Plant Cell Cultures: Biofactories for the Production of Bioactive Compounds
, Naravula Jalaja 3, Shri Mohan Jain 4,* and Suprasanna Penna 5,*
Round 1
Reviewer 1 Report
Comments / recommendations
28, 53, 56, 229 rows - in vitro, in situ – as Latin words should be in Italic - in vitro, in situ
29 row - keyword - remove the word written twice
68-69 rows and thereafter in the whole text … 135, – all the names of the species listed have to be in Italic.
66-67 and thereafter … – chemicals’ names have to be written with capital letters
191 row (page 5) and – when liter (l) is the unit it is preferred to be in capital letter – L according to a suggestion in the SI system of units. This is only a recommendation.
[56] – kilogram can be shortened – kg
[63] - Oryza sativacv.Dongjin - Oryza sativa cv. Dongjin
The numbers in the same table – table 1. Have to be written by the same way – if you use 2 signs after the coma use them for each number.
196 – N tabacum – N. tabacum – use Italic, please.
198 – E. coli – E. coli
227 r., 230 - [98,99] – use space, please [98, 99] everywhere like this example
290, 291, 294, 296, 300, 356 r. – use Italic please for the spp. names.
291 r. – a space {…, …]
295 r. – Is it necessary to use the name of author together with the number I he List of citations?
[121] (Lim et al. 2011) . You can remove the year.
303 r. – add a space, please before 252.4 mg/L
313 - used by many – who?
320, 324, 349 – spaces and Italic - Ophiorrhiza rugosa [128] .Besides; [129,130] . and so on…
Many other similar corrections are needed in the manuscript
References 32 and 33 are not completed.
Reference 35 – wrong name of author
I did not see information about carotenoids, except an example on tomato lycopene production given in the manuscript. I suppose, you can find. At least you can find mutations that stopped the biosynthetic pathway and transformed the mutants into a factory for producing carotenes. There are publications on achievements in potato beta-carotene production. You can find references in mdpi journal Foods.
If you can’t find at least describe the deficit in carotenoids in vitro production because they are important bioactive compounds in plants.
Author Response
Reviewer 1:
28, 53, 56, 229 rows - in vitro, in situ – as Latin words should be in Italic - in vitro, in situ
Ans: Thanks. The suggested corrections have been done.
29 row - keyword - remove the word written twice
Ans: Thanks. The suggested correction has been done.
68-69 rows and thereafter in the whole text … 135, – all the names of the species listed have to be in Italic.
Ans: Thanks. The suggested correction has been done.
66-67 and thereafter … – chemicals’ names have to be written with capital letters
Ans: Thanks. The suggested correction has been done.
191 row (page 5) and – when liter (l) is the unit it is preferred to be in capital letter – L according to a suggestion in the SI system of units. This is only a recommendation.
Ans: Thanks. The suggested correction has been done.
[56] – kilogram can be shortened – kg
Ans: Thanks. The suggested correction has been done.
[63] - Oryza sativacv.Dongjin - Oryza sativa cv. Dongjin
The numbers in the same table – table 1. Have to be written by the same way – if you use 2 signs after the coma use them for each number.
Ans: Thanks. The suggested correction has been done.
196 – N tabacum – N. tabacum – use Italic, please.
Ans: Thanks. The suggested correction has been done.
198 – E. coli – E. coli
Ans: Thanks. The suggested correction has been done.
227 r., 230 - [98,99] – use space, please [98, 99] everywhere like this example
Ans:. The suggested correction has been done.
290, 291, 294, 296, 300, 356 r. – use Italic please for the spp. names.
Ans: Thanks. Italics correction has been done.
291 r. – a space {…, …]
295 r. – Is it necessary to use the name of author together with the number I he List of citations?
Ans: No. it is not required; we have corrected.
[121] (Lim et al. 2011) . You can remove the year.
Ans: Thanks. The suggested correction has been done.
303 r. – add a space, please before 252.4 mg/L
Ans: Thanks. The suggested correction has been done.
313 - used by many – who?
Ans: The correction has been done.
320, 324, 349 – spaces and Italic - Ophiorrhiza rugosa [128] .Besides; [129,130] . and so on…
Ans: Thanks. The suggested correction has been done.
Many other similar corrections are needed in the manuscript
References 32 and 33 are not completed.
Ans: Thanks. References are added.
Reference 35 – wrong name of author
Ans: Thanks. The correction has been done.
I did not see information about carotenoids, except an example on tomato lycopene production given in the manuscript. I suppose, you can find. At least you can find mutations that stopped the biosynthetic pathway and transformed the mutants into a factory for producing carotenes. There are publications on achievements in potato beta-carotene production. You can find references in mdpi journal Foods.
If you can’t find at least describe the deficit in carotenoids in vitro production because they are important bioactive compounds in plants.
Ans: Thanks for your suggestion. We would like to point out that the review is aimed at plant cell culture-based production. As the expert reviewer suggested, there are several reviews/reports on production of carotenoids using plants or microbes. Report on use of plant cell culture based production has been added on page 2.
Author Response File: 
Author Response.docx
Reviewer 2 Report
The manuscript by Bapat et al. deals with the potential applications of plant cell cultures as promising platforms for the synthesis and accumulation of a wide range of bioactive compounds. In this short review the authors summarize the data currently available about the advantages of using plant cell cultures for basic and applied research.
The information presented can be of interest for many readers and offers an updated overview of the relevant literature in the field. Nevertheless, a careful and extensive English revision is absolutely required, in order to make the meaning of sentences clearer.
Please find below a list of comments, that should be taken into considerations by the authors in order to improve the quality of the manuscript.
Major criticisms:
- The title should be slightly amended, from “Plant cell cultures – biofactories for the production of bioactive compounds” into “Plant cell cultures: biofactories for the production of bioactive compounds”
- Lines 82-85, 96-98, 101-103, 128-131, 337-342, 491-493: please rephrase the sentences, because the meaning is not clear.
- - In the Introduction, the importance of plant cell cultures in basic research, for example to elucidate biochemical and physiological pathways in simplified experimental systems, should be better underlined. A reference for plant cell cultures derived from the model plant Arabidopsis thaliana should be added (https://pubmed.ncbi.nlm.nih.gov/33175378/).
- Legend to Fig. 1 must be more explanatory: the authors must include a description of all the terms contained in the picture (and not mentioned in the text), such as “germplasm preservation” and “in vitro mutagenesis”.
- Lines 188-190: concerning edible vaccines, the notion of their increased acceptability, as well as the lack of need of the cold chain, should be mentioned among their potential advantages in comparison with traditional vaccines.
- Lines 218-222: why should CRISPR/Cas9 help the production of recombinant proteins with different glycosylation pattern? It has to be noted that, for example, plant cells do not possess sialylated proteins, but CRISPR/Cas9 cannot help resolving this issue. Please explain.
- Fig. 4: the resolution of the picture is too low, please improve the quality.
- For clarity sake, some sentences (lines 440-461) in the Bioprinting paragraph should be rephrased.
Minor comments:
- - Line 54: please rephrase, because “are a good as a source” is not clear.
- - Line 66: change “signaling molecules” into “phytohormones”.
- - Lines 68-70 and 290: the name of bacteria must be written in italics.
- - Line 88: change “can” into “must”.
- - Line 107: delete “the”.
- - Line 126 and line 186: the name of plants must be written in italics.
- - Line 150: please explain what you mean with “free cells”.
- - Line 232: change “Cell cultures are being used now as colorants” into “Cell cultures are being used for the production of colorants”.
- - Lines 256 and 223: please call the molecules either “specialty” or “speciality” (same term, instead of different terms).
- - Line 470-473. The sentence “Stem cells are now being extracted from cultured plant cells” is not clear. Do you mean “isolated”?
- - Line 479: change “understating” into “understanding”.
- - Fig. 2: what does “Hassel free” mean?
- - Paragraph 5 (line 358): Please explain better how plant stem cell cultures can be obtained in vitro. Do they derive from primary meristems of the plants?
- - Table 2: correct “Vitis vinefera” into “Vitis vinifera”
- - Table 2: what does “meristem cell culture vacuole extract” mean?
- - Paragraph 6: To my knowledge, it is not necessary to use plant stem cells to produce food ingredients. Please clarify this point in the text.
Author Response
Reviewer 2
The manuscript by Bapat et al. deals with the potential applications of plant cell cultures as promising platforms for the synthesis and accumulation of a wide range of bioactive compounds. In this short review the authors summarize the data currently available about the advantages of using plant cell cultures for basic and applied research.
The information presented can be of interest for many readers and offers an updated overview of the relevant literature in the field. Nevertheless, a careful and extensive English revision is absolutely required, in order to make the meaning of sentences clearer.
Please find below a list of comments, that should be taken into considerations by the authors in order to improve the quality of the manuscript.
Major criticisms:
- The title should be slightly amended, from “Plant cell cultures – biofactories for the production of bioactive compounds” into “Plant cell cultures: biofactories for the production of bioactive compounds”
Ans: Title has been changed as per the suggestion.
- Lines 82-85, 96-98, 101-103, 128-131, 337-342, 491-493: please rephrase the sentences, because the meaning is not clear.
Ans: The suggested lines have been rephrased.
- - In the Introduction, the importance of plant cell cultures in basic research, for example to elucidate biochemical and physiological pathways in simplified experimental systems, should be better underlined. A reference for plant cell cultures derived from the model plant Arabidopsis thaliana should be added (https://pubmed.ncbi.nlm.nih.gov/33175378/).
Ans: Biochemical and physiological pathways associated with secondary plant products have been elucidated by integrating the data generated through multiomic techniques in many plant systems including the model plant A. thaliana (Zhu et al. 2023). (https://pubmed.ncbi.nlm.nih.gov/33175378/). This is now added in the Introduction.
Zhu F, Wen W, Cheng Y. et al. (2023). Integrating multiomics data accelerates elucidation of plant primary and secondary metabolic pathways. aBIOTECH. https://doi.org/10.1007/s42994-022-00091-4.
- Legend to Fig. 1 must be more explanatory: the authors must include a description of all the terms contained in the picture (and not mentioned in the text), such as “germplasm preservation” and “in vitro mutagenesis”.
Ans: Legend to Fig. 1 has been explained in the revised version.
- Lines 188-190: concerning edible vaccines, the notion of their increased acceptability, as well as the lack of need of the cold chain, should be mentioned among their potential advantages in comparison with traditional vaccines.
Ans: The suggested changes have been made in the revised version of the MS. (Acceptability of edible vaccines has increased now in many countries. Traditionally, vaccines are stored at low temperatures. But, such a need for the cold chain for storing the vaccine can be avoided especially in the rural areas if edible vaccines are promoted.)
- Lines 218-222: why should CRISPR/Cas9 help the production of recombinant proteins with different glycosylation pattern? It has to be noted that, for example, plant cells do not possess sialylated proteins, but CRISPR/Cas9 cannot help resolving this issue. Please explain.
Ans: The sentence has been rephrased keeping in view of the reviewer’s suggestion. The sentence now reads as “Genome editing technologies such as CRISPR/Cas9 are being utilized to create plant cells with desired characters that can aid in the production of recombinant therapeutic proteins”.
- Fig. 4: the resolution of the picture is too low, please improve the quality.
Ans: The resolution of the Fig. 4 has been improved.
- For clarity sake, some sentences (lines 440-461) in the Bioprinting paragraph should be rephrased.
Ans: Thanks for the suggestions. The entire section has been rewritten with the relevant portions.
Minor comments:
- - Line 54: please rephrase, because “are a good as a source” is not clear.
Ans: The sentence is rephrased as per the suggestion.
- - Line 66: change “signaling molecules” into “phytohormones”.
Ans: Signaling molecules is replaced with the word “phytohormones”.
- - Lines 68-70 and 290: the name of bacteria must be written in italics.
Ans: The names of bacteria have now been written in italics.
- - Line 88: change “can” into “must”.
Ans: Yes, the word is replaced with “must”.
- - Line 107: delete “the”.
Ans: The word “the” is deleted in the revised version.
- - Line 126 and line 186: the name of plants must be written in italics.
Ans: All botanical names of plants have now been put in italics.
- - Line 150: please explain what you mean with “free cells”.
Ans: The word “free cells” is now replaced with “suspensions”.
- - Line 232: change “Cell cultures are being used now as colorants” into “Cell cultures are being used for the production of colorants”.
Ans: The sentence has been rewritten as per the suggestion.
- - Lines 256 and 223: please call the molecules either “specialty” or “speciality” (same term, instead of different terms).
Ans: Thanks for the suggestion. The word is changed to “specialty”.
- - Line 470-473. The sentence “Stem cells are now being extracted from cultured plant cells” is not clear. Do you mean “isolated”?
Ans: Yes, it is “isolated”. (Stem cells are now being isolated from cultured plant cells).
- - Line 479: change “understating” into “understanding”.
Ans: “Understating” is replaced with “understanding”.
- - Fig. 2: what does “Hassel free” mean?
Ans: The words “Hassel free” are removed from the Figure 2.
- - Paragraph 5 (line 358): Please explain better how plant stem cell cultures can be obtained in vitro. Do they derive from primary meristems of the plants?
Ans: Revised version includes the following statement for stem cell production. Plant stem cells are undifferentiated cells present in the meristematic tissues of plants. Both apical and lateral meristems are the vital sources of stem cells. The stem cells are self-renewed, do not undergo ageing, and supply precursor cells which differentiate into diverse tissues or organs based on location. In vitro, callus is first initiated, suspension cultures are derived from callus, scaled up, and then cells are analysed through different analytical techniques. For details, the review article by Aggarwal et al. (2020) can be referred.
- - Table 2: correct “Vitis vinefera” into “Vitis vinifera”
Ans: It is corrected in the revised version.
- - Table 2: what does “meristem cell culture vacuole extract” mean?
Ans: Sorry, it is meristem cell culture extract (not vacuole extract). It is corrected in Table 2.
- - Paragraph 6: To my knowledge, it is not necessary to use plant stem cells to produce food ingredients. Please clarify this point in the text.
Ans: Yes, it is not necessary to use plant stem cells to produce food ingredients. But stem cells isolated from Citrus sinensis, Syringa vulgaris, Vitis vinifera, and others have good antioxidant and anti-inflammatory properties and such cell extracts are being used for cosmetic applications like skin cleansing, de-tanning, moisturizing, and whitening. Further, there is a renewed interest in using stem cells and plant suspensions also to produce food ingredients. For example, Theobroma cacao and Lippia citriodora cell extracts are being marketed as food supplements (Georgiev 2015, Freemont 2017).
Georgiev V (2015). Mass propagation of plant cells-an emerging technology platform for sustainable production of biopharmaceuticals. Biochem. Pharmacol. Open Access. 4: 1-3. doi:10.4172/2167-0501.1000e180.
Fremont F (2017) Cell culture: an innovative approach for production of plant actives. https://www.newfoodmagazine.com/article/33682/cell-culture-plant-actives/. Accessed 5 Jan 2018.
Reviewer 3 Report
This review covers a large number of topics. A review could be written for each chapter. As a result, one of the drawbacks is that this work frequently skims the surface of issues rather than delving deeply into them. The main advantage, however, is that it provides a clear overview of the current plant cell culture possibilities. After reading it, I believe that the benefit clearly outweighs the disadvantage, and this work will undoubtedly be very useful for students, teachers, but also industrialists or the curious who want a clear and concise vision of these PCC possibilities.
1. However, a methodological paragraph indicating the key words used in the selection of the articles presented here, as well as the criteria for selecting these articles, would be required. The period, databases, and other details must also be specified.
2. It would be important to include some regulatory considerations about the different types of PCC products reported in this review.
3. Plant Latin names should be in italic
4. The quality of the figures is low, please improve it.
Author Response
Reviewer 3
This review covers a large number of topics. A review could be written for each chapter. As a result, one of the drawbacks is that this work frequently skims the surface of issues rather than delving deeply into them. The main advantage, however, is that it provides a clear overview of the current plant cell culture possibilities. After reading it, I believe that the benefit clearly outweighs the disadvantage, and this work will undoubtedly be very useful for students, teachers, but also industrialists or the curious who want a clear and concise vision of these PCC possibilities.
- However, a methodological paragraph indicating the key words used in the selection of the articles presented here, as well as the criteria for selecting these articles, would be required. The period, databases, and other details must also be specified.
Ans: Initiation of suspension cultures is usually carried out from callus (can be obtained from any explant). Such suspensions may be used in cosmetic and food industries as mentioned elsewhere in the text. This preliminary information has been briefly mentioned in the section “stem cells”.
Keywords have been given at the bottom of the abstract. The articles presented in the manuscript or the databases referred in the manuscript have been given in the form of references.
- It would be important to include some regulatory considerations about the different types of PCC products reported in this review.
Ans: Plant cell culture-derived products and also stem cell extracts being used in food and cosmetic industries need to undergo rigorous regulatory issues stipulated by the regulatory authorities. We have well-laid down procedures and the companies test these products on human volunteers before marketing them. So, they are not mentioned in this article.
- Plant Latin names should be in italic
Ans: All botanical/Latin names are given in italics.
- The quality of the figures is low, please improve it.
Ans: The quality of the Figures 2 and 4 has been improved with high resolutions in the revised version of the manuscript.
Round 2
Reviewer 2 Report
The authors revised the manuscript taking into careful consideration the Reviewers’ criticisms. However, in the revised ms there are still some minor mistakes that need to be corrected, as listed below:
1) Lines 55, 79, 171: “In vitro” must be changed into “in vitro” (no capital letter).
- 2) In the Reference section (lines 589-590), Ref. 12 (Trehan et al 2017) must be cancelled and substituted by: Cortese E, Carraretto L, Baldan B, Navazio L (2021). Arabidopsis photosynthetic and heterotrophic cell suspension cultures. Methods Mol Biol 2200: 167-185. In: Arabidopsis Protocols. 4th Edition. Chapter 8. Sanchez-Serrano JJ, Salinas J eds. Springer Science+Business Media, New York, USA. https://pubmed.ncbi.nlm.nih.gov/33175378/
- 3) Line 61: here, the above reference must be cited only as [12], not providing the link in the text.
- 4) Line 72: change “the phytohormones” into “phytohormones” (delete “the”).
- 5) Legend to Fig. 1: change “Rare and endangered plant suspensions” into “Rare and endangered plants”.
- 6) Table 1: change “Nicotiana benthmiana” into “Nicotiana benthamiana”
- 7) Line 306: change “importants” into “importance”
- 8) Line 477: change “biocmpatible” into “biocompatible”
- 9) Line 505: delete the comma at the end of the line
- 10) Line 509: change “biorinted” into “bioprinted”
- 11) Fig. 4: the picture still looks of poor quality. Please improve the resolution (this issue was already raised before, and not solved yet).
Author Response
The authors revised the manuscript taking into careful consideration the Reviewers’ criticisms. However, in the revised ms there are still some minor mistakes that need to be corrected, as listed below:
1) Lines 55, 79, 171: “In vitro” must be changed into “in vitro” (no capital letter).
- 2) In the Reference section (lines 589-590), Ref. 12 (Trehan et al 2017) must be cancelled and substituted by: Cortese E, Carraretto L, Baldan B, Navazio L (2021). Arabidopsis photosynthetic and heterotrophic cell suspension cultures. Methods Mol Biol 2200: 167-185. In: Arabidopsis Protocols. 4th Edition. Chapter 8. Sanchez-Serrano JJ, Salinas J eds. Springer Science+Business Media, New York, USA. https://pubmed.ncbi.nlm.nih.gov/33175378/
- 3) Line 61: here, the above reference must be cited only as [12], not providing the link in the text.
- 4) Line 72: change “the phytohormones” into “phytohormones” (delete “the”).
- 5) Legend to Fig. 1: change “Rare and endangered plant suspensions” into “Rare and endangered plants”.
- 6) Table 1: change “Nicotiana benthmiana” into “Nicotiana benthamiana”
- 7) Line 306: change “importants” into “importance”
- 8) Line 477: change “biocmpatible” into “biocompatible”
- 9) Line 505: delete the comma at the end of the line
- 10) Line 509: change “biorinted” into “bioprinted”
- 11) Fig. 4: the picture still looks of poor quality. Please improve the resolution (this issue was already raised before, and not solved yet).
Answers: Authors thank reviewer for his encouragement and suggestions to improve the ms. Apologies for the typing errors. We have carefully checked and corrected the text. Figures have been drawn and added. Hope this version will meet your approval.
Author Response File: Author Response.docx
Reviewer 3 Report
The Authors ignored my requests, and the figure quality remains poor.
Before the paper can be considered for publication, these requests must be considered and answered.
Author Response
Reviewer 3:
- However, a methodological paragraph indicating the key words used in the selection of the articles presented here, as well as the criteria for selecting these articles, would be required. The period, databases, and other details must also be specified.
Ans: A paragraph indicating the key words used for the selection of articles presented here as well as the criteria for selecting these articles is given in the revised version.
Following key words were used for searching the articles:
bioactive compounds, cosmetics, foods and food ingredients, hairy roots, industrial production, phytochemicals, plant cell cultures, suspension cultures
Following webpages were also searched to obtain the information included in this article.
(a). https://www.abres.it/echinacoside.php
(b). https://www.abres.it/teupolioside.php
(c). https://www.abres.it/verbascoside-acteoside.php
(d). https://mibellebiochemistry.com/phytocelltectm-nunatakr
(e). https://mibellebiochemistry.com/phytocelltectm-symphytum
- It would be important to include some regulatory considerations about the different types of PCC products reported in this review.
Regulatory issues related to food ingredients or foods prepared through plant suspension cultures
The regulatory issues are meant for ensuring the safety of the food and food ingredients to the consumers, though they may differ from country to country. Foods derived from plant suspension cultures are considered and come under the novel food category as per the definition of the European Union Regulation 2015/2283 (Gubser et al. 2021). Regulatory landscape with a complex framework of plant suspension or hairy root culture-derived novel food and health-related products is undeniably a limiting factor for early commercialization of the products. Any delays in the approval will escalate the production costs. But food colorants and flavourings do not fall within the scope of European Union Regulation 2015/2283 (Gubser et al. 2021). Within India, Food Safety and Standards Act (FSS) 2006, is the law and takes care of the regulation of all food ingredients. Generally, Food Safety Standards Act of India (FSSAI) looks after and enforces the safety standards within India. On the other hand, in USA, addition of any substance to the food is considered as a food additive and needs approval from the Food and Drug Administration (FDA). But if the substance is proved as safe through scientific data generated under the supervision of qualified scientists or traditional history of safe use for a long period of time or if it meets the procedures laid down by Federal Food, Drug and Cosmetic Act, then approvals may not be necessary before such products are marketed. In the case of traditional foods, long history of its safe usage is taken into consideration, but not in plant tissue culture-derived foods and food ingredients since these are labeled as “novel”. Novel foods derived from plant suspensions and hairy root cultures may vary in their constituent profile also in comparison with that of traditional foods. Therefore, such novel foods need safety assessment performed by the European Food safety Authority (EFSA) as per the guidelines laid down with all the evidence of the scientific data (Turck et al. 2016). Extracts derived from the suspensions of plants such as Ajuga reptans and Echinaceae angustifolia are considered as novel food supplements under EU Regulation 2015/2283. But food colorants such as anthocyanins and betalains have not been granted any authorizations by EFSA for use in the food though they are common food ingredients in grapes, beet root and others (Stintzing and Carle 2004, Gubser et al. 2021). Lastly, it is vital to take sensory perception of the consumers into account since this aspect is also crucial.
References included:
- Gubser, G.; Vollenweider, S.; Eibl, D.; Eibl, R. Food ingredients and food made with plant cell and tissue cultures: State-of-the art and future trends. Eng Life Sci. 2021, 21, 87-98. doi:10.1002/elsc.202000077.
- Turck, D.; Bresson, J.; Burlingame, B.; Dean, T.; et al. Guidance on the preparation and presentation of an application for authorisation of a novel food in the context of regulation (EU) 2015/2283. EFSA J. 2016, 14, 1-24.
- Stintzing, FC.; Carle R. Functional properties of anthocyanins and betalains in plants, food, and in human nutrition. Trends Food Science Technol. 2004, 15, 19-38. https://doi.org/10.1016/j.tifs.2003.07.004.
Author Response File: Author Response.docx
