Enhancement of Phenolic and Polyacetylene Accumulation in Lobelia chinensis (Chinese lobelia) Plantlet Cultures Through Yeast Extract and Salicylic Acid Elicitation
and So-Young Park 1,*Round 1
Reviewer 1 Report
Comments and Suggestions for AuthorsIt is a very interesting work
I have some suggestions
Comments for author File: 
Comments.pdf
Author Response
Response to Reviewers Comments
Ref: Horticulturae-3613162
Authors are thankful to anonymous reviewers for their valuable comments on the manuscript and we have revised manuscript as per the instruction’s reviewers. All corrections are highlighted in blue colour. Following are the specific changes made in the revised manuscript.
Reviewer #1
Query #1. In vitro; must be in italics, in vitro
Answer: The words in vitro is presented in italics.
Query #2. The experiment was stated with axenic material? (if, so mention it)
Answer: Correction is incorporated.
Query #3. 16/8 photoperiod
Answer: The 16 h photoperiod is rewritten as 16/8 h photoperiod.
Query #4. The fresh weight (FW) of the biomass was estimated. The dry weight (DW)
Answer: Correction is incorporated.
Query #5. The plant material was only elicited for one week?
Answer: Yes, initially for four weeks cultures were maintained without elicitor and elicitor was introduced at the end three weeks.
Query #5. Figure 1. Lobelia chinensis biomass…
Answer: Correction is incorporated.
Query #6. L. chinensis (In the Table 1, You used L. chinenesis,)
Answer: Correction is incorporated.
Query #7. Is it necessary to mention in the discussion why only one week of elicitors is used?
Answer: It is well established with in vitro cultures that if elicitors are added in the initial stage the growth of the cultures will be hindered. To achieve growth of cultures and biomass, the cultures were maintained initial three weeks without elicitors and then cultures were treated with elicitors for one week.
Query #8. Discuss at what level the elicitors act in L. chinensis, a at what level within the biosynthetic pathway. The researcher’s opinion is important for readers.
Answer: Entire discussion has been revised in the light of reviewer’s comments.
Author Response File: Author Response.pdf
Reviewer 2 Report
Comments and Suggestions for AuthorsTitle
Enhancement of phenolic and polyacetylene accumulation in Lobelia chinensis plantlet cultures through yeast extract and salicylic acid elicitation
The common name of this species could be added.
Abstract
Line 14: Write in the third person.
Line 14: 3% sucrose (w/v)
Overall, the abstract is well-developed; however, a better justification is needed. For example, existing biotechnological methods for generating these compounds are not efficient, which is why increased production of these compounds through bioelicitors is being sought.
Keywords
Do not repeat the words used in the title.
Introduction
Lines 37-38: Support this idea with a citation.
The introduction is concrete and well-written, providing a solid justification for the purpose of the study.
Materials and Methods
Line 66 Characteristics of the plants used (in vitro, number of subcultures, etc.)
Line 69 3% sucrose (w/v)
Line 25 200.0 mL. If a decimal is to be used, it must be used throughout the text.
Line 76: Equipment characteristics and brand (check throughout the text).
Line 77: 70% RH in the environment? Inside the flask, it is at 100% because it is a liquid medium.
Lines 1013-104: Equipment characteristics and brand.
Line 136 and line 142: Check the numbering.
Line 163: Why was Duncan's used instead of Tukey's?
Materials and methods should provide as much detail as possible to be reproducible. For example, when taking a sample for physiological analysis, it is necessary to clarify what type of plant material was used (callus, leaf, stem, root).
Results
Table 1: Revise the dry weight and fresh weight units. In my opinion, they are only "g."
Figure 1: Add the unit mg L-1.
Lines 201-202: This is part of the materials and methods section.
Table 2: Revise the dry weight and fresh weight units. In my opinion, they are only "g."
Figure 4: Improve the quality and add the unit mg L-1.
The results are well organized and presented; they are commendable and provide valuable information.
Discussion
Lines 258-262: A citation is missing.
Lines 270-272: This is part of the materials and methods section.
Lines 285-288: Detail the studies mentioned (concentration of YE used, main observations, etc.) so they can be compared with the studies conducted.
Line 288: "In this study." Clearly delineate which part of the discussion is a result of this study and which is part of the available literature.
Lines 294-295: What advantages does this study have over the previous ones?
Lines 297-301: Why is this? When discussing results contained in the literature with those obtained in your study, an inference should be given as to why things happen (based on available scientific knowledge). For example, has SA been investigated to influence cellular oxidation pathways, is external (exogenous) addition involved in a metabolic pathway, etc.
Lines 303-305: At what concentrations of SA were these effects seen?
In general, the discussion should be improved. Only previous studies where there is equal experimental conditions should be discussed. The results obtained from similar studies need to be thoroughly discussed, not just a list of previous studies without providing details.
Conclusion
The conclusion is the list of the most important findings of the study and an interpretation by the authors. If two bioelicitors were compared, a conclusion should be drawn as to which of the two was better. Note that this is in vitro.
Author Response
Response to Reviewers Comments
Ref: Horticulturae-3613162
Authors are thankful to anonymous reviewers for their valuable comments on the manuscript and we have revised manuscript as per the instruction’s reviewers. All corrections are highlighted in blue colour. Following are the specific changes made in the revised manuscript.
Reviewer #2
Query #1. Enhancement of phenolic and polyacetylene accumulation in Lobelia chinensis plantlet cultures through yeast extract and salicylic acid elicitation.
The common name of this species could be added.
Answer: As per the suggestion the common name is added in the title of manuscript.
Query #2. Write in the third person
Answer: Correction is incorporated.
Query #3. 3% sucrose (w/v)
Answer: Correction is incorporated.
Query #4. Do not repeat the words used in the title.
Answer: The new keywords are incorporated.
Query #5. Lines 37-38: Support this idea with citation.
Answer: Citation is added as per suggestion.
Query #6. Line 66 Characteristics of the plant used (in vitro, number of cultures, etc.)
Answer: The axenic cultures of Lobelia chinensis which were maintained at the Department of Horticulture Science, Chungbuk National University was used as plant material. The cultures were maintained in vitro every six weeks of subculture for three months.
Query #7. Line 59: 3% sucrose (w/v)
Answer: Correction is incorporated.
Query #8. Line 75: 200.0 mL. If decimal is to be used, it must be used throughout the text.
Answer: The decimal is removed.
Query #9. Lien 76. Equipment characteristics and brand (check throughout the text).
Answer: Equipment details are provided.
Query #10. Line 77. 70% RH in the environment? Inside the flask, it at 100% because of liquid medium.
Answer: The RH humidity was that of culture room (70%).
Query #11. Lines 103-104: Equipment characteristics and brand.
Answer: Equipment details are provided.
Query #12. Line 136 and line 142: Check the numbering.
Answer: The numbering is okay. 2.9 Antioxidant activities under that 2.9.1 was analysis of antioxidant activity through DPPH assay and 2.9.2 was analysis of antioxidant activity using ABTS assay. Therefore, sub numbering is given.
Query #13. Line 163. Why was Ducan’s used instead of Tukey’s?
Answer: Tukey’s method is also equally good statistical method. Since we have Duncans software in our laboratory, we applied the same for analysis of data.
Query #14. Materials and methods should provide as much as possible to be reproducible. For example, when taking a sample for physiological analysis, it is necessary to clarify what type of plant material used (callus, leaf, stem, root).
Answer: We have used entire plantlet biomass for physiological analysis and this information is incorporated in the revised manuscript.
Query #15. Table 1: Revise the dry weight and fresh weight units. In my opinion, they are only “g”.
Answer: The fresh weight and dry weight values presented in the Table 1 were g L-1.
Query #16. Figure 1. Add the unit mg L-1.
Answer: It is not mg L-1 and it is g L-1 and it is provided in the legend of the figure.
Query #17. Table 2. Revise the dry weight and fresh weight units. In my opinion, they are only “g”.
Answer: The fresh weight and dry weight values presented in the Table 1 were g L-1.
Query #18. Figure the quality and add the unit mg L-1.
Answer: Improved figure is provided. It is not mg L-1 and it is g L-1 and it is provided in the legend of the figure.
Query #19. Lines 258-262. A citation is missing.
Answer: Citations are provided.
Query #20. 270-272. This is part of the materials and method section.
Answer: The said content is removed from discussion section.
Query #21. Lines 285-288: Detail the studies mentioned (concentration of YE used, main observation, etc.) so they can be compared with the studies conducted.
Answer: The discussion has been improved as suggested.
Query #22. Line 288. “In this study”, clearly delineate which part of the discussion is result of this study and which is part of the available literature.
Answer: The vague sentences/statements have been removed and discussion part is rewritten thoroughly.
Query #23. What advantages does this study have over the previous ones?
Answer: The discussion part is improved to show advancement over the previous published results.
Query #24. Lines 297-301: Why is this? When discussing results contained in the literature with those obtained in your study, an inference should be given as to why things happen (based on available scientific knowledge). For example, has SA has been investigated to influence cellular oxidation pathways, is external (exogenous) addition involved in a metabolic pathway, etc.
Answer: Discussion has been re-written as per suggestion.
Query #25. Lines 303-305: At what concentrations of SA were these effects?
Answer: At 25 µM SA has shown such effects.
Query #26. In general, the discussion should be improved. Only previous studies where there is equal experimental conditions should be discussed. The results obtained from similar studies need to be thoroughly discussed, not jus a list of previous studies without providing details.
Answer: Discussion has been re-written as per suggestion.
Query #27. The conclusion is the list of the most important findings of the study and an interpretation by the authors. If two bioelicitors were compared, a conclusion should be drawn as to which of the to was better. Note that this is in vitro.
Answer: Conclusion section is rephrased as per the suggestion.
Author Response File: Author Response.pdf
Reviewer 3 Report
Comments and Suggestions for AuthorsThis work shows the in vitro culture of Lobelia chinensis plantlets elicited with different concentrations of yeast extract and salicylic acid to induce the accumulation of phenolic compounds and polyacetylenes. This fact is relevant since it contributes to a biotechnological strategy to obtain major amounts of these secondary metabolites, reported as important for their potential healthy effects, including antioxidant activities.
However, there are methodological aspects that could be described or clarified to ensure reproducible results. Furthermore, a deeper discussion on the significance of results obtained is desirable.
To improve the manuscript, suggestions are attached into pdf file.
My best regards
Comments for author File: Comments.pdf
Author Response
Response to Reviewers Comments
Ref: Horticulturae-3613162
Authors are thankful to anonymous reviewers for their valuable comments on the manuscript and we have revised manuscript as per the instruction’s reviewers. All corrections are highlighted in blue colour. Following are the specific changes made in the revised manuscript.
Reviewer #3
Query #1. Line 65. Plant material and culture conditions: if there is a reference about the generation of in vitro plantlets of L. chinensis, please include it, or simply mention the strategy to get them (embryogenesis, organogenesis).
Answer: The nodal segments of Lobelia chinensis upon culturing in vitro in MS medium were involved in regeneration of axillary shoots (organogenesis) and simultaneous rooting at the base shoots (Reference 10. Bai, X.; Lee, H.S.; Murthy, H.N.; Kwon, H.J.; Yeon, S.H.; Ju, J.Y.; Park, S.Y. Micropropagation of Lobelia chinensis Lour.: In-fluence of medium parameters on plant regeneration, antioxidant activity and secondary metabolite accumulation. Korean J Plant Res 2024; 37, 225-234. https://doi.org/10.7732/kjpr.2024.37.3.225).
Query # 2. Were the explants immersed in liquid MS medium or how were they supported? What were the criteria for the concentration of elicitors?
Answer: The explants were immersed in liquid MS medium and the cultures were maintained on a rotary shaker (OS 4000 orbital shaker, Jeio Tech, Daejon, Republic of Korea) at 100 rpm. Thus, explants were aerated and subsequently immersed in the liquid medium. The elicitor concentration was fixed based on literature survey.
Query #3. Line 94. Does the gram of plantlet used during the extraction include leaves, stems or both? Please specify.
Answer: Plantlets with stem, leaves, and roots together were used for extraction.
Query #4. Lines 94. Please, indicate the type of extraction that was made; describe briefly. Line 99, Eliminate the “reagent” word. Only use “Foling-Ciocalteu method” Line 113 Line: Please, change the word “pressure” for “performance”.
Answer: The dried samples were pulverized using pestle and mortar and 1 g of powdered sample was used for extraction using extraction apparatus (LS-2050-S10; LS-TECH, Republic of Korea) with 30 mL 80% ethanol at 80 °C for 1 h. Other suggested corrections are incorporated.
Query #4. Line 115, I suggest: “Fresh plant samples were stirred”. Were the fresh plants powdered during the extraction?
Answer: Dried pulverized plant samples were used for extraction. The necessary corrections are incorporated.
Query #4. Lines 117 and 129, It is not necessary to mention that the analysis were performed in triplicate, since it is already mentioned in Statistical analysis.
Answer: Correction is incorporated.
Query #5. Lines 118-119, “. . . liquid chromatography system”
Answer: Correction is incorporated.
Query # 6, 7 and 8: Line 122, Please, indicate the type and trade of C18 column.
Line 123, Indicate the concentration of acetic acid in the eluent A).
Please, describe the elution gradient and the wavelength used for the identification of chromatographic peaks.
Answer: A C18 reversed-phased column (4.6 mm × 250 mm, 5 µm particle size, Waters XBridge) was used. Gradient elution was conducted with water (0.1% formic acid) (A) and acetonitrile (0.1% formic acid) (B): 0 min, 95% A; 25 min, 72%, and 40 min, 72% A, at a flow rate of 1.2 mL/min. The detection wavelengths was 300 nm.
Query #9: Line 127, Write “performance” instead of “pressure”. Line 130, Indicate type and trade of C18 column.
Answer: Correction is incorporated. A C18 reversed-phased column (4.6 mm × 250 mm, 5 µm particle size, Waters XBridge) was used.
Query #10: Line 149, Please, define control solution.
Answer: Ethanol was used blank (control solution).
Query #11: Table 1, Row “Fresh Weight” Arrange the letters of the significant differences in order from greatest to lower value.
Answer: Corrections are incorporated.
Query #12: Significant effect of YE on fresh and dry weight was evidenced, in fact, at the lowest concentration tested.
Answer: Corrections are incorporated.
Query #13: Line 203, DPPH scavenging activity of YE-elicited plants were higher than control plant only at 50 mg per L.
Answer: Yes, DPPH scavenging activity of YE-elicited plants were only at 50 mg L-1 treatment.
Query #14: Figure 3. ABTS radical scavenging and FRAP are reported as percentage and Fe+2/mg DW, respectively but not as GAE and AAE as described in the Material and Methods sections. Please, check it.
Answer: ABTS and DPPH gives relative measure and then absolute antioxidant content. In contrast FRAP which provides quantitative measure of antioxidant capacity based on standard culture. The results have been checked and found correct.
Query #15: Line 217, Treatment with 50 µM SA was similar to the control plant
Answer: Data has been presented as per results.
Query #16: Line 240, Lobetyolinin decreased at 25 µM SA, but it is remarkable that its concentration was greater at 100 and 200 µM.
Answer: Data has been presented as per results.
Query #17: Figure 6, As mentioned for figure 3, ABTS radical scavenging and FRAP are reported as percentage and Fe+2/mg DW, respectively but not as GAE and AAE as described in the Material and Methods sections. Please, check it.
Answer: The results have been checked and found correct.
Query #18: Is it possible to explain how SA and the components present in YE might act as elicitors or promoters of growing in the plantlets cultivated in vitro? Is there any hypothesis about the mechanisms that explain this effect? Could a particular carbohydrate, proteins or nucleotides content in YE have this effect? Is it possible that the results presented here are constant regarding the composition of YE??
Answer: YE functions as an elicitor, promoting cellular growth, division, and biomass accumulation in in vitro cultures through its abundance in proteins, carbohydrates, amino acids, nucleotides, and several trace elements [35]. Several studies have documented a high accumulation of secondary metabolites and the activation of the PAL enzyme by yeast extract, in addition to the in vitro cultured cell suspension or plant tissue culture.
Whereas, salicylic acid (SA) as a plant hormone affects the growth and development of plants, enhances secondary metabolites in in vitro plant cultures of different medicinal plants, and takes part in certain signal transduction pathways to stimulate specific enzymes. Furthermore, the modulation of the defence system and redox signalling are two major mechanisms by which SA works. Generally speaking, SA has been demonstrated to stimulate the production of antioxidants like SOD, APX, and glutathione in vitro cultures of a variety of plants.
The discussion has been revised and we tried to incorporate possible mechanism of YE effects in the light of available literature.
Query #19: Does the comparison of the DDPH and ABTS scavenging activity reveal information about the nature of secondary metabolites induced?
Answer: Yes, the DPPH and ABTS scavenging activity reveal about the nature of secondary metabolites produced in the cultures. It has been shown in several plants.
Query #20: According to the induced or repressed compounds, is it possible to identify the biochemicals pathways affected??
Answer: It has been demonstrated through molecular and gene expression investigations that in some plants, the elicitor (type and concentration) can cause the repression and enhancement of specific genes involved in biosynthetic pathways, which are responsible for the induced and repressed accumulation of chemicals.
Query #21: How can the repressive effect or lower tissue weight be explained at higher concentration of YE and SA?
Answer: The repressive effect or decreased tissue weight effect of elicitors, particularly YE and SA, in in vitro cultures is hard to explain. However, it has been shown that in a variety of plant systems, a particular elicitor concentration (YE or SA) is responsible for the promotive impact. In addition, either higher or lower concentrations are responsible for the reduction in biomass accumulation. For instance, YE has been used at concentrations of 50, 100, and 200 mg L-1. In some medicinal plants, such as Plumbago indica [39] and Arnica motana [38], YE boosted biomass accumulation and shoot multiplication at a dosage of 100 mg L-1. The negative effects on biomass production in Curcuma mangga, Glehnia littralis, and Knautia sarjevensis were reported by a few researchers.
Query #22: According to the results obtained, please conclude with a perspective on this work.
Answer: Conclusion has been revised as per the suggestion.
Author Response File: Author Response.pdf
Reviewer 4 Report
Comments and Suggestions for AuthorsManuscript „Enhancement of phenolic and polyacetylene accumulation in Lobelia chinensis plantlet cultures through yeast extract and salicylic acid elicitation” need to be improved. The comments are below.
Introduction:
- Please explain why the authors decided to investigate phenolic compounds and polyacetylenes? What are their health properties?
2. Please emphasize and clarify the purpose of the research
Materials and methods
- paragraph 2.1. Does the medium contain phytohormones? They should be listed along with their concentrations.
2. Paragraph 2.2. The procedure for preparing elicitors and their application should be described
3. Paragraph 2.4. How were the dry samples prepared for extraction?
4. Paragraph 2.5., 2.6. 2.9 State what spectrophotometer was used for the tests. State the equation of the calibration curve.
5. Paragraph 2.7 At what wavelength were the determinations made?
6. Paragraph 2.8. Briefly describe the method of polyacetylene extraction
Disscussion
- Compare the content of the tested compounds with their level in the extracts of other in vitro cultures of Lobelia chinensis and extracts from ground plants.
2. Rate whether the antioxidant properties were strong? Compare with extracts of other plants
3. Explain why YE at a concentration of 50 mg/L increases the content of selected compounds? How to link the obtained results with the biosynthesis pathway of the tested compounds?. How to explain that the content of some compounds is increased and others decreased under the influence of YE?
4. What mechanism is responsible for the effect of SA on increasing or inhibiting the production of selected metabolites? Explain this phenomenon on the physiological and genetic level
Conclusion
1. Line 315-317- too strong a statement, especially since it is not known what concentrations of the tested compounds are in ground plants. The authors did not address this issue in the manuscript
Author Response
Response to Reviewers Comments
Ref: Horticulturae-3613162
Authors are thankful to anonymous reviewers for their valuable comments on the manuscript and we have revised manuscript as per the instruction’s reviewers. All corrections are highlighted in blue colour. Following are the specific changes made in the revised manuscript.
Reviewer #4
Query #1. Please explain why the authors decided to investigate phenolic compounds and polyacetylenes? What are their health properties.
Answer: It has been demonstrated that flavonoids with anti-inflammatory and antioxidant properties include diosmin, diosmetin, and linarin (Elkhawas et al. 2024). Similarly, catechins, phloretic acid, and linarin are compounds with various potential health benefits, primarily related to their antioxidant, anti-inflammatory, and anti-carcinogenic properties. Furthermore, liver cancer has been treated with quercetin that was extracted from Lobelia chinensis (Luo et al. 2024). In addition, it has been found that polyacetylene lobetyolinin possesses antioxidant qualities (Elkhawas et al., 2024). L. chinensis is utilized as a raw material to make nutraceuticals and cosmeceuticals. Usually, in vitro cultures are used to produce biomass and multiply plants. Due to the inefficiency of the present biotechnological methods for generating these substances, bioelicitors are being employed to increase the synthesis of these molecules.
Query #2. Please emphasize and clarify purpose of the research.
Answer: Typically, Lobelia chinensis plants are multiplied and biomass is produced in vitro. Because the current biotechnological methods for producing these chemicals are inefficient, bioelicitors are being used to boost the synthesis of these molecules.
Query #3. Paragraph 2.1. Does the medium contain phytohormones? They should be listed along with their concentrations.
Answer: No, Murashige and Skoog medium basal medium supplemented with 3% (w/v) sucrose was used for multiplication of plants. The medium is devoid of phytohormones.
Query #4. Paragraph 2.2. Procedures for preparing elicitors and their application should be described.
Answer: The stock solution of YE (Duchefa, Haarlem, Netherlands) was prepared in distilled water, whereas, 10 mM of SA (Sigma, California, USA) was prepared in ethanol and solutions were filter sterilized through a 0.22 µm syringe Millipore filter (Sartorius, Gottingen, Germany) and then added to cultures.
Query #5. Paragraph 2.4. How dry samples prepared for extraction.
Answer: The plant material dry weight (DW) was estimated after desiccating the biomass at 50 °C for 72 h in an oven (Sanyo, MoV-112V, Aichi, Japan). The dried samples were pulverized using pestle and mortar and 1 g of powdered sample was used for extraction using extraction apparatus (LS-2050-S10; LS-TECH, Republic of Korea) with 30.0 mL 80% ethanol at 80 °C for 1 h and passed through filter paper (Advantec 110 mm; Toyo Roshi Kaisha Ltd., Japan). Final volume of the solution was adjusted at 30 mL using 80% ethanol.
Query #6. 2.5., 2.6., 2.9 state what spectrophotometer was sued for the tests. State the equation of calibration.
Answer: The spectrophotometer make was Libra S22, Biochrome Ltd., Cambridge, UK. The equation of the calibration curve for phenolic, flavonoid and FRAP analysis were y = 0.0023x + 0.0022 R2 = 0.9985; y = 0.0024x + 0.0562 R2 = 0.995; and y = 0.0028x + 0.5795 R2 = 0.7853, respectively. The equation for DPPH assay was (I%): I% = [(Ablank − Asample)/Ablank] × 100 where I% is the percent inhibition of the DPPH• radical, Ablank and Asample—the absorbance of the control reaction (containing all reagents except the extract) and the extract, respectively. Whereas, the equation for ABTS assay was (I%): I% = [(Ablank − Asample)/Ablank] × 100 where I% is the percent inhibition of the ABTS radical, Ablank and Asample—the absorbance of the control reaction (containing all reagents except the extract) and the extract, respectively.
Query #7. Paragraph 2.7. At what wavelength the determinations made?
Answer: The detection wavelengths was 300 nm.
Query #8. Paragraph 2.8. Briefly describe the method of polyacetylene extraction?
Answer: Samples were pulverized and the powder was screened through 180 μm sieves. Fine powder (1 g) was accurately weighed, 25 mL of methanol was added and the mixture was weighed again. The powder was then extracted by refluxing for 2 hr. After cooling, methanol was added to make up to the initial weight. The supernatant was filtered through a syringe filter (0.45 μm).
Query #9. Compare the content of the tested compounds with their level in the extracts of other in vitro cultures of Lobelia chinensis and extract of ground plants.
Answer: Discussion is improved as per the suggestion.
Query #10. Rate whether the antioxidant properties were strong? Compare the extracts of other plants.
Answer: Discussion is improved as per the suggestion.
Query #11. Explain why YE at a concentration of 50 mg/L increase the content of selected compounds? How to link the obtained results with biosynthetic pathway of the tested compounds? How to explain that the content of some compounds is increased and others decreased under the influence of YE?
Answer: Several studies have documented a high accumulation of secondary metabolites and the activation of the PAL enzyme by yeast extract, in addition to the in vitro cultured cell suspension or plant tissue culture. Further, differential accumulation of phenolics might be differential expression specific enzymes which are involved in final steps biosynthetic pathway.
Query #12. What mechanism is responsible for the effect of SA on increasing or inhibiting the production of selected metabolite? Explain the phenomenon on the physiological and genetic level.
Answer: The plant hormone salicylic acid (SA) affects the growth and development of plants, enhances secondary metabolites in in vitro plant cultures of different medicinal plants, and takes part in certain signal transduction pathways to stimulate specific enzymes. Furthermore, the modulation of the defence system and redox signalling are two major mechanisms by which SA works. Generally speaking, SA has been demonstrated to stimulate the production of antioxidants like SOD, APX, and glutathione in vitro cultures of a variety of plants. This is in charge of the differential expression of genes and enzymes that may have contributed to the buildup of phenolics in L. chinensis plantlet cultures.
Query #13. Line 315-317 – too strong a statement, especially since it is not known what concentrations of the tested compound are in ground plant. The authors did not address this issue in the manuscript.
Answer: Conclusion has been revised suitably.
Author Response File: Author Response.pdf
Round 2
Reviewer 3 Report
Comments and Suggestions for AuthorsI thank the authors for considering my suggestions to improve the message of their work.
The changes to the description of the work, mainly in the "Methodology" section are clearer in key details to ensure reproducibility.
In section 2.7. and 2.8., I consider that is more appropiate to mention as subheadings "Quantification of phenolic compounds" and "Quantification of polyacetylenes", respectively.
I have reviewed the manuscript again and I consider that appropiate corrections, improve in Discussion and modifications have been made. It could be accepted for publication.
Author Response
Response to Reviewers Comments
Ref: Horticulturae-3613162-R1
Authors are thankful to anonymous reviewers for their valuable comments on the manuscript and we have revised manuscript as per the instruction’s reviewers. All corrections are highlighted in blue colour. Following are the specific changes made in the revised manuscript.
Query #1: In section 2.7. and 2.8., I consider that is more appropriate to mention as subheadings "Quantification of phenolic compounds" and "Quantification of polyacetylenes", respectively.
Answer: As per the suggestion the section 2.7 and 2.8 are presented as “"Quantification of phenolic compounds" and "Quantification of polyacetylenes", respectively.
Query of Assistant Editor: During the technical check of your manuscript, we noticed that a high proportion of the cited references belong to you or your co-authors' references: 9,10,11,17,12,13,14,15,16,18,32,33,34 making a self-citation rate of higher than 22%. Self-citation rate should be less than 15%. Therefore, we kindly ask you if you could check whether the inclusion of each of these references is appropriate. Please replace or revise at least 7 of the references listed above, if that is agreeable to you.
Answer: As per the suggestion assistant editor seven references (self-citations) have been removed and rest of the references are rearranged. All the rearranged references as shown blue colour.
