Protocol for ethyl methanesulphonate (EMS) mutagenesis application in rice [version 3; peer review: 2 approved]

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 (0.0%, 0.5%, 1.0%, and 2.0%), and four EMS exposure periods (6, 12, 24, and 48 hours). Germination rate, plumula and radicle length, seedling survival, LD50 (Lethal Dose) determination, shoot length, root length and fresh seedling weight were evaluated. Results: Results showed that a 12-hour presoaking duration, 0.5% EMS dose, and six hours of EMS exposure were the best practices for the optimum number of mutant plants. 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: 0.5% dose EMS and six hours, (3) Final washing: six hours, (4) Drying: 72 hours at 38°C. A user-friendly protocol has been presented for utilization by researchers.