Propagation of Saffron (Crocus sativus L.) Using Cross-Cuttings under a Controlled Environment

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

Crocus  sativus  L is a  male  sterile  vegetatively  propagated plant. It characterizes by slow vegetative propagation and diseases that limit the production and the development of saffron; 3–4 daughter corms per mother corm are obtained each season under natural field conditions. Development of effective methods to overcome these limitations is very interesting. And the aim of this study is propagation of saffron  by cross cutting under controlled conditions. This is a simple manuscript describing the achievement of saffron propagation by cutting.

 

Comments and suggestions

•   Justification of this study was based on limitation of saffron production by low vegetative propagation, low number of high quality of corms and that in vitro propagation may not be the best solution to improve plant propagation (ligne 80, 81). For the last point, Conversely, tissue culture methods offer great potential for large-scale multiplication. Morata et al., (2013) reported high in vitro propagation efficiency from low-temperature storage- produced corms that are induced from non-planted saffron corms; up to 400 shoot primordia were produced from one initial mother corm. These shoot primordia elongated and 90% produced corms with good quality. In vitro regeneration and development of shoots and corms of Moroccan saffron cultivars was also reported (Lagram et al., 2016; 2023). The best adventitious shoot initiation rate of 80% with 10.2 ± 0.23 shoots per explant was obtained using 0.5 mg/L NAA and 2.75 mg/L BAP. Remarkably, the critical step of mini-corm regeneration was improved using ½ MS, 6% sucrose, 1 mg/L NAA, and dark incubation. The corms produced weighed 7.9 ± 0.8 g. So, this argument should be to reconsider 

•   Materiel and methods: Control wasn’t mentioned in this part. And I think is better to have two controls; one present corms without treatment under conditions controlled and the other present corms without treatment in field conditions. It's better to have photos of explant type used

•   Results:  In this study, the authors investigated Safron cutting in controlled conditions (greenhouse) and they produced corms through used technique. However, for all steps, the control was better except for shoot production by corm and the number of daughter corms formed. The lowest diameter and weight were also observed when cutting CTB and BC compared to the control group. My following question is: with these results, this technique used (vegetative propagation by cross cutting) could be considered as a promising highly economical and effective approach?  The authors might want to explain what is the advantage of propagating saffron from BC or CTB corms, instead from uncut corm, which seems to be easier and ensures a significant number of corms with good quality and size uniformity. If not, the conclusion (lines 335-337) reported in this manuscript should be change

Comments on the Quality of English Language

Good

Author Response

Thank you so much for taking the time to review this manuscript; enclosed are the detailed responses and corresponding revisions/corrections, which have been highlighted changes in the re-submitted files.

Comments and suggestions

• Justification of this study was based on limitation of saffron production by low vegetative propagation, low number of high quality of corms and that in vitro propagation may not be the best solution to improve plant propagation (ligne 80, 81). For the last point, Conversely, tissue culture methods offer great potential for large-scale multiplication. Morata et al., (2013) reported high in vitro propagation efficiency from low-temperature storage- produced corms that are induced from non-planted saffron corms; up to 400 shoot primordia were produced from one initial mother corm. These shoot primordia elongated and 90% produced corms with good quality. In vitro regeneration and development of shoots and corms of Moroccan saffron cultivars was also reported (Lagram et al., 2016; 2023). The best adventitious shoot initiation rate of 80% with 10.2 ± 0.23 shoots per explant was obtained using 0.5 mg/L NAA and 2.75 mg/L BAP. Remarkably, the critical step of mini-corm regeneration was improved using ½ MS, 6% sucrose, 1 mg/L NAA, and dark incubation. The corms produced weighed 7.9 ± 0.8 g. So, this argument should be to reconsider

Regarding the justification mentioned above, in vitro propagation could be considered as an alternative propagation method. However, the optimization and commercialization of this method is not simple because of the cost of production of corm, and the limited corm produced by this method could be a second limitation. Thus, it is crucial to investigate other vegetative methods. In this study, we present a promising method for saffron propagation and the cost-effective formation of a high number of corms.

• Materiel and methods: Control wasn’t mentioned in this part. And I think is better to have two controls; one present corms without treatment under conditions controlled and the other present corms without treatment in field conditions. It's better to have photos of explant type used.

In this study, the control group used was planted under the same condition, in which all cutting treatments and the control group were maintained under controlled environmental conditions, and light intensity of 500 μmol/m²/s with relative humidity (RH)of 70% at 15 °C for the flowering period (for three months) and 28 °C for corm formation. The details were added ( line 107). 

Regarding The use of the uncut group (controls) under controlled conditions was based on the objective of this study, which was the propagation of Crocus sativus under controlled conditions. Thus, there is no need for field growth of saffron. Moreover, field growth could lead to the same growth parameters being recorded, as we selected the optimal conditions for saffron flowering and growth. furthermore, the evaluation of the field conditions for the saffron uncut group and cutting treatment could be tested from the perspective of this work.

• Results: In this study, the authors investigated Safron cutting in controlled conditions (greenhouse) and they produced corms through used technique. However, for all steps, the control was better except for shoot production by corm and the number of daughter corms formed. The lowest diameter and weight were also observed when cutting CTB and BC compared to the control group. My following question is: with these results, this technique used (vegetative propagation by cross cutting) could be considered as a promising highly economical and effective approach?  The authors might want to explain what is the advantage of propagating saffron from BC or CTB corms, instead from uncut corm, which seems to be easier and ensures a significant number of corms with good quality and size uniformity. If not, the conclusion (lines 335-337) reported in this manuscript should be change

For the justification of the cost-effective method for saffron production, we believed that method could be a promising method for enhancing corm formation, as explained in the introduction, one of the main limitations of saffron culture is the limited number of planting materials. thus, the cutting method offers a higher cormlet formation compared to the control. However, as we mentioned in the lines, the is a need for further improvement, specifically in the saffron daughter cormlet size.

 

Author Response File: Author Response.docx

Reviewer 2 Report

Comments and Suggestions for Authors the current study aimed to investigate the effect of saffron cross-cutting on shoot, leaf, and corm formation. it is helplful to  improve the propagation of saffron.

 

Comments for author File: Comments.pdf

Author Response

Thank you so much for taking the time to review this manuscript; all the requested modifications have been made in the re-submitted files.

Author Response File: Author Response.docx

Reviewer 3 Report

Comments and Suggestions for Authors

The manuscript is well written. The results can be more adequately presented. In conclusion, some remarks should be addressed.

Abstract:

- It would be preferable if the research findings were referenced with greater precision, regardless of whether the cutting method had a positive or negative impact on the specific parameter under investigation.

Introduction :

- Line 39 eliminate repetition of the word Iran

- The aim study is better referred in the abstract and discussion rather than the introduction, which necessitates revision of this paragraph.

- Why you used (PGPR) as an abbreviation for growth-promoting rhizobacteria instead of using GPRB or GPR

- Line 78 gives the scientific vocabulary of the abbreviation PGRs

2-1-Plant material :

-Identify the specific year or years during which the collections were conducted, determine the coordinates of the collection sites, and provide detailed information about the control treatment (uncut) instead of referencing it in line 302.

2-2- In vivo cross-cuttings

- You used as abbreviations: sterilized distilled water (SDW), Basal cutting (BC) for which you used the abbreviation (CTB) for top-to-bottom cutting instead of (TBC)

2.3. Statistical analysis  

- Why did you choose the Duncan post hoc test and not another test such as the Tukey post hoc test?

3- Results:

- The boxplot of the BC group concerning the number of saffron shoots formed under the control treatment shows intense heterogeneity within this group. How can you explain this result?

4. Discussion

- Replace this: “The main objective of the present study was to improve saffron production by vegetative propagation. Two different cutting methods were evaluated” in the introduction

- The impact of the cutting technique on the parameters under investigation holds considerable significance, either in a positive or negative manner. It is imperative to specify the nature of this impact for each parameter that has been studied.

5. Conclusions

- The scientific vocabulary of the abbreviations PGRs and PGPRs is not predefined in the elements of the research document preceding the conclusions.

Author Response

Thank you so much for taking the time to review this manuscript; enclosed are the detailed responses and corresponding revisions/corrections, which have been highlighted changes in the re-submitted files.

Abstract:

- It would be preferable if the research findings were referenced with greater precision, regardless of whether the cutting method had a positive or negative impact on the specific parameter under investigation.

Based on our findings, the cutting treatment had a positive effect on the number of daughter corms formed. The highest number of daughter corms was obtained after cutting BC (Table 2). The information mentioned has been precise in the abstract (line 26)

Introduction:

- Line 39 eliminate repetition of the word Iran

The repetitions have been deleted

- The aim study is better referred in the abstract and discussion rather than the introduction, which necessitates revision of this paragraph.

The aim of this present study was to improve saffron production by vegetative propagation. Two different cross-cutting methods were evaluated, and it was explained in the abstract (line20) and in the discussion line (line 272). The introduction was also revised, and the objective was clearly explained in lines (87-89).

- Why you used (PGPR) as an abbreviation for growth-promoting rhizobacteria instead of using GPRB or GPR

PGPB is the abbreviation for Plant Growth-Promoting Bacteria, and PGPR is the abbreviation for Plant Growth-Promoting Rhizobacteria. They both mean bacteria provide multiple benefits to plants by increasing the nutrient content and yield of crops. PGPR is more specific for Rhizobacteria (bacteria root-associated bacteria: rhizosphere bacteria), and they are more used in increasing plant production because they have a crucial role in promoting plant growth by producing hormones, solubilizing phosphates, and fixing nitrogen).

- Line 78 gives the scientific vocabulary of the abbreviation PGRs

For the plant growth regulators, the scientific name of PGR was added

2-1-Plant material:

-Identify the specific year or years during which the collections were conducted, determine the coordinates of the collection sites, and provide detailed information about the control treatment (uncut) instead of referencing it in line 302.

Plant material was collected from the Taliouine-Taznakht Region in Morocco during the dormancy period (July-August 2022) (line 95). All tests were performed under controlled conditions. The control group and cross-cutting corm were planted in nursery pots and maintained under controlled environmental conditions, and light intensity of 500 μmol/m²/s with relative humidity (RH)of 70% at 15 °C for the flowering period (for 3 months) and 28 °C for corm formation (line106)

2-2- In vivo cross-cuttings

- You used as abbreviations sterilized distilled water (SDW), Basal cutting (BC) for which you used the abbreviation (CTB) for top-to-bottom cutting instead of (TBC)

The "CTB" and "TBC" both signify the concept of "top-to-bottom cutting." The choice of the abbreviation was based on a study reported by (Haspolat,et al 2018) cited in this study

2.3. Statistical analysis  

- Why did you choose the Duncan post hoc test and not another test such as the Tukey post hoc test?

Duncan test was used to study the difference between means, and it is the better choice to be used in our study.

3- Results:

- The boxplot of the BC group concerning the number of saffron shoots formed under the control treatment shows intense heterogeneity within this group. How can you explain this result?

For shoot emergence, we could explain the higher number of shoots formed compared to that in the control group, that the cutting treatment had overcome the apical dominance of the apical meristem (line 291). However, the heterogeneity of this group could be explained by the number of axillary buds stimulated by the cutting treatment. Moreover, group BC had a higher number of shoots, which could mean that the removal of dominance apical was better by the BC cutting rather than CTB cutting. 

4. Discussion

- Replace this: “The main objective of the present study was to improve saffron production by vegetative propagation. Two different cutting methods were evaluated” in the introduction

The phrase was replaced (line 88)

- The impact of the cutting technique on the parameters under investigation holds considerable significance, either in a positive or negative manner. It is imperative to specify the nature of this impact for each parameter that has been studied.

5. Conclusions

- The scientific vocabulary of the abbreviations PGRs and PGPRs is not predefined in the elements of the research document preceding the conclusions.

growth-promoting rhizobacteria (PGPR) and plant growth parameters, both scientific vocabularies had been in the introduction (lines 66-79)

 

 

 

Author Response File: Author Response.docx

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

GOOD

Comments on the Quality of English Language

GOOD