Effect of γ-Irradiation on the Growth and Yield Response of Three Varieties of Pea (Pisum spp.)

Comments 1: The author should stress the novelty of this research since many similar researcher were done?

Response 1: Thank you for this comment. Our research provides a fundamental description of the M1 generation of irradiated pea plants. While previous studies have typically focused on single varieties, our study uniquely examines three different pea varieties (including one Greek with no previous studies reported), providing a broader range of analysis and comparison. Moreover, despite being the first generation, our study has already detected significant differences in traits among the irradiated plants which serve as a foundation for future breeding programs. By combining our results with other “omics” technologies, there is significant potential for enhancing yield parameters and other desirable traits in pea varieties. Our study not only expands the existing knowledge on pea genetic variability but also provides practical insights and tools for future breeding programs aimed at meeting the increasing consumer demand for non-GMO, plant-based products. These points are also highlighted further in the manuscript.

Comments 2: In the abstract, the author should stress the importance why you do this research work, what is the problem and how do you tackle the problem with your research? Otherwise, what are the points to do these research?

Response 2: Thank you for your suggestion. The points you are mentioning have been highlighted in the abstract.

Comments 3: For the abstract, the first letter should be capital for each key word?

Response 3: Thank you for your observation. We have revised the abstract.

Comments 4:  There are no references cited from 2023-2024.

Response 4: Thank you for your comment. We have reviewed the latest literature and have added relevant references.

Comments 1: Seeds that have experienced stress (such as irradiation) will not perform the same as unstressed seeds, even if the genetics are identical. The observed changes in plant height for ‘Early Onward’ and ‘Rondo’ is an example of this, which is likely stress induced.

Response 1: Thank you for your insightful comment. We acknowledge that seeds subjected to stress, such as irradiation, will exhibit different performance compared to unstressed seeds, even with identical genetics. The observed changes in plant height for ‘Early Onward’ and ‘Rondo’ are indeed likely induced by stress, which aligns with the primary objective of our research. Our study aims to explore the effects of gamma irradiation on pea plants to understand how induced stress can contribute to genetic variability and improvement in qualitative and quantitative traits. By documenting these stress-induced changes, we can identify beneficial traits that may not be present or detectable under normal, unstressed conditions. This approach allows us to leverage induced mutations to enhance the genetic diversity and performance of pea varieties in future breeding programs. Moreover, there are also other studies for different species trying to explore genetic variability induced by gamma radiation. For instance, Ramazan et al., 2016 studied the effects of gamma radiation on the physiological responses of the M1 sainfoin plants and showed that radiation stress in sainfoin results in changes in several physiological parameters. They also highlighted that their study provided basic information on the impact of gamma radiation on the physiological responses of sainfoin and its radiosensitivity and that these findings will be useful in the development of a mutation breeding program of sainfoin.

(http://dx.doi.org/10.1016/j.apradiso.2016.09.005)

Comments 2: The initial generation needs to be observed in further generations to make reasonable inferences about genetic changes. Moreover, many genetic changes will be of a recessive type that will only manifest when they are fixed through selfing in later generations.

Response 2: Thank you for the comment. Truly genetic changes can generally be observed and studied in later generations. In our experiment we focused our study on morphological rather than genome characteristics and molecular mechanisms. We tried to monitor not only the overall performance of the M1 generation of different pea varieties during their biological cycle but also the performance of individual plants. Then we will further examine them into M2 generation and study the appearance of the desired characteristic and then investigate the cause of this new desired characteristic at genome level. However, there are studies at genome level focusing on γ-Radiation-Induced DNA Polymorphisms in the M1 Population (Zhang et al 2020), in this work, they analyzed the genomic diversity of the japonica rice cultivar Gaogengnuo, through genome resequencing, they have described the distribution of SNPs, InDels, structural variations (SVs), and copy number variants (CNVs) and they provided novel insights for γ-radiation mutation research from the study of the very first generation. 

Comments 3: The experimental design seems sketchy to me because the treatment will not be uniform across plants in a block since mutation is random by definition so each plant will have experienced unique random mutations not shared by any other plant in the block

Response 3: Thank you for your comment. There are many studies in which the same experimental design is being used for M1 generation of mutagenized plants (i.e Girija and Dhanavel 2013; Sharma et al. 2020). Moreover, an experiment that was conducted very recently at the Botanic Garden of the Botany and Microbiology Department, Suez Canal University, regarding the effects of gamma irradiation on fenugreek plants used a completely randomized block design (CRBD) (El-Gazzar et al. 2023).

Comments 4: The authors should follow up on their mutagenesis experiments and identify individual lines presenting unique mutations of importance to breeding. This can only be accomplished in later generations.

Response 4: Thank you for your comment. In this study, our primary objective was to assess the yield traits and morphological characteristics in the M1 generation of irradiated pea plants. Given the significant variability induced by gamma irradiation, it was essential to first establish a comprehensive baseline of these traits such as documented also by Bhattu et al., 2022 and Chimdi et al., 2022 did. (https://www.ijtsrd.com/other-scientific-research-byarea/other/49327/induced-mutagenesis-of-flowering-phenology-and-yield-in-m1-generation-of-bambara-groundnut-vigna-subterrnea-l-verdc/grace-o-chimdi, https://www.ijair.org/index.php/issues?view=publication&task=show&id=1435) and highlight the importance of the careful identification of promising plants (genotypes) at this early stage.

4. Response to Comments on the Quality of English Language

Point 1:

Response 1:    (in red)

5. Additional clarifications

[Here, mention any other clarifications you would like to provide to the journal editor/reviewer.]

Response 1: Thank you for your valuable feedback. In this study, our primary objective was to assess the yield traits and morphological characteristics in the M1 generation of irradiated pea plants. Given the significant variability induced by gamma irradiation, it was essential to first establish a comprehensive baseline of these traits as documented also by Bhattu et al., 2022 and Chimdi et al., 2022. (https://www.ijtsrd.com/other-scientific-research-byarea/other/49327/induced-mutagenesis-of-flowering-phenology-and-yield-in-m1-generation-of-bambara-groundnut-vigna-subterrnea-l-verdc/grace-o-chimdi, https://www.ijair.org/index.php/issues?view=publication&task=show&id=1435)

The findings from this initial phase provide crucial insights that will guide our future research. We fully intend to incorporate further studies in subsequent generations (M2 and beyond).

Comments 2: There are many reports on γ-irradiation on growth and yield response, please include more relevant studies.

Response 2: Thank you for your suggestion. More relevant studies were added to the manuscript.

4. Response to Comments on the Quality of English Language

Point 1:

Response 1:    (in red)

5. Additional clarifications

[Here, mention any other clarifications you would like to provide to the journal editor/reviewer.]

Comments 1: The research work concerning γ-irradiation on the ground is well described in your manuscript; however, the methodology section shows the results. Please shift the results from the methods section to the result section. The abstract must be more precise, and the same is applicable throughout the manuscript. There are several irradiation-based mutation studies available in various crops, including groundnut; hence, authors need to describe how your study is different from other studies and what kind of novel hypothetical experimental validation was done with your research.

Response 1: Thank you for this comment. Our research offers a detailed analysis of the M1 generation of irradiated pea plants. Unlike previous studies that typically focus on single varieties, we examine three distinct pea varieties, including a Greek variety with no prior studies, offering a wider scope for comparison. There aren’t many studies that emphasize on the M1 generation for pea varieties. Furthermore it was a field experiment with the seeds sown directly to the soilthus the plants were tested under natural environmental conditions for their whole cycle, and our observations were based on their full potential without external modifications. Even in this initial generation, we've identified significant trait differences among the irradiated plants, establishing a basis for future breeding programs. Integrating our findings with other “omics” technologies holds great promise for improving yield and other desirable traits in pea varieties. This study not only broadens the understanding of pea genetic variability but also provides valuable insights and tools for breeding programs, addressing the growing consumer demand for non-GMO, plant-based products. These points are also clarified better in our manuscript.

Comments 2: Please mention as Pisum spp. hence you are using more than one variety in title and wherever required.

Response 2: Thank you for your suggestion. We have updated the title and the manuscript.

Comments 3: Please rewrite as "In this study, three pea varieties (Pisum sativum), including one field pea variety ‘Dodoni’, and two varieties of garden peas ‘Early Onward’ and ‘Rondo’, were irradiated with 100 Gy gamma rays"

Response 3: Thank you for your suggestion. We have revised the text accordingly.

Comments 4: Please write mutagens as physical and chemical modes or something similar instead of writing as natural and chemical means.

Response 4: Thank you for your comment. We have updated the manuscript accordingly.

Comments 5: Please mention as Physical mutations caused by physical mutagens. Please check this article: Kodym A, Afza R. Physical and chemical mutagenesis. Methods Mol Biol. 2003;236:189-204. doi: 10.1385/1-59259-413-1:189. PMID: 14501066

Response 5: Thank you for your comment. We have adjusted the manuscript accordingly.

Comments 6: Please mention the wave length of γ rays.

Response 6: Thank you for your suggestion. We have included the wavelength of γ rays in the revised manuscript.

Comments 7: Please write as, it has a mutagenic effect on DNA through breaking a double strand or a single strand through introducing lesions at line 62

Response 7: Thank you for the comment. The correction is made in the manuscript.

Comments 8: Does fresh bean consumption not have antigenic epitopes that may cause allergies, etc.? Please describe some allergic properties as well.

Response 8: Thank you for your comment. We have elaborated on these points in the revised manuscript.

Comments 9:  Why have you used the term pre-breeding here? Have you used any wild relative introgression lines? If you have a variety derived from a wild cross, then you could discuss aspects of pre-breeding; if not, please mention that this methodology can be used for marker-assisted selections and for further marker-assisted breeding for the improvement of yield parameters, etc

Response 9: Thank you for your comment. Indeed, we acknowledge the oversight. We have now corrected this point and made the necessary adjustments in the manuscript.

Comments 10: Please change 59,19% to 59.19% at line 124

Response 10: Thank you for pointing that out. We have updated the text.

Comments 11: Have you applied any bio-pesticide during the experiment? If not, have you done the data normalization for the data of missing seeds or plants? Please check this article: https://plantmethods.biomedcentral.com/articles/10.1186/s13007-020-00653-x

Response 11: Thank you for this comment. We recognize the importance of data normalization in many cases, but we believe it was not necessary for our work because the values of our variables are already on a similar scale without significant variations.

Comments 12: Please add the field pictures if you have from these devastating effects.

Response 12: Thank you for your suggestion. Unfortunately, we do not have any pictures available to include.

Comments 13:  Please clarify: In Figure 3, HSD test-derived letters are missing to observe whether these traits show differences among the varieties or mutations.

Response 13: Thank you for pointing this out. In this figure where the letters of multiple comparisons are absent, there were no statistically significant differences observed between the treatments. Moreover, we clarified this note in the manuscript.

Comments 14: There must be some meaning to the negative correlation as well. For instance, pods per plant and seeds per pod were negatively correlated in mutant plants; however, a weak positive correlation was found in the control plant. Please slightly discuss this kind of comparison with appropriate references.

Response 14: Thank you for the comment. We proceeded to highlight and discuss the comparisons more in this paragraph.

Comments 15: Please rewrite: Seeds exposed to 200 Gy exhibited notable increases in shoot fresh weight (123.3%), root fresh weight (69.8%), number 292 of pods (46%), 100-seed weight (47.7%), and total yield (65.6%) [28]. as it derived from the following article: Rephrasing is important although you have cited from other article:

https://www.researchgate.net/publication/377076072_Effects_of_Different_Levels_of_Gamma_Radiation_on_Growth_and_Yield_Characteristics_of_Groundnut

Response 15: Thank you for this comment. We have revised the text accordingly.

Comments 16: Please specify instead of writing a lot to be investigated. Based on the current findings, what kind of feasible future perspectives can be included?

Response 16: Thank you for the comment. Based on our current findings, we plan to focus our future research on specific areas in the M2 and later generations of “Dodoni” variety. These include identifying plants with high productivity but also with enhanced seed quality traits such as protein content, mineral content(i.e Zn and Fe fortified plats), and total antioxidant capacity. Additionally, we will study the plants' tolerance to biotic and abiotic stresses. These targeted studies aim to enhance our understanding and improvement of pea varieties in subsequent generations. We also highlighted these points in our conclusions.

4. Response to Comments on the Quality of English Language

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Response 1:    (in red)

5. Additional clarifications

[Here, mention any other clarifications you would like to provide to the journal editor/reviewer.]