Development of a multi‐resistance and high‐yield rice variety using multigene transformation and gene editing

Approximately one-third of the total annual food production in the world is lost owing to pests, diseases and weeds. Therefore, the challenges posed by crop losses and population growth have emphasized the need for better breeding techniques (FAO et al., 2023). Practical experience has demonstrated that the utilization of existing resistance genes to breed and cultivate herbicide- and pest-resistant rice cultivars is the most economical, safe and effective method for preventing and controlling weeds and pests (Zhang, 2007). The incorporation of a single or few resistance genes during rice breeding is no longer adequate for in-demand production. In addition, hybridization and backcrossing involve a long breeding cycle, and the issue of linkage drag may occur. The multi-gene transformation strategy can be utilized for the rapid and accurate incorporation of multiple resistance genes into rice (Zhu et al., 2017). The fact that a trade-off between growth and defence generally exists in crops is universally accepted. Therefore, the overexpression of multi-resistance genes in rice causes considerable changes to the agronomic traits of crops, especially yield. The crop yield is positively correlated with the flowering stage within a certain range. For example, editing Ehd1 or overexpressing Ghd7 to appropriately extend the basic vegetative growth period of rice may be possible, and ultimately promote rice yield and quality (Eshed and Lippman, 2019; Zhou et al., 2023). This strategy is more effective for rice varieties with shorter growth periods. For some rice varieties with longer growth periods, we can use editing other yield related genes (grain type or grain weight), such as GS3 and GS5 (Ren et al., 2023). The herbicide resistance gene I. variabilis-EPSPS*, brown planthopper resistance genes Bph14* and OsLecRK1*, borer resistance gene Cry1C*, bacterial blight resistance gene Xa23* and blast resistance gene Pi9* are resistance gene resources in rice that have been extensively validated for use in rice breeding (Appendix S1). In our work, a highly efficient transgene system was used to construct an assembly of six resistance genes (about 26 Kb) mentioned earlier (380-6G) and Ehd1 CRISPR/Cas9 editing vector (Cas9-Ehd1) (Figure 1a; Appendix S2 and S3). We expect to extend the basic vegetative growth period of multi-resistance gene transgenic rice by editing Ehd1 to improve the agronomic traits (especially yield) and obtain a new multi-resistance and high-yield rice germplasm resource, termed MR&HY rice. We transformed two vectors, 380-6G and Cas9-Ehd1, into ZH11 rice varieties using Agrobacterium-mediated dual-strain transformation and screened using glyphosate and hygromycin simultaneously. When T0 transgenic plants were obtained, single-copy families with correct expression of resistance genes and correct editing of Ehd1 were screened out. Subsequently, the lines with homozygous single copy of multi-resistance genes and the Cas9-free family with Ehd1 mutation were screened in the T1 generation for further study (Figures 1b and S2). According to the process shown in Figure 1b, we obtained MR&HY-3 and MR&HY-5 with six resistance genes single copy homozygous, Ehd1-editing and Cas9-free. In MR&HY-3 and MR&HY-5 T2 generation, all six resistance genes were expressed normally and Ehd1 was mutated as expected (Figures 1c–g and S2). The MR&HY rice not only possessed resistance to herbicide (glyphosate), pests (brown planthopper and stem borer) and diseases (bacterial blight and blast), but also exhibited a considerable increase in yield (Figure 1h–m). In addition, the resistance rice transformed with multiple resistance genes had substantially better resistance to specific diseases and pests than single gene effects. In field experiments, although the MR&HY rice was cultivated without pesticide throughout the entire growth period, as compared with the ZH11 with pesticide, the yield increased by 20%; compared with ZH11 without pesticide throughout the entire growth period, the yield had almost increased by three times (Figure 1p,q). Although the growth period of MR&HY rice was extended by approximately 13 days, its yield and even quality had improved (Li et al., 2022a). Improving yield and disease resistance are important indicators of breeding, but the signals regulating yield and disease resistance often contrast each other. Currently, it is reported that only a few genes can simultaneously promote growth and resistance (Li et al., 2020, 2022b; Sun et al., 2019). In this study, we used multigene transformation techniques to enhance rice resistance against diseases, pests and herbicides and simultaneously performed CRISPR/Cas9 gene editing to adjust the heading stage of rice. This approach successfully balanced the tradeoff between rice growth and defence, and created rice germplasm with resistance to diseases, pests and weeds as well as increased yield. The multiple resistance of the resultant rice germplasm decreases the use of pesticide, which lowers rice production cost, reduces environmental pollution, enhances rice quality and renders the rice safe for human consumption. Its high yield effectively increases grain production, thereby addressing the global food crisis that has become increasingly severe with continuous population growth. This research was supported by the Biological Breeding-Major Projects (2023ZD04074), the Hubei Province Outstanding Youth Project (2024AFA088) and the Science and Technology Major Program of Hubei Province (2022ABA001 and 2021ABA011). The authors declare no competing interests. A.Q.Y., Y.J.L., L.Z. and C.Y.L. designed the research; C.Y.L., Z.H.Z., X.Z.X., C.X.L., C.H.L., E.-L. and J.Y.W. performed the research; C.Y.L, H.C., W.Z. and B.W. analysed the data; C.Y.L., L.Z., Y.J.L. and A.Q.Y. wrote the paper. The data that support the findings of this study are available in TIGR at http://rice.uga.edu/analyses_search_locus.shtml. These data were derived from the following resources available in the public domain: – LOC_Os03g63150, http://rice.uga.edu/cgi-bin/sequence_display.cgi?orf=LOC_Os03g63150.2 – LOC_Os04g12540, http://rice.uga.edu/cgi-bin/sequence_display.cgi?orf=LOC_Os04g12540.1 – LOC_Os11g37620, http://rice.uga.edu/cgi-bin/sequence_display.cgi?orf=LOC_Os11g37620.1 – LOC_Os06g17900, http://rice.uga.edu/cgi-bin/sequence_display.cgi?orf=LOC_Os06g17900.1. Appendix S1 Six resistance gene sequences optimized for codons. Appendix S2 The process of obtaining Multi-resistance and high-yield rice. Appendix S3 Primers used in this study. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.