: Thank you for your observation. The English language has been reviewed and improved.

C8-C9: This is a repetition of the sentence highlighted in blue above

R: Thank you for your observation. The repetitions have been revised and avoided as suggested.

C10: In my opinion, this is not cost effective….

R: Thank you for your observation, you are right. However, in the scientific research is conducted in small units with a low number of shoots. These bioreactors can be scaled up to 4 liters. In 4-liter bioreactors, this corresponds to 40 explants per container.

C11: Which was the purpose on determining the chlorophyll content?

R: Thank you for your observation. The purpose of determining chlorophyll content is to photosynthetic pigments also promote in vitro shoots development (growth and/or differentiation). This clarification was included in the manuscript.

C12: NO picture were provided in the results section for the microscopic observation. I recommend to put pictures, or delete the section

R: I appreciate your comment. In section 2.6. evaluation of stomatal index and closed stomata was deleted as suggested.

C13: I understand that mass production is done first in small containers. Still these numbers do not correspond with the laboratory scale production. On the other hand, 5 explants/bioreactor is a very small number to perform such statistical tests.

R: Thank you for your observation. Working with a small number of explants per bioreactor is a common practice in research, particularly when optimizing protocols or evaluating physiological responses under controlled conditions. The use of five explants per TIB was selected to ensure uniform exposure to immersion cycles and reduce competition effects. For that reason, the experimental design included four repetitions, each with four replicated TIBs per treatment, resulting in a total of 80 explants per treatment (four repetitions × replicated four times × five explants=80 explants in total).

C14: As an answer to your reply in the cover letter: I know that is necessary to perform subcultures to have a high stock of explants to carry out the experiments. But, if you don’t provide any data in the results for this part, then why is necessary to add the subsection 2.1. in the Material and methods section? I would suggest to delete it and just say that in vitro stabilized cultures were used.

C15-C16: I strongly suggest you to delete this section from your manuscript since it lacks details, results and pictures.

R: I appreciate your comment. The 2.6. Evaluation of stomatal index and closed stomata section was deleted as suggested.

C17: These sentences are repetitions. Try to maintain one sentence only and put all the references you have provided

R: I appreciate your comment. These repeated sentences were revised and rewritten as suggested.

C18: This sentence perfectly aligns with my comments for the number of explants in 250 ml of medium.

R: I appreciate your comment. It is true, as noted by Bello-Bello et al. [37], that using a low number of explants per bioreactor can lead to inefficient use of the culture medium and increase production costs. In our case, we used five explants with 250 mL medium per bioreactor to reduce hyperhydricity and improve shoot vigorous under our specific conditions. We acknowledge that for commercial applications, a higher explant density should be evaluated to maximize the use of resources and reduce costs, without compromising plantlet quality.

C19: Delete ‘passive’ please. The water absorption is only passive, not active, and this process is called ‘osmosis’.

R: Thank you for your observation. The term ‘passive’ has been deleted and rewritten as suggested. Increase the absorption of water by osmosis.

C20-21: I suggest to shorten this subsection…. It’s too long and pedantic, I strongly recommend to delete this paragraph. Moreover, the chlorophyll itself does not convert the light energy to chemical energy. These pigments only absorb wavelength, and after a series of events during the light reactions in photosynthesis including photophosphorylation happen due to the potent energy from the H+ gradient created.

R: Thank you for your observation. The paragraph has been revised and deleted this subsection for a better understanding as suggested.

C22: I would suggest to say “to lower the level of mixotrophic growth….’ Or something similar

R: Thank you for your suggestion. The paragraph has been revised and rewritten as suggested.

C23: I strongly suggest to delete all this and everything related to the stomatal evaluations. These parts are not supported by results and pictures.

R: Thank you for your observation. The section has been revised and delete as suggested.

Yours sincerely,

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

Dear Authors,

I still have a problem with the methodology regarding the effect of the volume of the medium. Figure 1 shows identical containers. The methodology states that 500 ml containers were used in experiments I and II, and 1000 ml containers were used in experiment III. Also, 7 times 75 ml is 525 ml, not 500 ml as the authors state. This part of the paper still requires clarification and improvement.

Best regards,

Reviewer

Author Response

May 22, 2025

Dear Reviewer

C1: I still have a problem with the methodology regarding the effect of the volume of the medium. Figure 1 shows identical containers. The methodology states that 500 ml containers were used in experiments I and II and 1000 ml containers were used in experiment III. Also, 7 times 75 ml is 525 ml, not 500 ml as the authors state. This part of the paper still requires clarification and improvement.

R: Thank you for your observation, you are right! 1000 ml containers were used in experiment III, this is because in small containers it is not possible to evaluate the effect of the volume of the medium per explant in TIBs. However, in these containers, equal conditions were respected. On the other hand, 7 times 75 ml is 525 ml, not 500 ml as the authors state. For this clarification, the correction was made in the manuscript, the experiments under this design correspond to 25, 50, 71, and 100 mL per explant, respectively. The volume of 500 ml of culture medium was always constant for this experiment. The correct volume is 71 ml and not 75 ml. This observation was already included in the document, including tables and figures.

Yours sincerely,

Author Response File: Author Response.pdf

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Temporary Immersion Bioreactor for In Vitro Multiplication of Raspberry (Rubus idaeus L.)

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