Callus Induction and Plant Regeneration from Carum copticum and Assessment of Antioxidant Responses and Phytochemical Profiling by In Vitro Salinity Stress
Round 1
Reviewer 1 Report
The paper entitled: "Callus Induction and Plant Regeneration from Carum copticum and Assessment of Antioxidant Responses and Phytochemical Profiling by in vitro Salinity Stress", submitted to Horticulturae, is an interesting paper that falls within the scope of the journal. The aim of this study is to determine the potentialities of Carum copticum to regenerate after in vitro tissue culture. The study also has as a major objective the study of the biochemical response of the studied plant to in vitro salt stress. determine if in vitro salt stress. Although the paper is important some major revisions are required before considering it to publication.
* As an overall comment about the introduction section, I find it clearly written, but I suggest to the authors to developpe more the part discussing the studied plant (C. copticum), Its ethnobotanical and ethnopharmacological properties and why not the toxicological profile of the plant if found.
* Regarding the methods and Results sections, please add a space between the number and its unity of measure. the results must be as the example: 5 ± 1 (consider the spaces); Culture media as the example: MS + 0 mg.L-1 2,4-D + 0.25 mg.L-1 BAP.
* Figure 3: each picture must be with a scale bar as in 0 mM NaCl treatment (control) callus picture.
* Line 555: para-cymene must be p-cymene.
* Why the authors used 100 mM NaCl treated plant material (seedlings, calli, regenerated seedlings) in the biochemical part of the work, why the authors did not consider working on the plant material (calli and seedlings) from 150 mM NaCl treatment, as they contain more phenolics and the regenerated seedlings from 100 mM NaCl treatment ?
* "Overall, this study suggests addition of salinity to the growth medium at the certain concentration as an efficient strategy to enhance the synthesis of the secondary metabolites.", This is a very interesting idea, I invite the authors to develop it more in the discussion.
* Please reformat the paper correctly according to Horticulturae template.
Author Response
Reviewer 1
The paper entitled: "Callus Induction and Plant Regeneration from Carum copticum and Assessment of Antioxidant Responses and Phytochemical Profiling by in vitro Salinity Stress", submitted to Horticulturae, is an interesting paper that falls within the scope of the journal. The aim of this study is to determine the potentialities of Carum copticum to regenerate after in vitro tissue culture. The study also has as a major objective the study of the biochemical response of the studied plant to in vitro salt stress. Determine if in vitro salt stress. Although the paper is important some major revisions are required before considering it to publication.
* As an overall comment about the introduction section, I find it clearly written, but I suggest to the authors to develop more the part discussing the studied plant (C. copticum), Its ethnobotanical and ethno pharmacological properties and why not the toxicological profile of the plant if found.
Answer: As suggested, more explanations have been added to the introduction.
* Regarding the methods and Results sections, please add a space between the number and its unity of measure. The results must be as the example: 5 ± 1 (consider the spaces); Culture media as the example: MS + 0 mg.L-1 2,4-D + 0.25 mg.L-1 BAP.
Answer: Thank you very much for your correction. Corrections have been made in numbers and spaces in the section of methods and results.
* Figure 3: each picture must be with a scale bar as in 0 mM NaCl treatment (control) callus picture.
Answer: The scales have been added to the pictures in Figure 3.
* Line 555: para-cymene must be p-cymene.
Answer: It has been edited.
* Why the authors used 100 mM NaCl treated plant material (seedlings, calli, regenerated seedlings) in the biochemical part of the work, why the authors did not consider working on the plant material (calli and seedlings) from 150 mM NaCl treatment, as they contain more phenolics and the regenerated seedlings from 100 mM NaCl treatment ?
Answer: In this experiment, the effects of different concentrations of NaCl from 0 to 200 mM were first examined on growth parameters and chlorophyll contents in all three samples of C. copticum. Then, according to the obtained results, these concentrations were optimized and other physiological and biochemical parameters were evaluated. Since, 100 mM NaCl exhibited the least negative effect on growth with increased production of valuable secondary metabolites, it was considered as the optimal concentration for determination of essential oils.
* "Overall, this study suggests addition of salinity to the growth medium at the certain concentration as an efficient strategy to enhance the synthesis of the secondary metabolites.", This is a very interesting idea, I invite the authors to develop it more in the discussion.
Answer: More explanation and information have been added to this part of discussion.
* Please reformat the paper correctly according to Horticulturae template
Answer: The manuscript and the references have been reformatted according the journal instruction.
Reviewer 2 Report
Razavizadeh et al. describe the study that shows induction of biosynthesis of various secondary metabolites by salt stress in regenerated seedlings from callus cultures of Carum copticum. In this study, the regenerated seedlings exhibited accumulation of osmolytes and different activities of some antioxidant enzymes. Gas chromatography analysis displayed formation of various metabolites as components of oil extracts. The authors concluded that in vitro plant cultures under salt stress could be a valuable source of secondary metabolites.
However, this study might be valuable, some points should be improved and explained.
Material and methods/Results: APX, CAT, SOD activities are expressed as unit/mg of protein. Did you use unit as U (umol min -1) ?
It would be very interesting to show analysis of the enzymatic activity (SOD, APX, CAT) by native electrophoresis (zymography), because various isoforms of these enzymes are activated durind stress conditions.
Legend of Tables 1-5: Values presented in the Tables 1-5 are expressed as mean +/- SD or SE. This information should be added to the legend.
Table 4: Secondary metabolites in oil extracts. The unit (mg, umol,???) of metabolite amount should be added. Why these values do not contain SD or SE ?
Comment for future research: Determination of total amount of reducing sugars. Colorimetric method of Miller with DNS is more specific that Somogyi-Nelson method.
Author Response
Reviewer 2
Razavizadeh et al. describe the study that shows induction of biosynthesis of various secondary metabolites by salt stress in regenerated seedlings from callus cultures of Carum copticum. In this study, the regenerated seedlings exhibited accumulation of osmolytes and different activities of some antioxidant enzymes. Gas chromatography analysis displayed formation of various metabolites as components of oil extracts. The authors concluded that in vitro plant cultures under salt stress could be a valuable source of secondary metabolites. However, this study might be valuable, some points should be improved and explained.
Material and methods/Results: APX, CAT, SOD activities are expressed as unit/mg of protein. Did you use unit as U (umol min -1) ?
Answer: Yes, one unit of CAT activity was defined as 1 μmol H2O2 decomposed per 1 min at 25 °C.
One unit of APX activity was defined as the amount of enzyme oxidising 1 μmol of AsA per 1 min.
One unit of SOD was identified as the amount of enzyme needed to prevent 50% (w/v) NBT photo-reduction rate in 1 min.
It would be very interesting to show analysis of the enzymatic activity (SOD, APX, CAT) by native electrophoresis (zymography), because various isoforms of these enzymes are activated durind stress conditions.
Answer: Thank you for your valuable comment. Unfortunately, in our laboratory, it is only possible to measure the activities of enzymes by spectrophotometric method. Of course, we will consider this comment for our future research.
Legend of Tables 1-5: Values presented in the Tables 1-5 are expressed as mean +/- SD or SE. This information should be added to the legend.
Answer: Values represented the mean of three replicates ± SD. It has been added to the legend.
Table 4: Secondary metabolites in oil extracts. The unit (mg, umol,???) of metabolite amount should be added. Why these values do not contain SD or SE ?
Answer: Percentage was considered as the unit of metabolite amount. The values have been corrected with ± SD and It was added to the legend.
Comment for future research: Determination of total amount of reducing sugars. Colorimetric method of Miller with DNS is more specific that Somogyi-Nelson method.
Answer: We appreciate your valuable comment and we will definitely consider it in the next tests.
