Review Reports

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The work deals with in-vitro culture studies of Vitellaria paradoxa nilotica, an important economic species, in its initial stages of cultivation using leaf explants and callus formation in relation to the physiological state of the leaves in combination with appropriate combinations of plant hormones. Also interesting is the histological analysis which describes the characteristics of the various sections relating to the physiological stages of the leaves. The work is well-written and in appropriate English. There are some suggestions that could improve the manuscript. First of all, the authors should unify the term callus (Latin noun), which in the plural means calli. It should be modified throughout the text, avoiding calluses, which is inappropriate. In the abstract I would avoid citing proembryo structure (row 26 and 27) because they are not explained or described in the text. Furthermore, the term embryogenic callus should not be cited in the tables because it is not described or verified in the text. In the materials and methods section, the type of data recorded must be mentioned, for example: the fresh weight obtained after (30?) days of treatment is considered. Again, in the mat and met section, it should be noted that the statistical analysis of the percentages was performed after transformation into angular values. If this has not been done, it should be done before comparing the means and therefore also modified in the relevant table. The following section will be Results and Discussion since, correctly, data from relevant publications are discussed and compared.  In figure 3 you need to describe the part from which the leaf explant comes, if it is cumulative you need to specify it. In the legend of Table 2, the statistical analysis performed and the P value must be specified. In row 189 please insert the reference for this sentence. In conclusion, I believe that this work can provide interesting insights and information for better understanding the phenomenon of recalcitrance in woody species.

Author Response

Comment 1: The authors should unify the term callus (Latin noun), which in the plural means calli. It should be modified throughout the text, avoiding calluses, which is inappropriate. 

Response 1: The term “calluses” has been modified to “calli” throughout the manuscript. 

Comment 2: In the abstract, I would avoid citing proembryo structure (row 26 and 27) because they are not explained or described in the text. 

Response 2: The original text explaining the formation of pro-embryo structures has been modified (line 334-335). A description of the pro-embryo structures has been provided in the text (row 340-341). 

Comment 3: The term embryogenic callus should not be cited in the tables because it is not described or verified in the text.

Response 3: The term has been removed from the tables.  

Comment 4: In the materials and methods section, the type of data recorded must be mentioned, for example: the fresh weight obtained after (30?) days of treatment is considered. 

Response 4: Types of data recorded in the tables but previously missing in the methodology have been included i.e. callus weight and percentage callusing (lines 138-140); callus ranking (145-146). 

Comment 5: Again, in the mat and met section, it should be noted that the statistical analysis of the percentages was performed after transformation into angular values. If this has not been done, it should be done before comparing the means and therefore also modified in the relevant table.

Response 5: Angular transformation of percentage data for ANOVA testing has been included in the mat and met section (line 151-152). Consequently, the means have been modified in Table 2.

Comment 6: In figure 3 you need to describe the part from which the leaf explant comes, if it is cumulative you need to specify it. 

Response 6: A description of the leaf part has been specified in the mat and met section (line 132-133).  The legend of Figure 3 has been corrected. 

Comment 7: In the legend of Table 2, the statistical analysis performed and the P value must be specified.

Response 7: The statistical analysis performed has been specified and the P value of (7.98x10^-6) has been appropriately reported. Table 2 has been changed into a bar graph (Figure 5). 

Comment 8: In row 189 please insert the reference for this sentence.

Response 8: Row 189 has moved to line 203-204. The appropriate reference has been inserted in line 205. 

 

 

Reviewer 2 Report

Comments and Suggestions for Authors

The manuscript authors “Effect of explant physiology and media composition on callogenesis of Vitellaria paradoxa leaf explants.” used tissue culture techniques to developed the method to induct callus of shea tree, an economically important crop. The description is clear and well presented. The results provide a foundation for the solution of the plant propagations. There are a few small issues can be addressed.

1. How big is the seed? Can the seeds be germinated on sterile cultures rather than soil. Because the leave can bypass the sterilization process  which could potentially regeneration from callus. 

2. The source (company names etc) of the products used can detailed in the method.

3. What is the liquid detergent used? 

4. Is there other reports in the related plants in the genus (family). This could be discussed.

 

 

Although it is a pity of that the regeneration process can not be demonstrated in this study while it could be fulfilled in the future. 

Author Response

Comment 1: How big is the seed? Can the seeds be germinated on sterile cultures rather than soil. Because the leave can bypass the sterilization process which could potentially regeneration from callus. 

Response 1: The seed measures approximately 2–3 cm in diameter. Although germination on sterile culture media is possible, leaf emergence takes up to three months, even though the radicle emerges within one week of sowing. This prolonged process requires frequent transfer of germinating seeds to fresh media, which is cumbersome. Under natural field conditions, leaves may reach lengths of up to 30 cm, but in greenhouse environments this is reduced to 3–10 cm. In vitro germination is likely to further reduce leaf size, making it unsuitable for long-term studies where a large number of leaf explants is required. Using the protocol outlined in the manuscript, sterile cultures can be achieved. 

Comment 2: The source (company names etc) of the products used can be detailed in the method.

Response 2: The source of the PGRs used has been specified in the methods section (line 137-138). 

Comment 3: What is the liquid detergent used? 

Response 3: The liquid detergent used is JAR dishwashing detergent. A brand of Procter and Gamble (P&G), an American multinational consumer goods company.

Comment 4: Is there other reports in the related plants in the genus (family)? This could be discussed.

Response 4: Reports from related plants have been included (Line 351-359). 

Reviewer 3 Report

Comments and Suggestions for Authors

This manuscript presents a valuable contribution to tropical plant biotechnology by examining callogenesis in Vitellaria paradoxa subsp. nilotica. The novelty lies in the use of leaf explants from an underexplored subspecies, combined with histological analysis to link physiology with morphogenic competence. The experimental approach is sound; however, improvements in data visualization, deeper analysis, and a more critical discussion of limitations would substantially strengthen the manuscript.

 

** The manuscript does not explicitly clarify whether explants were sourced from multiple genotypes or only one mother plant. (Since genotype strongly influences tissue culture responses, this should be clearly stated).

** Microscopic images (e.g., Figure 4) lack sufficient labeling, scale bars, and clear indication of anatomical features such as chloroplasts, trichomes, and vascular tissues. Improved annotations would make the histological analysis more"scientific".

** Results are largely presented in tables. For stronger impact, callogenesis rates and callus weights should also be shown graphically (bar graphs or heatmaps). This will help readers quickly interpret stage × treatment effects. (Graphical representation highlights key patterns and makes complex datasets easier to interpret).

** While callus induction was successful, no somatic embryos or organogenesis were obtained. The authors should explicitly state this as a limitation and propose potential future strategies (e.g., ABA supplementation, osmotic pre-treatments, activated charcoal, molecular markers like SERK/LEC).

** The discussion on why BAP failed to induce callus could be expanded by referencing auxin–cytokinin antagonism in woody perennials. This would contextualize the finding beyond empirical observation.

** In the Abstract, include exact induction rates (e.g., “up to 100% callus induction at Stage III–IV”) to emphasize the study’s success quantitatively.

** The sterilization protocol is well described but overly long. Consider adding a flowchart or schematic representation.

** I highly recommend adding a color-coded matrix or heatmap showing callogenesis across stages and PGR combinations would enhance interpretation of the results in section 3.3 (which is the most critical section). (A visual summary in form of a heatmap to easily grasp stage × treatment interactions and improves manuscript impact)

 

Author Response

Comment 1: The manuscript does not explicitly clarify whether explants were sourced from multiple genotypes or only one mother plant. (Since genotype strongly influences tissue culture responses, this should be clearly stated). 

Response 1: This has been clarified in the manuscript (line 64-65).

Comment 2: Microscopic images (e.g., Figure 4) lack sufficient labeling, scale bars, and clear indication of anatomical features such as chloroplasts, trichomes, and vascular tissues. Improved annotations would make the histological analysis more scientific. 

Response 2: The microscopic images have been labelled accordingly (Figure 6). 

Comment 3: Results are largely presented in tables. For stronger impact, callogenesis rates and callus weights should also be shown graphically (bar graphs or heatmaps). This will help readers quickly interpret stage × treatment effects. (Graphical representation highlights key patterns and makes complex datasets easier to interpret).

Response 3: Table 2 has been transformed into a bar graph (Fig. 5). 

Comment 4: While callus induction was successful, no somatic embryos or organogenesis were obtained. The authors should explicitly state this as a limitation and propose potential future strategies (e.g., ABA supplementation, osmotic pre-treatments, activated charcoal, molecular markers like SERK/LEC). 

Response 4: Potential strategies for obtaining embryogenesis or organogenesis have been included (line 347-350). 

Comment 5: The discussion on why BAP failed to induce callus could be expanded by referencing auxin–cytokinin antagonism in woody perennials. This would contextualize the finding beyond empirical observation. 

Response 5: The discussion has been expanded accordingly (line 232-242). 

Comment 6: In the Abstract, include exact induction rates (e.g., “up to 100% callus induction at Stage III–IV”) to emphasize the study’s success quantitatively.

Response 6: This concern has already been emphasized in the abstract (line 22-24). 

Comment 7: The sterilization protocol is well described but overly long. Consider adding a flowchart or schematic representation.

Response 7: The sterilization protocol has been summarized in a flow chart (Figure 2). 

Comment 8: I highly recommend adding a color-coded matrix or heatmap showing callogenesis across stages and PGR combinations would enhance interpretation of the results in section 3.3 (which is the most critical section). (A visual summary in form of a heatmap to easily grasp stage × treatment interactions and improves manuscript impact). 

Response 8: A color-coded table has been used to delineate callogenesis at different stages of growth (Table 2).