An Effective Protocol for Callus Induction and Plant Regeneration in an Indica Rice Cultivar RD43

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The authors provide an efficient protocol for regeneration of the indica rice cultivar RD43. Some comments to clarify and improve the presentation are provided below:

The authors describe callus induction, callus proliferation and plantlet regeneration. It seems, however, that the sequence of steps is to go from 'induction' to 'regeneration' without using the 'proliferation' protocol. In that case, what is the intended value of knowing the callus proliferation protocol? If the authors intend to describe two different end points in their - callus proliferation versus plantlet regeneration - they should be clear what the value of callus proliferation is.

The authors use the term 'optimization' that suggests a much more robust data analysis than what was actually done here. Their experimental designs are not described nor analyzed in terms of a response surface (for example) that would allow for true optimization. Instead, what they have is an 'efficient' or 'effective' protocol. I would strongly discourage the use of optimal or optimization in the description of outcomes as well as the title. They are simply aligning their suggested protocol with maximal values at the chosen experimental points. The authors should also state that they are simply looking at a main effects model (no interactions or quadratic or higher terms) so the only valid statements are related to selection of highest responses.

The authors state (lines 196-199) 'Optimal callus induction for RD43 in this work required 4.0 mg/L 2,4-D. This value is somewhat higher than the range reported in other varieties (2.0–3.0 mg/L) [4, 11], suggesting that RD43 may require a unique auxin dosage. Genetic variability between rice cultivars likely explains differences in 2,4-D responsiveness.' Forgetting about the use of the word 'optimal', their data analysis in Table 1 shows there is no statistically significant difference for callus induction between 2-5 mg/L. In what sense is 4.0 mg/L 2,4-D required or unique? The other responses associated with diameter, fresh and dry weight have no difference as well with respect to the 'a' short letter designation. I think the sentences noted here are overstating the conclusions.

The authors may further strengthen their data analysis by noting that any normality assumptions around the responses tabulated are valid. The software may provide this. I mention this also in the context of the responses in Table 2 for NAA = 0, BA = 3 mg/L. The data points are likely outliers that should also be captured by the statistical software and properly included in the ANOVA. If possible, this would help.

 

 

Author Response

Reviewer 1

The authors provide an efficient protocol for regeneration of the indica rice cultivar RD43. Some comments to clarify and improve the presentation are provided below:

 

The authors describe callus induction, callus proliferation and plantlet regeneration. It seems, however, that the sequence of steps is to go from 'induction' to 'regeneration' without using the 'proliferation' protocol. In that case, what is the intended value of knowing the callus proliferation protocol? If the authors intend to describe two different end points in their - callus proliferation versus plantlet regeneration - they should be clear what the value of callus proliferation is.

- We agree that the sequence of steps in the Results section would benefit from clearer alignment with the actual tissue culture workflow. Accordingly, we have moved the callus proliferation results to follow callus induction and precede plantlet regeneration.

 

The authors use the term 'optimization' that suggests a much more robust data analysis than what was actually done here. Their experimental designs are not described nor analyzed in terms of a response surface (for example) that would allow for true optimization. Instead, what they have is an 'efficient' or 'effective' protocol. I would strongly discourage the use of optimal or optimization in the description of outcomes as well as the title. They are simply aligning their suggested protocol with maximal values at the chosen experimental points. The authors should also state that they are simply looking at a main effects model (no interactions or quadratic or higher terms) so the only valid statements are related to selection of highest responses.

- We agree with the suggestion. We have modified the title to an effective protocol for callus induction and plant regeneration in an indica rice cultivar RD43. We also modified the text to avoid using the term “optimization” and replaced with “effective”. We have also clarified in the Material and Methods in “2.5 Statistical” section that the study is based on a main effects model only, and no factorial design or interaction terms were assessed.

 

The authors state (lines 196-199) 'Optimal callus induction for RD43 in this work required 4.0 mg/L 2,4-D. This value is somewhat higher than the range reported in other varieties (2.0–3.0 mg/L) [4, 11], suggesting that RD43 may require a unique auxin dosage. Genetic variability between rice cultivars likely explains differences in 2,4-D responsiveness.' Forgetting about the use of the word 'optimal', their data analysis in Table 1 shows there is no statistically significant difference for callus induction between 2-5 mg/L. In what sense is 4.0 mg/L 2,4-D required or unique? The other responses associated with diameter, fresh and dry weight have no difference as well with respect to the 'a' short letter designation. I think the sentences noted here are overstating the conclusions.

-We agree that the term "optimal" may overstate the statistical interpretation, especially since there were no significant differences (P ≤ 0.05) among 2.0, 3.0, 4.0, and 5.0 mg/L 2,4-D in terms of callus induction frequency and biomass accumulation based on Table 1. We have revised the manuscript (At page 7 line 211-215) to clarify this point and avoid overstating the conclusion. Specifically, we now state that 4.0 mg/L 2,4-D was selected as the most suitable concentration for callus induction of rice cv. RD43 because it consistently produced the largest and healthy calli. Although the differences were not statistically significant, the calli induced at 4.0 mg/L were more actively proliferating, which we considered important for supporting subsequent callus proliferation and regeneration stages.

 

The authors may further strengthen their data analysis by noting that any normality assumptions around the responses tabulated are valid. The software may provide this. I mention this also in the context of the responses in Table 2 for NAA = 0, BA = 3 mg/L. The data points are likely outliers that should also be captured by the statistical software and properly included in the ANOVA. If possible, this would help.

- As suggested, we have conducted a Shapiro–Wilk test to assess the normality of the dataset. The results indicated that the data were not normally distributed. Therefore, we applied the Kruskal–Wallis test to assess differences among treatments, followed by Fisher's least significant difference (LSD) test with Holm's method for p-value adjustment in post hoc comparisons at P ≤ 0.05. The results in Tables 1–3 have been revised accordingly to reflect the updated statistical analysis. The description in the Materials and Methods about the statistical analysis has been modified accordingly.

Reviewer 2 Report

Comments and Suggestions for Authors

This manuscript, by Pundanai Chitphet et al., aims to establish an ‌in vitro regeneration system‌ for ‌RD43 rice‌. The study investigated the effects of ‌2,4-D‌ and ‌BA‌ on ‌callus induction‌, ‌callus proliferation‌, and ‌callus-based shoot regeneration efficiency‌ in RD43 rice, determining the optimal ‌plant growth regulator‌ ratios for the response stages. The results lay a foundation for RD43 rice breeding research. However, ‌ambiguities‌ exist in the methodology description, and ‌unreasonable findings‌ appear in ‌Result 3.2‌. Specific issues include:

1. The ‌shoot regeneration method‌ for RD43 rice is overly simplified. For example, ‌how many calluses per treatment were statistically analyzed‌ to calculate shoot regeneration rates?

2. Table 2‌ shows that BA significantly affects the ‌plant regeneration rate‌: regeneration rates induced by ‌0, 1.0, 2.0, 4.0, and 5.0 mg/L BA‌ are ‌18.75%, 30.00%, 50.00%, 44.44%, and 12.50%‌, respectively. However, ‌3.0 mg/L BA yields a 0% regeneration rate‌, which is clearly illogical. ‌Is this due to the authors’ insufficient sample size in their statistical analysis or incorrect experimental operation?‌

3. Figure 3‌ lacks a ‌figure number‌.

Author Response

Reviewer 2

This manuscript, by Pundanai Chitphet et al., aims to establish an ‌in vitro regeneration system‌ for ‌RD43 rice‌. The study investigated the effects of ‌2,4-D‌ and ‌BA‌ on ‌callus induction‌, ‌callus proliferation‌, and ‌callus-based shoot regeneration efficiency‌ in RD43 rice, determining the optimal ‌plant growth regulator‌ ratios for the response stages. The results lay a foundation for RD43 rice breeding research. However, ‌ambiguities‌ exist in the methodology description, and ‌unreasonable findings‌ appear in ‌Result 3.2‌. Specific issues include:

 

1. The ‌shoot regeneration method‌ for RD43 rice is overly simplified. For example, ‌how many calluses per treatment were statistically analyzed‌ to calculate shoot regeneration rates?

- We have revised the manuscript to include the number of calli used for regeneration in each treatment. This detail has now been added in Table 2 (which is now Table 3).

 

2. Table 2‌ shows that BA significantly affects the ‌plant regeneration rate‌: regeneration rates induced by ‌0, 1.0, 2.0, 4.0, and 5.0 mg/L BA‌ are ‌18.75%, 30.00%, 50.00%, 44.44%, and 12.50%‌, respectively. However, ‌3.0 mg/L BA yields a 0% regeneration rate‌, which is clearly illogical. ‌Is this due to the authors’ insufficient sample size in their statistical analysis or incorrect experimental operation?‌

- We also initially found the 0% regeneration rate at 3.0 mg/L BA unexpected, given the generally trend observed with other BA concentrations. To confirm this result, we conducted an additional experiment using 24 calli under the same 3.0 mg/L BA treatment. Despite this increased sample size, no regeneration was observed in this treatment. Therefore, we believe this outcome is biologically reproducible rather than an artifact of insufficient replication or error in execution. This explanation has been added to page 6 line 171-174.

 

3. Figure 3‌ lacks a ‌figure number‌.

- The number has been added to Figure 3, which is now changed to Figure 2.

Reviewer 3 Report

Comments and Suggestions for Authors

Article „Optimization of Callus Induction and Regeneration in an indica Rice cultivar RD43" by Pundanai Chitphet et all. is the first report on callus induction and in vitro regeneration of shoots of a new highly perspective rice cultivar. Due to the good nutritive characteristic combined with difficulties in commercial propagation, development of an in vitro culture procedure enables better understanding of growth feature of this new cultivar. This work can be a solid foundation for further studies aimed to overcome heat and drought stress which are the main problems in cultivation. Authors have well designed heir treatments missing a few details which I have to mention which can improve presentation of this manuscript,

1. Title should be somewhat changed to read „Optimization of Callus Induction and PlantRegeneration in an Indica Rice cultivar RD43".
2. I believe that instead of "RD43 rice" it should be written "rice cultivar RD43" or "rice cv.RD43". However I am not an expert on English language.
3. In chapter 2.2. components of the MS medium need to be fully provided together with the full Murashige and Skoog reference. Authors should provide data of culture vessels use in specific stages.
4. In the whole text instead of p < 0.05 it should be p≤0.05 (line 98, 105, 112,136,167)
5. For every of the three treatments number of explants should be provided and number of replications. No valid conclusion can be made without at least two replications.
6. For Table 1. as there was no callus regeneration as there was no callus regeneration for control I suggest that you should for Callus diameter, Fresh weight and Dry weight instead of 0.00±0.00 write "-" and recalculate statistics.
7. In chapter 3.2. apart of the number of explants and replications you should state what was the composition of media from which explants were subcultured. Number of regenerated shoot or root buds is always expressed in relation to number of explants that produced regenerants and not for the initial number of explants. That should be noted in the legend text. You should stress the inhibitory effect of NAA on organogenesis.
8. Chapter 3.3. in line 145 instead "2,4-D (0, 2.0, 4.0 mg/L) and BA (0, 1.0 mg/L) " it should be "2,4-D (0, 2.0, 4.0 mg/L) alone or with BA (0, 1.0 mg/L) ".

   Article „Optimization of Callus Induction and Regeneration in an indica Rice cultivar RD43" by Pundanai Chitphet et all. is the first report on callus induction and in vitro regeneration of shoots of a new highly perspective rice cultivar. Due to the good nutritive characteristic combined with difficulties in commercial propagation, development of an in vitro culture procedure enables better understanding of growth feature of this new cultivar. This work can be a solid foundation for further studies aimed to overcome heat and drought stress which are the main problems in cultivation. Authors have well designed heir treatments missing a few details which I have to mention which can improve presentation of this manuscript,

   1. Title should be somewhat changed to read „Optimization of Callus Induction and PlantRegeneration in an Indica Rice cultivar RD43".
   2. I believe that instead of "RD43 rice" it should be written "rice cultivar RD43" or "rice cv.RD43". However I am not an expert on English language.
   3. In chapter 2.2. components of the MS medium need to be fully provided together with the full Murashige and Skoog reference. Authors should provide data of culture vessels use in specific stages.
   4. In the whole text instead of p < 0.05 it should be p≤0.05 (line 98, 105, 112,136,167)
   5. For every of the three treatments number of explants should be provided and number of replications. No valid conclusion can be made without at least two replications.
   6. For Table 1. as there was no callus regeneration as there was no callus regeneration for control I suggest that you should for Callus diameter, Fresh weight and Dry weight instead of 0.00±0.00 write "-" and recalculate statistics.
   7. In chapter 3.2. apart of the number of explants and replications you should state what was the composition of media from which explants were subcultured. Number of regenerated shoot or root buds is always expressed in relation to number of explants that produced regenerants and not for the initial number of explants. That should be noted in the legend text. You should stress the inhibitory effect of NAA on organogenesis.
   8. Chapter 3.3. in line 145 instead "2,4-D (0, 2.0, 4.0 mg/L) and BA (0, 1.0 mg/L) " it should be "2,4-D (0, 2.0, 4.0 mg/L) alone or with BA (0, 1.0 mg/L) ".

Comments on the Quality of English Language

-

Author Response

Reviewer 3

Article „Optimization of Callus Induction and Regeneration in an indica Rice cultivar RD43" by Pundanai Chitphet et all. is the first report on callus induction and in vitro regeneration of shoots of a new highly perspective rice cultivar. Due to the good nutritive characteristic combined with difficulties in commercial propagation, development of an in vitro culture procedure enables better understanding of growth feature of this new cultivar. This work can be a solid foundation for further studies aimed to overcome heat and drought stress which are the main problems in cultivation. Authors have well designed heir treatments missing a few details which I have to mention which can improve presentation of this manuscript,

 

1. Title should be somewhat changed to read „Optimization of Callus Induction and PlantRegeneration in an Indica Rice cultivar RD43".

- The title has been modified to “An effective protocol for callus induction and plant regeneration in an indica rice cultivar RD43”

 

2. I believe that instead of "RD43 rice" it should be written "rice cultivar RD43" or "rice cv.RD43". However I am not an expert on English language.

- "RD43 rice" has been replaced with "rice cv.RD43" throughout the manuscript.

 

3. In chapter 2.2. components of the MS medium need to be fully provided together with the full Murashige and Skoog reference. Authors should provide data of culture vessels use in specific stages.

- We have revised section 2.2 to include the reference to the original publication and details on the culture vessels used in all experiments as follows: “Sterilized seeds were placed on Murashige and Skoog (MS) medium pH 5.8 [8] supplemented with 30 g/L sucrose, 8 g/L agar, 2,4-dichlorophenoxyacetic acid (2,4-D) at concentrations of 0 (control), 1.0, 2.0, 3.0, 4.0, or 5.0 mg/L. One seed was cultured per bottle (120 ml glass bottle containing 20 mL of medium).”

 

4. In the whole text instead of p < 0.05 it should be p≤0.05 (line 98, 105, 112,136,167)

- “p≤0.05” has been used throughout.

 

5. For every of the three treatments number of explants should be provided and number of replications. No valid conclusion can be made without at least two replications.

- We have revised the relevant sections to clearly state the number of explants used for each treatment. Specifically, for Table 1, each treatment consisted of 10 seed explants. For Table 2, 15 calli were used for each treatment. For Table 3, the number of explants are now added in the table.

 

6. For Table 1. as there was no callus regeneration as there was no callus regeneration for control I suggest that you should for Callus diameter, Fresh weight and Dry weight instead of 0.00±0.00 write "-" and recalculate statistics.

- We have replaced the values of 0.00 ± 0.00 with “–” for the control treatment in Table 1, where no callus regeneration was observed. We have also recalculated the statistical analysis accordingly and updated the table to reflect these changes (now in Table 3).

 

7. In chapter 3.2. apart of the number of explants and replications you should state what was the composition of media from which explants were subcultured. Number of regenerated shoot or root buds is always expressed in relation to number of explants that produced regenerants and not for the initial number of explants. That should be noted in the legend text. You should stress the inhibitory effect of NAA on organogenesis.

- We clarified that the explants used in this experiment were subcultured from MS medium supplemented with 4.0 mg/L 2,4-D. We added information about the number of explants used in the Table 2. Noting that the numbers of tested calli per treatment were varied due to contamination. The regeneration data in Table 2 (now in Table 3) are expressed in relation to the number of explants that produced regenerants. A note has been added to the figure legend to reflect this clarification. We have also included a text stating the inhibitory effect of NAA on regeneration in the revised text at page 6 line 180-182.

 

8. Chapter 3.3. in line 145 instead "2,4-D (0, 2.0, 4.0 mg/L) and BA (0, 1.0 mg/L) " it should be "2,4-D (0, 2.0, 4.0 mg/L) alone or with BA (0, 1.0 mg/L) ".

- We have modified the text accordingly.

Round 2

Reviewer 2 Report

Comments and Suggestions for Authors

The revised manuscript has been improved. However, the key problem remains unsolved. BA significantly affects the ‌plant regeneration rate‌: regeneration rates induced by ‌0, 1.0, 2.0, 4.0, and 5.0 mg/L BA‌ are ‌18.75%, 30.00%, 50.00%, 44.44%, and 12.50%‌, respectively. However, ‌3.0 mg/L BA yields a 0% regeneration rate‌, which is clearly illogical (Table 3). 

Although the authors provided explanations in the revised manuscript, these results remain unexpected and contrary to established scientific principles. Additionally, the number of biological replicates used in this experimental setup is insufficient to meet statistical requirements (Line 203-211; Table 3).

If the author insists that this unexpected result is scientifically valid and reliable, they should investigate the underlying mechanisms behind it rather than merely report this preliminary phenomenon; otherwise, these incomplete findings could easily mislead readers.