Agrobacterium-Mediated Genetic Transformation Method in Long-Term Cell Culture Thalictrum minus L.

Abstract A long-term Thalictrum minus L. cell culture derived from the leaf petiole of this medicinal plant retains the ability to biosynthesize the alkaloid berberine. Berberine possesses a wide spectrum of biological activity. An Agrobacterium-mediated genetic transformation method was used to obtain new cell lines with increased levels of berberine. The modified Agrobacterium tumefaciens (C58) strain contains the cointegrative vector pGV3850. The plasmid carries the target ipt gene encoding a key enzyme in cytokinin biosynthesis under the control of the cauliflower mosaic virus 35S promoter, and the selectable nptII gene encoding the enzyme neomycin phosphotransferase under the control of the NOS promoter, conferring kanamycin resistance. The ipt gene was selected due to the known involvement of cytokinins in the regulation of berberine biosynthesis. Transformation optimization was evaluated at different phases of the Agrobacterium growth cycle and based on the duration of co-cultivation with plant cell cultures in liquid medium when shaken and on agar. The optimal approach for Agrobacterium-mediated genetic transformation involved diluting an overnight-grown bacterial suspension fivefold with mMS medium and performing co-cultivation in liquid medium with shaking during the first 24 h, corresponding to the exponential growth phase of the Agrobacterium. The transformation efficiency with kanamycine (sublethal concentration) is 10%. A high correlation (90%) was observed for the ipt gene in cell lines selected on kanamycin. Six of the nine cell lines were PCR-positive after one year of cultivation under standard conditions. Among them, only one exceeded the original strain in protoberberine alkaloid content by 58%.