A RUBY Reporter Enables Visual Identification of Transformed Calli for Efficient Genome Editing in Broccoli

Plant transformation and genome editing have become a key driving force for functional genomics research and crop improvement (Li et al. 2025). Broccoli (Brassica oleracea var. italica) is a high-value cruciferous vegetable enriched in vitamins, minerals, and bioactive compounds (James et al. 2012). In current broccoli studies, the hairy root system is mainly used for leaf-derived hairy root induction to investigate metabolite production, or for hypocotyl-induced composite plants for root functional analyses (Amer et al. 2024; Geng et al. 2022). However, for many crops including broccoli, the low efficiencies of regenerating whole plants from transgenic hairy roots remain major bottlenecks that restrict broader application of this system in functional gene studies and genome editing (Wang 2024a). To date, hairy root–based regeneration strategies have been validated in species such as apple and poplar (Liu et al. 2024; Wei 2025). Nevertheless, an efficient regeneration system has yet to be established in broccoli. In this study, we performed transformation using Rhizobium (Agrobacterium) rhizogenes strain K599 carrying the RUBY reporter (Figure 1A,B). Cotyledon-attached hypocotyl segments were excised from 7-day-old broccoli seedlings (B42) and immersed in bacterial suspension for 30 min (OD₆₀₀ = 0.6–0.8), followed by dark co-cultivation for 3 d on MS medium supplemented with 200 μM acetosyringone (AS). After co-cultivation, explants were transferred to callus induction medium (MS supplemented with 1 mg L⁻¹ 6-benzylaminopurine [6-BA], 0.1 mg L⁻¹ naphthaleneacetic acid [NAA], 4 mg L⁻¹ AgNO₃, 3% sucrose, and 300 mg L⁻¹ timentin; pH 5.8) for approximately 2 weeks. AgNO₃ is a commonly used additive in plant tissue culture to alleviate tissue browning. Enlarged calli were then excised and transferred to shoot induction medium (MS supplemented with 2 mg L⁻¹ 6-BA, 0.1 mg L⁻¹ NAA, 4 mg L⁻¹ AgNO₃, 3% sucrose, and 300 mg L⁻¹ timentin) for 2–3 weeks until shoot regeneration. Regenerated shoots were further transferred to shoot elongation medium (Figure 1B). Because this workflow does not include a hairy root induction stage, the time from infection to regenerated transgenic plants is typically ~8–10 weeks. Moreover, the RUBY reporter enables visual identification of transformation events (Hu et al. 2025), thereby simplifying operations and reducing screening workload, with a transformation efficiency of 16.4% (Table S1). Efficient callus induction is a key determinant of regeneration performance. To optimise induction conditions, we compared callus induction across different 6-BA (cytokinin) concentrations (1, 2, and 5 mg L⁻¹). After ~2 weeks of culture, calli gradually formed and expanded at the basal region of explants (Figure 1C,D). Callus induction at 1 mg L⁻¹ 6-BA (86.6%) outperformed that at 2 mg L⁻¹ (47.6%) and 5 mg L⁻¹ (25%) (Figure 1C,D). Notably, on hormone-free control medium, explants primarily produced abundant hairy roots but failed to generate callus (Figure 1C). These results indicate that 1 mg L⁻¹ 6-BA is more favourable for callus induction and subsequent regeneration. After transfer to soil, regenerated plants maintained an overall red pigmentation and exhibited slightly curled leaves, indicating stable expression of the RUBY reporter after acclimation (Figure 1E). The regeneration efficiency, which was calculated by dividing the number of transgenic plants by the total number of calli used for regeneration, was 24.2%. PCR amplification using Cas9 and RUBY specific primers confirmed stable integration of the transgene in all eight lines (Figures 1F and S1A). RT–qPCR analysis showed high RUBY transcript levels in the transgenic plants (Figure S1B). Sanger sequencing chromatograms with mixed peaks indicated chimeric mutations at the BoARF7 (BolC2t07069H) target sites (Figure S2). PCR amplicons were TA-cloned and individual clones were Sanger-sequenced, revealing that most edits were base substitutions or short insertions/deletions (Figures S2 and S3). To evaluate potential off-target effects, we predicted candidate off-target sites for each sgRNA using CRISPOR and selected the top 2–5 sites with the highest off-target scores for validation. PCR amplification followed by sequencing detected no off-target mutations at any of the examined sites (Figure S1C; Table S2). We further applied this system to another broccoli cultivar, B1 (Figure S4). Among 80 infected explants, nine independent callus lines were obtained, and one regenerated into a plant with a regeneration efficiency of 11.1%. Differences in infection and transformation efficiency among cultivars may influence regeneration outcomes. Overall, our results indicate that this system is applicable across different broccoli genotypes. In summary, we established a R. rhizogenes–mediated callus-to-shoot regeneration workflow in broccoli, and achieved rapid visual identification of transformed calli using the RUBY reporter (Figure 1B). By bypassing the hairy-root induction step prior to callus formation, our approach can shorten the timeline to regenerated plants (Abdullah et al. 2021). Recent studies have likewise achieved whole-plant regeneration in Chinese cabbage through co-expression of developmental factors (Wang 2024b). This system provides an ideal option for functional gene studies and molecular breeding in broccoli. This study was supported by grants from the Beijing Natural Science Foundation (6262025), the China Agriculture Research System (CARS-23-A05), and the Science and Technology Innovation Program of the Chinese Academy of Agricultural Sciences (CAAS-ASTIP-IVFCAAS). The authors declare no conflicts of interest. The data that supports the findings of this study are available in the supplementary material of this article. 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.