Establishment of an efficient and genetically stable Agrobacterium-mediated transformation protocol for Beta vulgaris L. (sugar beet) based on direct shoot regeneration

Background Beta vulgaris (sugar beet) is a crop of great economic value that has limited biotechnological enhancement despite being notoriously recalcitrant to in vitro regeneration and genetic engineering. Therefore, a consistent regeneration and transformation system should be developed to improve this species’ functional genomics and trait improvement. Results In this study, we developed A protocol for B. vulgaris based on a direct organogenesis system, thereby simplifying the Agrobacterium tumefaciens -mediated transformation procedure. Leaf explants were cultured on media containing 3.1 µM BAP and 0.54 µM NAA to generate organogenesis. No callus phase was required, and regenerated plants exhibited low somaclonal variation. The system exhibited the highest regeneration efficiency (91.67%) with an average of 10.33 shoots per explant. The transformation parameters were optimized in a systematic manner, and the best conditions were a bacterial density of OD 600 = 0.6, acetosyringone concentration of 75 µM, and infection duration of 20 min. The use of low kanamycin selection pressure (60 mg/L) was critical. This effectively reduced phytotoxicity without compromising selection effectiveness. Successful transgene integration and expression were established by polymerase chain reaction (PCR), RT-qPCR, and histochemical GUS assays. Acclimatization was successful in 70% of the transgenic plants, which was much higher than the rates previously reported for B. vulgaris . Conclusion This study proposes a powerful regeneration and transformation procedure for sugar beet. This system has the potential to be useful in functional genomics studies and other applications aimed at enhancing the genetic value of B. vulgaris due to the high regeneration efficiency, low selection toxicity, and high survival rate of transgenic plants.