The ENABLE project, funded under the KMU‑innovativ‑20 (031B0801B) programme, ran from 1 April 2019 to 30 September 2022 and was led by Dr Andreas Müller of Strube Research GmbH & Co. KG. The consortium comprised the small‑business partner Strube Research, the coordination partner Crop Genetic Systems Hamburg, and the academic partners the University of Hamburg and Christian‑Albrechts‑Universität Kiel. The main objective was to establish genome‑editing methods based on site‑specific endonucleases in sugar beet, with a focus on transgene‑free approaches that could accelerate breeding for traits such as resistance to beet cyst nematodes and reduced leaf angle.

Strube Research supplied a diverse set of sugar‑beet genotypes and developed regeneration protocols. From an initial pool of 140 lines, 25 were selected for tissue‑culture trials based on SNP‑based genetic distance, ensuring representation of both closely related and genetically distant lines. In the first phase (AP ST2.1) explants from leaf and petiole were cultured on media containing four different phytohormones. Cytokinin 6‑benzylaminopurine (BAP) proved effective for both direct regeneration and callus induction, whereas the auxin 2,4‑D consistently inhibited callus formation and was therefore excluded. Callus induction rates reached 83–96 % for leaf and petiole explants, and regeneration efficiencies varied between 0.1 and 1.0 % across the most promising genotypes.

The core of the technical work involved Agrobacterium‑mediated transformation of callus cultures from three genotypes (GT31, GT58, GT78). Two binary plasmids were used: a reporter construct (P35S::GUS; AtUbq10::GUS; P35S::nptII) and an endonuclease construct carrying two sgRNAs (NSL). Three successive transformation series (V1–V3) were performed with a constant paromomycin concentration. Growth rates of callus after co‑cultivation varied from 20 % to 65 % depending on genotype and explant type. Transformation frequencies ranged from 0 % to 100 %, with the highest value observed for leaf explants transformed with the GUS plasmid in series V2. Regeneration frequencies were generally low; only the third series yielded data, with regeneration rates of 31 % for leaf‑derived callus and 58 % for petiole‑derived callus in the GUS‑transformed groups. From the transformed callus, 5 regenerants were obtained from GT31 and 26 from GT78, while no regenerants emerged from GT58. Histochemical GUS staining and PCR confirmed transgene presence in several regenerants, but all were non‑transgenic in the endonuclease‑transformed groups.

To assess genome editing, DNA from endonuclease‑transformed callus was extracted and pooled for next‑generation sequencing. Each pool yielded an average of 90 000 reads, with a total of 7.2 million read pairs across 15 pools. Mean quality scores ranged from 37.27 to 37.5, and 96.5–97.6 % of bases had a quality score above 30. Despite the high sequencing depth and quality, no evidence of targeted mutagenesis was detected, indicating that the current transformation and selection conditions were insufficient to recover edited cells.

Overall, the project demonstrated the feasibility of Agrobacterium‑mediated delivery of endonucleases into sugar beet callus, identified suitable genotypes and hormone regimes for callus induction and regeneration, and highlighted the challenges of achieving efficient genome editing in this species. The collaborative framework, combining expertise in plant tissue culture, molecular genetics, and breeding, provided a solid foundation for future optimisation of transgene‑free editing strategies in sugar beet.