Periodic Reporting for period 1 - HerbaRice (Herbicide Resistant Rice Development For European Continental Project)
Okres sprawozdawczy: 2020-11-16 do 2022-11-15
Rice is a staple food for more than half of the world's population. The rice farming system has problems such as weed management, disease control, abiotic stress tolerance, etc. The issue of weed and weedy rice (red rice) is one of the most important problems in terms of reducing both yield and quality. Chemical application as herbicide is a common technique to control weeds, but herbicides are losing their effectiveness due to various reasons day by day. Chemical weed control is a laborious and costly business, in some cases it is repeated several times, increasing the negative effects on the environment as well as costly. On the other hand, since weedy rice is a close relative of cultivated rice, its chemical control is very difficult. Two herbicide resistance systems, Clearfield and Provisia, were developed so far for the use of rice farmers. After Clearfield was used, it started to lose its effectiveness due to reasons such as gene flow and misuse. The second one, Provisia, entered the farming system in 2018 but is not yet available in Europe. With the refrain from these systems will be damaged over time, it is important to make new resilience resources ready for use by European countries. We aim to develop a new herbicide resistance source in rice and put it at the disposal of European farmers. The developed resistance source will not only be used directly but also EU countries will be able to improve their breeding programs according to their needs via this base material.
In order to develop a rice base material resistant to the clethodim herbicide, firstly, mutation application was carried out. Then low dose resistance study, high-dose resistance study, and determination of mutation points study were carried out. In the mutation generation study, the standard protocol for EMS mutagenesis application in rice was developed. It is useful to obtain a sufficient number of mutant plants instead of working with a severe mutation in a few plants. This application protocol contains presoaking for 12 hours, 0.5% dose EMS application for six hours, final washing for six hours, and dry for 72 hours. According to the created protocol, the chemical mutation was applied to 5 rice varieties and a low-dose clethodim resistance study was started. Chemical mutation applied seeds 10,000 seeds each of 5 rice varieties, were planted in the plant growth chamber. It was known that clethodim herbicide can be killed the rice, however, the doses needed to be standardized. Therefore, a dose-response study was carried out with clethodim in rice. As a result of the study, the dose that killed 90% (ED90) of the rice was determined in the range of 70-80 g ai ha-1. Clethodim 1x was utilized 150 gr ai ha-1 in the experiments. In the low-dose experiment, herbicide was applied at 1/2x doses on the m1 plants. Clethodim was applied when the plants were at the 3-4 leaf stage in the spray chamber. Visual injury ratings were conducted 28 days after treatment (DAT) based on 0=not injury and 100=plant killed. A total of 30 plants survived. The frequency of survival was found as 0.06%. Then, the mutation determination process started. Clethodim herbicide binding point was on the ACCase carboxtransferase region on the 5th chromosome in rice. Five primers were designed to cut this region. With this primer, the target region was cut and amplified by PCR and sequenced by the Sanger method. Although the plants were found to be resistant, it was observed that the original sequence was not changed in the target region. In the high dose resistance study, the mutation was applied to 10000 seeds of 5 varieties according to the determined protocol. First-year mutant rice was planted and generation advancement was achieved from M1 to M2 plant. The M2 generation was grown in the field and harvested for herbicide scanning. The second-year M2 mutant rice plant was grown up 3-4 leaf stage on the field. Double doses of clethodim were sprayed on mutant rice varieties two times when first at the 3-4 leaf stage and second 14 days later the first application, and 750 surviving plants were selected. The clethodim resistance was tested in the greenhouse at fully controlled conditions, such as an automatic herbicide sprayer and arranged climatic conditions, and 3 out of 750 M3 plant genotypes survived high-dose clethodim treatment. At the next stage observed that these 3 genotypes had no resistance exceeding ½ × dose detected in the herbicide dose-response study and they had no mutation in the ACCase gene region according to the DNA sequence study. As a result, 30 low-dose resistant genotypes were developed, but no material was determined at high-dose resistance for commercial resistance. The dynamic structure of the study allows us to test the newly produced material again until the study is terminated. High-dose resistance study will be re-implemented to achieve success.
The state-of-the-art of project is to develop Non-GMO resistant clethodim herbicide for rice. Up to now, 30 low-dose clethodim-resistant plants selected but no one high-dose resistant plant was developed. A dynamic structure has been created in the study, that is, the mutant material used in the experiment is constantly supported. This gives us the opportunity to test the material again until the study is terminated. Although high-dose resistance could not be developed from the data obtained at the end of the two-year study, this work package will be re-implemented and the possibility of achieving success will be increased. The project aimed to develop a patentable technology. The patent application requires the confidentiality of some information, therefore, no scientific or nonscientific article has been published directly on the subject. On the other hand to make a standardization use of clethodim herbicide some results were published in scientific journals and congresses. The publications included a series of studies such as clethodim and dose-response experiments on rice, red rice, and some weeds, application time, and application method. The website was released and updates were made to increase the visibility of the project. The effects of the project are to obtain scientific literature, standardize the use of the chemical, and raise awareness subject so far. The main effects of the project will be seen in the years after the project is completed. Releasing the patented product is closely related to rice seed companies, herbicide companies, and end-user farmers. This product, which is clethodim herbicide-resistant rice seed, can be used directly by sowing the base material, or it can be utilized by the different countries' own seeds by gene transfer via classical breeding. The product was developed primarily for the use of the European rice farmer. Starting from here, it can be predicted to spread to other countries and continents.