Development of transgenic sugar beet (Beta vulgaris L.) plants via chloroplast transformation technology

Chloroplast transformation is a powerful tool in plant biotechnology, offering major advantages for genetic engineering through features like genetic stability, high levels of gene expression, and maternal inheritance. In this study, our objective was to develop a transplastomic sugar beet plant by utilizing the pExpPN-phaA-SB vector, which was engineered to include the phaA, adaA, and ampR genes, along with the trnA and trnI sequences serving as homologous recombination sites. A total of 66 independent transformation experiments were conducted using the biolistic bombardment method to insert the pExpPN-phaA-SB vector. Putative transplastomic explants were initially subjected to selection on MS medium with 0.5 mg/L BAP, 0.1 mg/L NAA, and 50 mg/L spectinomycin. The induced calli were then transferred to MS medium containing 1 mg/L BAP, and 0.5 mg/L ABA for further development. Regenerated plants were planted in MS medium with 0.25 mg/L KIN medium. Long-term antibiotic treatments were found to negatively impact both callus formation and regeneration efficiency. Additionally, it was observed that MS medium with 0.25 mg/L KIN effectively reduced the incidence of vitrification, a common issue in sugar beet tissue culture studies. Following the biolistic bombardment of 5846 explants, regeneration of 516 putative transplastomic plants was successfully achieved. Genomic DNA was isolated from these plants, and PCR analysis confirmed the partial integration of the pExpPN-phaA-SB vector, resulting in the generation of 10 transplastomic plants. These results provide a significant reference for chloroplast transformation in difficult-to-transform species such as sugar beet. Moreover, the data generated from this project are invaluable, offering key insights and experience that will be instrumental for future genome editing initiatives in sugar beet and related crops.