Gene flow and spontaneous mutations are responsible for imidazolinone herbicide-resistant weedy rice (Oryza sativa L.).

For more than two decades, weedy rice (Oryza sativa L.) has been controlled in rice fields by using imidazolinone (IMI) herbicide-resistant rice technology (Clearfield®). Outcrossing in weedy rice populations and spontaneous mutations are potential problems with herbicide-resistant crop management technologies, such as the IMI-resistant rice. The aim of this study was to characterize the mechanism of IMI herbicide resistance in weedy rice through dose-response bioassay study and evaluating amino acid substitutions in acetolactate synthase (ALS) protein. A total of 118 suspected IMI-resistant weedy rice samples, which survived in the field after an IMI herbicide application, were collected at harvest time from Türkiye in 2020 and 2021. Single-dose imazamox application experiment revealed that 38 plants survived herbicide treatment. The imazamox resistance of the surviving plants was confirmed by dose-response experiment. ALS gene region underwent a sanger DNA partial sequencing. No substitution was found in 10 samples, however, amino acid substitutions were found in 26 samples with S563N, one sample with S653T, and one sample with E630D. The S653N point is the same substitution point that serves as the origin of resistance for the Clearfield® rice varieties that are commonly cultivated in the region. It has been hypothesized that the gene flow from IMI-resistant rice may be the cause of resistance in the IMI resistant weedy rice samples with S653N. The other substitution, S653T, were considered spontaneous mutation to IMI resistance. Interestingly, the S653T mutation was detected for the first time in weedy rice. The mechanism of resistance of 10 resistant weedy rice was not confirmed in this study, however, it may be a non-target resistance or another mutation point in target site, but evidently, they did not acquire resistance by gene flow from IMI-resistant rice. It has been concluded that the effectiveness of IMI-resistant rice technology in controlling weedy rice has drastically decreased due to possible gene flow, spontaneous mutation and non-target resistance. In addition to cultural controls like clean seed, clean machinery and crop rotation, other herbicide-tolerant rice systems such as Provisia® and Roxy-RPS® rice are needed to create a diverse weedy rice management ensemble available for rice production and move towards sustainable rice farming.

Protocol for ethyl methanesulphonate (EMS) mutagenesis application in rice.

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, LD 50 (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.