Glyphosate-resistant crops developed by the CP4-EPSPS gene from Agrobacterium have been planted on a massive scale globally, which benefits from the high efficiency and broad spectrum of glyphosate in weed control. Some glyphosate-resistant (GR) genes from microbes have been reported, which might raise biosafety concerns. Most of them were obtained through a hygromycin-HPT transformation system. Here we reported the plant source with 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) gene from goosegrass endowed rice with high resistance to glyphosate. The integrations and inheritability of the transgenes in the rice genome were investigated within two generations. The EiEPSPS transgenic plants displayed similar growth and development to wild type under no glyphosate selection pressure but better reproductive performance under lower glyphosate selection pressure. Furthermore, we reconstructed a binary vector pCEiEPSPS and established the whole stage glyphosate selection using the vector. The Glyphosate-pCEiEPSPS selection system showed a significantly higher transformation efficiency compared with the hygromycin-HPT transformation system. Our results provided a promising alternative gene resource to the development of GR plants and also extended the plant transformation toolbox.

Herbicides are pivotal for modern agriculture, but challenges like weed resistance and crop rotation issues necessitate the development of herbicide-resistant genetic resources. This study focused on acetolactate synthase (ALS), a key enzyme targeted by numerous herbicides. Using CRISPR/Cas9-mediated non-homologous end joining (NHEJ) and combining with whole-stage selection, we induced mutations in the OsALS gene of indica rice and identified novel in-frame mutations at the P171 and S627 sites, respectively. Among them, one mutation at the P171 site, the triple mutation P171T/R172G/M174L (ALS-TM) conferred broad-spectrum resistance to Imidazolinones Pyrimidinylthiobenzoates Sulfonylaminocarbonyltriazolinones and Sulfonylureas herbicides. Compared to wild-type (WT) rice, ALS-TM showed 1153-fold higher resistance to imazethapyr (IMT) than WT based on GR50 values (The herbicide dose causing a 50% reduction in growth), with minimal growth inhibition at 10-fold IMT treatment. Enzymatic assays revealed that ALS-TM maintained catalytic efficiency while reducing herbicide binding, which validated the resistance at the protein level. Field trials showed that ALS-TM mutant retained normal agronomic traits even after IMT spraying, indicating no yield penalty. Additionally, ALS mutations were validated as effective transgenic selection markers, enabling efficient rice transformation under different selection systems. These results demonstrated that ALS-TM could also serve as a reliable tool in basic research, facilitating the selection and identification of transgenic materials in laboratory studies. This study provided a robust method for generating herbicide-resistant rice germplasm and highlighted the potential of CRISPR-mediated NHEJ for creating novel resistant mutations.

Oil palm (Elaeis guineensis Jacq.) is the most efficient oil-producing crop globally, yet progress in its research has been hampered by the lack of effective genetic transformation systems. The EPSPS gene, encoding 5-enolpyruvylshikimate-3-phosphate synthase, has been used as a transgenic selection marker in various crops, including rice and soybean. This study evaluated the EPSPS/glyphosate selection system for oil palm transformation. We constructed a binary expression vector, pCGlyDESCLI-C, containing the TIPS-EiEPSPS selection marker from goosegrass and the mScarlet-I red fluorescent reporter gene. This vector was introduced into oil palm embryonic callus (EC) via Agrobacterium-mediated transformation. After optimizing the transformation steps, positive calli were obtained, and integration of the foreign gene into the oil palm genome was confirmed through molecular analysis. Notably, the selection efficiency of the EPSPS/glyphosate selection system exceeded that of the traditional hpt/hygromycin selection system, demonstrating its advantages. Our findings support the effectiveness of the TIPS-EiEPSPS/glyphosate selection system for oil palm genetic transformation, marking its first application in this species and offering a valuable tool for advancing research on this economically significant crop.

Palmitoleic acid (C16:1Δ9) has high nutritional and industrial value, but enriching it in rice is hindered by low endogenous levels and poorly underst