An efficient transformation and gene editing system for Lilium species

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A transformation system exploits lily bulb scale propagation and de novo formation of bulblets, integrating tissue culture with non-tissue culture approaches. Through optimized scale propagation and disinfection treatments, it achieves efficient stable gene overexpression and editing in lily. Lilium spp. are globally significant due to their ornamental, edible, and medicinal value; with the release of lily genomes, Lilium has gradually emerged as a model species for functional studies in bulbous plants (Liang et al., 2025). Gene function analysis is essential for research and molecular breeding; however, genetic transformation in lily still primarily relies on somatic embryogenesis, which has limitations, including long periods (10 to 18 months), complex procedures, and strict reliance on sterile tissue culture and has unstable, low efficiency (less than 3%), which hinder its application (Fan and Sun, 2024). With the advent of tissue culture-independent transgenic methods, plants that are recalcitrant to somatic embryogenesis-based transformation have access to more efficient genetic transformation strategies (Hu et al., 2025). Notably, lily scales possess a strong propagative capacity; within 1–2 months after excision, the bulblets can form de novo at the basal part of detached scales (Figure S1; Wu et al., 2025). Based on this trait, our study developed an efficient protocol for directly obtaining transgenic bulblets by leveraging Agrobacterium-mediated genetic transformation, and further optimized hormone treatments and disinfection procedures using a non-sterile tissue culture system. The Agrobacterium-mediated method is a classic approach used for plant genetic transformation, with transformation efficiency closely dependent on the strain (Hao et al., 2024). To identify a highly efficient strain for lily stable transformation, we conducted an experiment utilizing L. davidii var. unicolor (hereafter L. davidii), a cultivar with broad application potential, strong propagative ability, and published genomic data. We first used the bacterial infection buffer of four Agrobacterium strains carrying the pCAMBIA2300-GFP vector to infiltrate middle layers' scales under vacuum pressure and smeared the bacterial infection buffer on the basal wounds of the scales, followed by culture in moist cotton under dark conditions (Figures 1A, S2A). Here, we used a matrix-free approach with de-fatted cotton, which is highly water-retentive, easy to manage, and allows for regular observation of scale growth and bulblet formation while maintaining a clean environment and reducing scale rot (Figure S3). Broad applicable transformation and gene editing system in Lilium species (A) Schematic workflow of broad-spectrum genetic transformation in lilies. (B) GFP fluorescence in P1-generation transgenic L. davidii bulblets. Scale bar: 2 mm. (C) GFP fluorescence in P2-generation transgenic L. davidii bulblets. Scale bar: 1 mm. P2: Bulblets generated from P1 plants' scales. (D, E) GFP fluorescence in leaves of P2-generation transgenic L. davidii bulblets. In (D), scale bar: 2 mm. In (E), scale bar: 50 μm. (F) RT-PCR detection of eight independent GFP-positive P2 transgenic bulblets using GFP-specific primers. FP (F-box protein) primers served as a reference. PC: Positive control, pCAMBIA2300-GFP vector. (G) Western blot analysis of eight independent GFP-positive P2 transgenic bulblets using an anti-GFP antibody; anti-H3 served as a loading control. (H–J) Transgenic bulblets obtained from ‘Siberia’ (H), ‘Tresor’ (I), and L. brownii (J). Scale bar: 5 mm. (K) Correlation analysis between transformation efficiency and propagation speed across cultivars. R2 > 0.6 represents a significant correlation between two variables. (L) IAA treatment enhanced transformation efficiency. Data represent means ± SD from three independent experiments. *P < 0.05 (Student's t-test). (M) Sterilization procedure significantly increased transformation efficiency. Data are presented as means ± SD of three replicates, **P < 0.01 (Student's t-test). (N) GFP fluorescence transformed with pBUE414 (GFP)-ABI5. Scale bar: 2 mm. (O, P) Genotyping analysis of abi5 mutants. In (P), red arrows show the SNP and purple arrows show Indel. (Q) Phenotypic characterization of P2 abi5 mutants. Scale bar: 1 cm. (R) Statistics of leaf length of P2 abi5 mutants. Data are presented as means ± SD of three individual lines, **P < 0.01 (Student's t-test). After 40 d of culture, we successfully observed GFP-positive bulblets by fluorescence microscopy, with strain C58C1 (pRiA4b) achieving the highest transformation efficiency (Table S1). Furthermore, GFP fluorescence was stronger in the primary generation (P1) bulblets (Figure 1B). To ensure the survival of the valuable P1 transformants and to achieve efficient and stable retention of their transgenic traits, we established subculture propagation of transgenic bulblets under tissue culture conditions (Figure 1A; Table S2). Ten independent P1 GFP-positive transgenic plants generated by C58C1-mediated transformation were selected for next-generation propagation. We sterilized these fluorescent scales and placed them in 1 g/L mancozeb (Figure 1A; Table S2). This is a standard but necessary and effective sterilization procedure that promotes aseptic germination when handled under open conditions (Figure S4). After 30 d, second-generation bulblets (P2) were produced on P1 scales (Table S3). Moreover, fluorescence signals were stably inherited in more than half of the P2 bulblets and continuously expressed in subsequent leaf development (Figure 1C–E). By RT-PCR and Western blot analyses of P2 GFP-positive bulblets, we confirmed the stable expression of exogenous protein, indicating successful genetic transformation (Figure 1F, G). To further validate the universality of this protocol across different lilies, we selected cultivars with varying propagation speeds, including ‘Tresor’, L. brownii, L. davidii, and ‘Siberia’. Following the above process, transgenic plants were successfully obtained from all tested cultivars, demonstrating the broad adaptability of this system (Figure 1H–J). Additionally, a significant positive correlation was observed between propagation speed and transformation efficiency (Figure 1K). Thus, we hypothesized that shortening the duration of bulblet formation may improve transformation efficiency. On adding supplemented indole-3-acetic acid (IAA), a hormone known to promote bulblet formation, to bacterial infection buffer, we found that it significantly shortened the bulblet formation, but increased the propagation coefficient and transformation efficiency (Figure 1L; Table S4; Wang et al., 2025). Due to the high starch and soluble sugar content, lily scales are highly susceptible to decay, often resulting in substantial loss of explants. To address this issue, we treated scales with carbendazim before bacterial infection, which significantly reduced decay rates, thereby improving explant survival and transformation efficiency (Figure 1M; Table S5). Finally, we evaluated the application of this system for genome editing in lily ‘Tresor’. We cloned a growth-inhibitory factor gene ABI5 and constructed it with the modified CRISPR/Cas9 editing vector pBUE414(GFP) (Figure S2B). Using fluorescence detection and nanopore-based haplotype sequencing, we identified more than 10 edited P1 lines. No canonical mutation was observed in target 1, while both indel and SNP mutations were detected in target 2. These lines could be classified into three recurrent editing patterns, represented by variants #1, #4, and #9; these editing patterns were consistently detected in the P2 generation (Figure 1N–P; Table S6). However, heterozygous and chimeric mutation patterns were still observed in P2 edited lines (Figure 1N–P; Tables S6–S8). In lily, regenerated bulblets often originate from multiple cell layers, which may harbor differential Cas9 activity, resulting in different mutati

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