Development of efficient Agrobacterium-mediated transformation of a second-generation biofuel crop, Brassica carinata

Brassica carinata (B. carinata) is a promising non-food biofuel crop with the potential for low carbon intensity biofuel production under suitable management conditions. However, B. carinata requires extensive genetic improvement of many agronomic traits for it to be widely deployed into agriculture systems. Its rapid improvement via gene editing or genetic modification requires robust, genotype-flexible transformation protocols, as current methods are limited and have only been applied to a narrow germplasm base, hindering broader genetic enhancement efforts. In this paper, cotyledonary petioles from five diverse B. carinata genotypes were evaluated for shoot regeneration under different cytokinin treatments. Among the genotypes tested, regeneration efficiency varied substantially across the five B. carinata genotypes. Transient expression of the GUS reporter gene in cotyledonary petioles confirmed high transformation proficiency, with expression frequencies varying from 96.4 to 100% among four genotypes. Consistently, high stable transformation efficiency was achieved for these four genotypes of at least 5%, with the highest efficiency being 12%. Interestingly, transformation and regeneration efficiencies did not correlate, indicating the latter does not serve as a reliable indicator of transformation efficiency when subjected to selection pressures. Transgene integration was validated by PCR and copy number analysis was performed using digital droplet PCR. Furthermore, T1 seedlings derived from selected transgenic events demonstrated resistance to glufosinate ammonium whereas wild-type seedlings exhibited mortality. Overall, this study presents a significantly improved and reproducible Agrobacterium-mediated transformation method for B.carinata, advancing the potential for rapid genetic improvement and trait development in this promising biofuel crop. We established an efficient and reproducible B. carinata regeneration and transformation system that works reliably across multiple genotypes.