Slight variations appeared in terms the most suitable combination of factors. However, results were obtained that could be used to make a standard protocol. Presoaking is an important stage for the EMS solution to diffuse into the seed and optimum presoaking duration is expressed as the presoak duration when the seed reaches full saturation.
The results illustrated that the rice seed reached full saturation after 24 hours presoaking. However, when the presoaking duration was evaluated on its own and with other conditions, it was determined that the hour period was the most suitable time for EMS application.
In addition, EMS exposure periods of more than six hours might be damaging to the seed. The seeds might tolerate a long exposure period of 12 hours or 24 hours. However, 48 hours of application caused the seed to irreversibly lose its germination ability. EMS application doses of 0. The LD 50 was determined as 0. In this study, 12 hours presoaking duration, a six-hours EMS exposure period, and 0.
The EMS application protocol might be successfully utilized in rice mutation research. The most suitable EMS application practice was determined to be 12 hours presoaking, 0. The protocol includes the following: 1 Presoaking: 12 hours, 2 EMS application: 0. In addition, the protocol sheets are presented as a user-friendly protocol as Extended data Unan, b. Zenodo: Dataset related paper "protocol for ems mutagenesis application in rice".
Zenodo: Factsheet related paper "protocol for ems mutagenesis application in rice". Data are available under the terms of the Creative Commons Attribution 4. Competing Interests: No competing interests were disclosed. Is the work clearly and accurately presented and does it cite the current literature?
Is the study design appropriate and does the work have academic merit? Are sufficient details of methods and analysis provided to allow replication by others? If applicable, is the statistical analysis and its interpretation appropriate? Are all the source data underlying the results available to ensure full reproducibility?
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ALL Metrics. Get PDF. Get XML. How to cite this article. NOTE: it is important to ensure the information in square brackets after the title is included in all citations of this article. Close Copy Citation Details. Revised Protocol for ethyl methanesulphonate EMS mutagenesis application in rice [version 2; peer review: 1 approved].
Abstract Background: Non-transgenic chemical mutagen application, particularly ethyl methanesulfonate EMS , is an important tool to create mutations and gain a new genetic makeup for plants. It is useful to obtain a sufficient number of mutant plants instead of working with a severe mutation in a few plants.
EMS dose and exposure period have been previously studied in several crops; however, EMS used to create point mutations in presoaked rice seeds has not been sufficiently studied and there is no standard protocol for such treatment. The aim of this study is to establish a standard protocol for EMS mutagenesis application in rice.
Methods: Two studies were conducted to evaluate the effect of four durations of rice seed presoaking 0, 6, 12, and 24 hours , four EMS concentration doses 0. Germination rate, plumula and radicle length, seedling survival, LD 50 Lethal Dose determination, shoot length, root length and fresh seedling weight were evaluated. Results: Results showed that a hour presoaking duration, 0. Conclusions: In light of both this study and the literature, a standard application protocol was established.
This application protocol, detailed in this article, contains the following guidelines: 1 Presoaking: 12 hours, 2 EMS application: 0. A user-friendly protocol has been presented for utilization by researchers. Keywords EMS, dose, mutagenesis, protocol, rice.
Corresponding Author s. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Methods Materials Osmancik is a Japonica type Turkish rice variety. EMS mutagenesis The experiment was carried out using a randomized parcel design with three replications for the germination experiment and four replications for the seedling experiment, and each replication used seeds under a fume hood in a phytotron growth chamber.
Experiment 1: Germination experiment The experiment was carried out using a randomized block design with three replications for germination. Experiment 2: Seedling experiment The experiment was carried out using a randomized block design with four replications. Factsheet and flowchart of protocol for EMS mutagenesis application in rice A one-page user protocol might be useful in laboratory studies.
Statistical analysis Three-way analysis of variance was used in order to detect any statistically significant differences between presoaking duration, EMS dose, and EMS exposure period.
Results Imbibition rate The imbibition rate was calculated for Osmancik rice at the start of the experiment. Figure 1. Water uptake measurement compared to imbibition time interval at six hours in Osmancik rice variety. Figure 2. Water uptake measurement compared to imbibition time hourly in Osmancik rice variety.
Germination experiment Germination is a crucial factor for EMS mutagenesis experiments. Table 1. Table 2. Table 3. Seedling experiment Germinated seeds might lose their vitality over time at the seedling stage. Table 4. Table 5. Effect of EMS application dose, EMS exposure period and presoaking duration on shoot length in rice seedling experiment mm.
Table 6. Effect of EMS application dose, EMS exposure period and presoaking duration on root length in rice seedling experiment cm. Table 7. Effect of EMS application dose, EMS exposure period and presoaking duration on fresh seedling weight in rice seedling experiment mg.
Table 8. Discussion The experimental results of both the germination experiment and the seedling experiment revealed that the presoaking duration, EMS dose, EMS exposure period, and their interactions were significant. Conclusion The most suitable EMS application practice was determined to be 12 hours presoaking, 0.
Data availability Underlying data Zenodo: Dataset related paper "protocol for ems mutagenesis application in rice".
Extended data Zenodo: Factsheet related paper "protocol for ems mutagenesis application in rice". This project contains the following extended data: - Factsheet of Protocol for EMS mutagenesis application in rice.
Proceeding of 37th Rice Technical Working Group. Upadhyaya, Ed. Springer, New York, ; — Weed Sci. Sci Agric. Crop Sci. Access date is Germination and duration of competition for integrated weed management in water-seeded rice. Weed Res. International Rice Research Institute, Philippines. Reference Source Karger G: Contribution to the collective treatment of pharmacological series tests.
Trends Biosci. Ekin Journal of Crop Breeding and Genetics. MR for Lethal Dose Determination. Am J Plant Sci. Seed Sci Technol. Turk J Agric For. Comments on this article Comments 0. Competing interests No competing interests were disclosed. Article Versions 2 version 2 Revised. This is an open access article distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Manager RIS Sente. Track an article to receive email alerts on any updates to this article. Approved with reservations A number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit. Not approved Fundamental flaws in the paper seriously undermine the findings and conclusions.
How to cite this report:. For data generated from M 2 population, only frequency distribution and the variance around the average of wild-type control plants with exceptional trait values was performed because the data were gathered from individual plants which were not replicated.
Our first step was to evaluate the EMS treatment conditions for optimal mutagenesis. We tested a range of EMS concentrations and soaking times, both of which showed significant effects on germination rate. The germination rate decreases as the soaking time and the EMS concentration increases Figure 1.
Although three soaking times 12, 18, and 24 h are not significantly different, 18 h was used in the experiment as it is within the range that Meksem et al. Statistical analysis of the effect of EMS concentrations on germination for three soaking time 12, 18, and 24 h Figure 2 showed that treatment with 30 mM is not significantly different from the control.
Plot of germination rate for various EMS concentrations mM and soaking time. Error bar represents the standard deviation of three replicates. Chart of the effect of EMS concentration mM on germination rate for three soaking time 12, 18, and 24 h.
Based on these results, we concluded that 60 mM 18 h was the optimum concentration for our bulk EMS mutagenesis of soybean. While higher concentrations of mutagen would produce higher mutation frequency Shah et al.
In contrast, if we use lower concentrations 30 mM of EMS the survival rate of treated plants would be higher but the mutation rate will be low Porch et al. Using the optimized treatment protocol, a total of 1, M 2 individuals were generated from 15, mutagenized seeds. There were two batches of mutagenesis, first batch with 10, mutagenized seeds generated 1, M 2 individuals while second batch have 5, mutagenized seed generated only M 2 individuals.
Batch 1 were planted in the greenhouse while Batch 2 were in the field. The survival of germinated seedling was reduced due damaged cotyledon and to poor development of root and shoot. This type of tissue damage results from EMS mutagenesis and has also been observed pepper Arisha et al.
In addition, some seedlings even grew until early vegetative stage but died before pod set. Previous studies have shown that EMS-induced mutation continues to affect germination and seedling survival of the M 2 generation of peppers Arisha et al. This is likely the result of lethal mutations present in the M 1 population becoming homozygous in the M 2 generation.
This result is similar to that observed for other experiments, suggesting that mutagenesis was sufficient enough to produce a high rate of mutations. Above ground visual phenotypic variation, including changes in leaf morphology, plant architecture, and changes in chlorophyll content, were also measured Table 1.
Statistical analysis was not performed in M 2 generation as each plant is considered as one sample, which cannot be replicated. Statistical analysis of observed phenotypes will be conducted in the next generation planting with proper experimental design and replication, and to assess heritability of mutants. Some of the most striking phenotypic variations observed were altered leaf including, tetra-foliate Figure 4A-b , penta-foliate Figure 4A-c , rough texture Figure 4A-d , and narrow leaf Figure 4A-e as compared to the wild-type JTN Figure 4A-a.
In some instances, tetra- and penta-foliate mutants were observed in just one or two leaves but did not penetrate the whole plant. Leaf phenotypes are important traits since they affect the leaf surface and the ability to perform photosynthesis.
Another leaf phenotype documented is short-petiole leaf Figures 4B-a—c. Abnormal chlorophyll phenotypes were also observed in several M 2 plants including mutants with Figure 4C-a chlorotic leaves, Figure 4C-b chimeric and rough-textured leaves, and Figure 4C-c compact plant with distinct yellow and green leaves. Some of the mutants died, while other mutants that exhibit chimeric yellow leaves survived and produced some pods.
TABLE 1. Incidence of phenotypic variants observed from 6, M 2 individuals. Phenotypic variation on leaf morphology observed in M 2 plants.
A , top Indicate phenotypic alterations observed in leaf morphology as compared to a wild-type JTN, including b tetra-foliate, c penta-foliate d rough-textured leaf and e narrow leaf. B , bottom left Indicate variations observed in petiole length, with a wild-type JTN compared to b,c short-petiole mutant. C , bottom right Represent the changes in chlorophyll content including mutants with a chlorotic leaves, b chimeric and rough-textured leaves, and c compact plant with distinct yellow and green leaves.
Several mutants with altered architecture and growth habit were also identified. Compared to wild-type Figure 5a , these changes include, lack of lateral branching Figure 5b , short internode and bushy type Figure 5c , increased height Figure 5f , unfilled pods Figure 5d and additional lateral branching Figure 6b vs.
This may be a desirable agronomic trait if it proves to be resistant to lodging and produces additional pods per plant. However, there were also mutants that displayed more pods and shorter internodes. Similarly, mutants with reduced height dwarf but similar seed set may be useful for reducing cost of cultivation since fewer seeds would need to be planted per unit area to achieve sufficient number of pods Hwang et al.
Phenotypic variations on plant architecture observed in M 2 mutants. Comparison between wild-type a and an mutant b with additional lateral branching.
Other than leaf and architecture traits, other phenotypes such as sterility, lodging, and shattering Figure 5e were also observed. Some sterile plants did not develop pods at all, while some developing pods went unfilled. There were also mutants that were prone to lodging and shattering. These phenotypes are not favorable variations, but were recorded for future reference as they may provide clues about the genes required for agronomically important traits.
For the mutants where interesting phenotypic variations were identified, they will be selected to be planted with replicates and appropriate experimental design in the next planting for further characterization and evaluation of heritability. Because multiple seeds were planted for each M 2 lines, we were able to harvest seeds from 5, M 2 plants. Individual plant yield was measured and categorized based on the total seed weight per plant Table 2. However, some mutants showed two to seven times higher yield than the controls.
There were three mutant plants JB1-M, , and that yielded more than g. Since single plant yield is strongly affected by plant density, efforts are underway to determine the heritability of these high yielding lines.
TABLE 2. For the mutants that yielded at least 12 g of seeds, we performed NIR analysis Table 3 to determine seed quality traits. The ranges of protein observed It is not clear if it is due to mutagenesis or if the control plants had lower protein content than normal due to location in the field or other variables.
The range of total oil content The individual plants and Figure 7 have highest protein and oil content, respectively. We also observed multiple mutants with decreased linoleic acid and linolenic acid Figure 8.
Figure 9 shows the variation observe for sucrose, raffinose, and stachyose levels. Three mutants , , and show almost double the sucrose content of controls. Similarly, a high number of mutants showed a decrease in raffinose and stachyose. We also found number of mutants with altered amino acid contents Figures 10 , Protein and oil content in mutant seeds.
Histograms left indicate variations in protein and oil content present in the population. Bold type indicates the average of protein and oil content in wild-type plants.
Bar graphs right represent the protein and oil content values for average wild-type JTN, top five and bottom five. Fatty acids profile in mutant seeds. Histograms left indicate variations of oleic acid, linoleic acid, and linolenic acid oil content present in the population.
Bold type indicates the average of oleic acid, linoleic acid and linolenic acid in wild-type plants. Bar graphs right represent the for average wild-type JTN top five mutants and bottom five mutants for each fatty acid. Sugar profile in mutant seeds. Histograms left indicate variations of sucrose, raffinose, and stachyose content present in the population.
Bold type indicates the average of sucrose, raffinose, and stachyose in wild-type plants. Bar graphs right represent the for average wild-type JTN top five mutants and bottom five mutants or each sugar. Variation of essential amino acid content in mutant seeds. Histograms indicate variations in methionine, lysine, valine, phenylalanine, leucine, threonine, tryptophan, isoleucine, and histidine content, respectively.
Bold type indicates the average amino acid content in wild-type JTN Essential amino acid profile of seeds from top and bottom five mutants. The average wild-type JTN, top five and bottom five of methionine, lysine, valine, phenylalanine, leucine, threonine, tryptophan, isoleucine, and histidine are shown.
The success of mutation breeding program depends first and foremost on the effectiveness and efficiency of the mutagen used Arisha et al. However, different mutagens have different effects based on the concentrations and the materials being treated. It is imperative to optimize the concentration of the mutagen before treating the bulk materials to ensure high mutation frequency and at the same time obtain enough viable seeds.
High concentrations of mutagen are detrimental to plants, however, higher concentrations can also give higher mutation frequency Shah et al. If the concentration of mutagen applied is too low, the survival rate of treated plants is higher although there is reduction in frequency of mutation Porch et al. According to Cooper et al. In case of EMS, an increase in concentration can significantly decreases the seed germination rate Talebi et al.
A total of 6, individual M 2 mutants were generated and DNA was collected for each plant. Several phenotypes were observed such as changes in leaf morphology, plant architecture, chlorophyll content, and germination rate. Leaf phenotypes such as narrow leaf, tetra-foliate, penta-foliate, and rough texture are important traits since they affect the leaf surface area.
Leaf is the main site of photosynthesis, so the larger leaf surface area increases the photosynthesis rate as the leaf receives maximized sunlight for photosynthesis. Similar phenotypic changes have been observed with other mutagenesis programs.
It has also been shown to be carcinogenic in mammals. While ethylation of DNA occurs principally at nitrogen positions in the bases, because of the partial SN1 character of the reaction, EMS is also able to produce significant levels of alkylation at oxygens such as the O6 of guanine and in the DNA phosphate groups.
There is also some evidence that EMS can cause base-pair insertions or deletions as well as more extensive intragenic deletions.
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