Efficient genetic transformation and genome editing via an Agrobacterium‐mediated in commercial oat (Avena sativa L.) cultivars

Oat (Avena sativa L.) is a versatile, annual herbaceous species widely cultivated for both grain and forage production. It is renowned for its high biomass yield, strong resistance to abiotic stress, superior nutritional quality, and excellent palatability (Wu et al., 2019; Zhang et al., 2023). Despite these advantages, oat's genetic transformation system lags behind that of major crops like rice, wheat, and maize. Traditionally, oat transformation has relied on biolistic bombardment of callus tissue derived from immature embryos, mature embryos, or leaf bases (Maqbool et al., 2002). Agrobacterium-mediated transformation of oats has been reported only once, with a maximum transformation rate of 12.3% using immature embryos (Gasparis et al., 2008). Although 79% of the putative transgenic plants were confirmed as transgenic, only 27.5% of the T1 generation tested positive (Gasparis et al., 2008). In this study, we optimized the Agrobacterium-mediated transformation protocol, and successfully obtained transgenic plants in multiple commercial oat varieties. Additionally, we developed highly efficient genome editing systems using clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) and CRISPR/Cas12i technologies, marking a significant milestone for functional genomics in oats. One of the critical factors influencing plant transformation is the plant's callus induction ability (Wang et al., 2022). We first examined the effect of seed sterilization time on callus induction efficiency for Longyan 3 seeds. A 1-min treatment with 75% ethanol followed by 5 min in sodium hypochlorite resulted in an 80.73% callus induction rate. However, a 7-min sodium hypochlorite treatment reduced the rate to 55.51%, and a 3-min treatment increased contamination to 45.26% (Table S1). We optimized the callus induction medium, finding that 2.5 mg/L 2,4-D in L3 medium with maltose resulted in the highest callus induction rates: 82.66% for Longyan 3, 76.99% for Qinghaitian, and 76.86% for Forage plus (Tables S2). We then tested the callus induction capacity of mature embryos from 33 oat cultivars, with only eight cultivars exceeding a 70% induction rate (Table S3). We also evaluated the effect of immature embryos at different developmental stages, finding that stage five yielded the highest callus induction rate at 100% (Figure 1A; Table S4). Further, we compared various medium compositions for callus induction and found that 2.5 mg/L 2,4-D in L3 medium with maltose again provided the highest callus induction rates for immature embryos: 92.68% for Longyan 3, 92.59% for Baiyan 7, and 83.97% for Qingyin 3 (Table S5). Finally, we tested immature embryos from eight oat cultivars under optimal conditions, achieving induction rates exceeding 70% (Figure 1B; Table S6). To compare the regeneration efficiency of mature and immature embryos, we selected nine varieties for analysis (Table S7). Our results demonstrated that immature embryos outperform mature embryos in regeneration efficiency (Table S7). For instance, in varieties such as Qingyin 3 and Qinghai 444, the regeneration rate of mature embryos was extremely low (0%), whereas the regeneration rate of immature embryos increased to 65.56% and 41.11%, respectively. Overall, immature embryos serve as superior explants for plant regeneration in oats. Agrobacterium-mediated genetic transformation systems for mature and immature embryos, and two efficient genome editing platforms in oats (A) The immature embryos at different developmental stages and calluses induced from these embryos. 1–6: The grains at 10, 14, 18, 22, 26, and 30 d-post-anthesis, respectively. (B) Callus induction and regeneration from immature embryos. (C) Progress of oat immature embryo genetic transformation system. (D) Progress of the oat immature embryo genetic transformation system using the RUBY reporter module. (E) Progress of oat mature embryo genetic transformation system, quickstix strip detection and histochemical β-glucuronidase (GUS) activity analysis. (F) Sequencing of editing types in CRISPR/Cas9-edited AsPDS plant and phenotype of the Aspds1 mutant. (G) Targeted mutagenesis type of the editing plants T0-1 in multiplex genome editing. (H) Phenotypes, target sites, and Sanger sequencing results for the Asare1 mutant, with phenotypic comparisons between wild type (WT) and Asare1 mutant plants under 0.1 mmol/L NH4NO3 treatment. We established a stable Agrobacterium-mediated genetic transformation system for oat immature embryos under optimized conditions (Figure 1C). Transgenic plants were obtained from nine oat varieties, Forage plus, Qinghaitian, Longyan 3, Baiyan 7, Qingyin 3, Ares, Mufeng, Zhongyan 1 and Monida, achieving transformation efficiencies ranging from 6.82% to 22.50%. Forage plus exhibited the highest efficiency (Table S8). These cultivars represent all current planting regions, demonstrating that the oat transformation system meets the breeding needs of researchers across diverse regions. Further, we evaluated the inheritance of T1 generation transgenic plants from six varieties, with heritability rates ranging from 44.44% to 100% (Table S9). Notably, the majority exceeded 70%, which is significantly higher than previously reported values (Gasparis et al., 2008). The RUBY system, based on three genes involved in the betalain biosynthetic pathway, has been used to track transgenic events in various species (Li et al., 2023; Chen et al., 2024). RUBY expression produces betalain pigments providing a clear visual marker for successful transformation. We used the maize (Zea mays) Ubi promoter to drive the RUBY gene (Chen et al., 2024) and introduced it into Mufeng and Zhongyan 1 embryos via Agrobacterium-mediated transformation. Red-colored calli were observed, with some explants showing red pigmentation in the leaves and roots (Figure 1D). A total of three transgenic plants were obtained from Mufeng and six from Zhongyan 1, including two and three red-colored plants, respectively. The green plants were positive, indicating that low RUBY expression may have been the cause of the absence of red coloration. The red plants displayed red-colored inflorescences and seeds. To assess inheritance, we analyzed three Zhongyan 1 lines (two red and one green) in the T1 generation. All red plants in the T1 generation were positive, while only three green plants tested negative, confirming the heritability of the transgene (Table S9). Overall, the RUBY reporter system is an effective, non-invasive and cost-efficient tool for confirming transformation and detecting transgene presence in oat plants. Due to seasonal limitations on immature embryo availability, we established a genetic transformation system for oat mature embryos (Figure 1E). The mature embryos, were soaked overnight, sterilized, scraped and then placed on callus induction medium. After approximately 2 weeks of culturing, the calluses were infected with Agrobacterium containing the pWMB110-GUS (β-glucuronidase) vector. Using the same transformation protocol as for immature embryos, we successfully obtained two transgenic plants from 85 mature embryos of Dingyan-2 and six transgenic plants from 165 mature embryos of Galileo, with transformation efficiencies of 2.35% and 3.64%, respectively. Putative T0 transgenic oat plants were identified through Quickstix strip detection for bar protein (Figure 1E), histochemical analysis for GUS activity (Figure 1E), and polymerase chain reaction (Figure S1). However, the heritability rate of two genotypes was only approximately 20%. While the transformation efficiency of mature embryos still requires optimization, this system provides a viable alternative to overcome the time constraints of using genetic transformation materials. Genome editing has become a powerful tool for genetic improvement in various species, and the high transformation efficiency in oats provides a strong foundation for advancing this research. To explore this, we constructed a CRISPR/Cas9 vector containing AsPDS-sgRNA (single-guide RNA) and transformed the oat callus. We successfully obtained three albino plants. TA cloning and Sanger sequencing confirmed that the AsPDS loci targeted by the sgRNA were edited in these albino plants. The Aspds1 had a 1-base pair deletion in the A genome, a 2-base pair deletion in the C genome, and a 1-base pair deletion in the d genome (Figure 1F). Moreover, we developed a multiplex genes editing vector incorporating 10 sgRNAs designed to target five genes (AsARE1, AsAPP1, AsIPA1, AsSPDT and AsAKT1), with two sgRNAs assigned to each gene to enhance the likelihood of successful editing. From this effort, we successfully generated 53 transgenic plants. Through TA cloning and subsequent Sanger sequencing, we identified that eight of these plants harbored the desired mutations, resulting in an overall editing efficiency of 15.09%. Delving deeper into the editing patterns, we discovered that two plants had edits in two genes simultaneously, another pair exhibited concurrent editing across three genes, and notably, two plants demonstrated successful editing in all five targeted genes at once, achieving a multiplex editing efficiency of 3.77% (as detailed in Table S10). Specifically, in the T0-1 plant, all six target sites across the five genes were edited concurrently (Figure 1G). In conclusion, we have established a robust and efficient CRISPR/Cas9-mediated gene editing system, enabling multiplex genome editing in oats. CRISPR-Cas12i is an editing system with independent intellectual property rights in China (Zhang et al., 2021). ARE1 plays a crucial role in enhancing nitrogen fertilizer utilization, while also influencing traits such as plant height and photosynthesis (Wang et al., 2018). It is highly conserved across plant species, and reducing its expression has been shown to boost yields, especially under low nitrogen conditions (Wang et al., 2021). We designed a CRISPR-Cas12i vector with an sgRNA targeting the promoter region of the AsARE1 gene. From a total of 16 transgenic plants, two successfully underwent editing at the sgRNA target site, resulting in an editing efficiency of 12.5%. One edited plant showed a 4-base pair deletion in the A genome and a 2-base pair deletion in the C genome (Figure 1H). The process of developing CRISPR-Cas12i system led to the creation of potential oat germplasm with improved low nitrogen tolerance. In this study, we established an Agrobacterium-mediated genetic transformation system for immature embryos in nine oat varieties and developed a visualized transformation system. To overcome the seasonal limitations of immature embryos, we also established a transformation system for mature embryos. Furthermore, we have achieved a significant milestone by successfully establishing, for the first time, two highly efficient genome editing systems—CRISPR-Cas9 and CRISPR-Cas12i—along with a multiplex genome editing system in oats. These advancements in oat transformation and gene editing would significantly accelerate the progress of oat biotechnology breeding and provide strong technical support for future breeding efforts. This research was funded by the National Center for Forestry and Grassland Genetic Resources (2005DKA21003). We thank Professor Hui Zhang (Shanghai Normal University) for providing the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) vector and Professor Jinsheng Lai (China Agricultural University) for providing the CRISPR/Cas12i vector. The authors declare there are no conflict of interest. S.K., H.W., and H.T. conceived the main experiments. Z.L., D.Z., and Z.C. performed the transformation experiments. Z.M. and H.T. constructed Cas12i vector and transformation. S.K. and W.K. contributed to the data analysis. T.S. and W.Z. designed the manuscript and wrote the manuscript. S.K., H.W., and H.T. contributed equally to this work. All authors read and approved the contents of this paper. 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