Abstract
Transient gene expression is far superior to stable gene expression in terms of labor time efficiency and is well suited for the study of plant promoters, protein localization, protein–protein interactions, or binary vectors for genome editing. This study investigated the factors affecting the efficiency of transient gene expression in Panax ginseng by analyzing green fluorescent protein (GFP) fluorescence intensity under a confocal laser microscope. The influences of variations in Agrobacterium strain and suspension density, acetosyringone concentration, number of vacuum applications, infiltration buffer composition, and shoot developmental stage on transformation efficiency of P. ginseng leaf parenchyma cells were investigated. We found that the A. tumefaciens EHA105 strain is best suited for P. ginseng transformation, and the highest expression level was observed at the suspension density OD600 = 1.0. The addition of acetosyringone greatly increases transformation efficiency, but only if it does not exceed a concentration of 100 μM. A special mode of physical treatment, which includes five consecutive applications of a relatively deep vacuum, further increased the transformation efficiency. In addition, the use of MES buffer at pH 5.6 and mature leaves for transformation allowed us to achieve the highest level of transit expression, while more than 80% of leaf parenchyma cells were transformed. The optimized transient transformation method allowed us, for the first time, to evaluate the effect of rol gene expression on the accumulation of ginsenosides in P. ginseng leaves. Our study is the first work aimed at the optimization of transient ginseng transformation, and the developed protocol will be of particular importance for ginseng physiology research and modulation of valuable secondary metabolite biosynthesis.
Key message
An optimized protocol for the transient transformation of Panax ginseng shoots is described, which allows fast transformation with high efficiency. The optimized method allowed us, for the first time, to evaluate the effect of rol gene expression on the accumulation of ginsenosides in P. ginseng leaves.
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Acknowledgements
Financial support was provided by the Russian Science Foundation, Grant No. 20-16-00016. The investigations were conducted using equipment from the Instrumental Centre for Biotechnology and Gene Engineering at the Federal Scientific Centre of the East Asia Terrestrial Biodiversity of the Far Eastern Branch of the Russian Academy of Sciences (Vladivostok).
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TYG provided the plant material and performed the confocal imaging analysis. VPG performed the ginsenoside content analysis. AID and YNS designed and performed all experiments, analyzed data and wrote the manuscript. YNS and VPB supervised the research. All the authors have read and approved the final manuscript.
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Degtyarenko, A.I., Gorpenchenko, T.Y., Grigorchuk, V.P. et al. Optimization of the transient Agrobacterium-mediated transformation of Panax ginseng shoots and its use to change the profile of ginsenoside production. Plant Cell Tiss Organ Cult 146, 357–373 (2021). https://doi.org/10.1007/s11240-021-02075-6
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DOI: https://doi.org/10.1007/s11240-021-02075-6