A vir binary vector system for Agrobacterium-mediated transient transformation in Nicotiana benthamiana

Aims: Achieving high efficiency in Agrobacterium-mediated plant transformation is an ongoing challenge in plant biotechnology. One key obstacle is the large size of native Tumour-Inducing (Ti) plasmid in Agrobacterium, which harbours essential virulence (vir) genes. To address this issue, we developed a novel strategy that combines a miniaturised helper Ti-plasmid (pRIDE101) with a complementary binary vector (pG103) carrying three key vir genes, namely virG-N54D, virD2, and virE2, regulated by an artificially constructed isopropyl β-D-thiogalactopyranoside (IPTG) inducible integron promoter, iPintV1, to enhance transformation efficiency. Methodology and results: With a T-DNA region inserted, the constructed plasmid pG103 functioned as a binary vector, simplifying the incorporation of a desired gene (E35S::sfGFP) through the Gateway® cloning method. Our inducible vector system exhibited an impressive 70% and 500% enhancement in superfolder Green Fluorescent Protein (sfGFP) fluorescence relative to the positive control (A. tumefaciens GV3101 with pMP90, pE35S::sfGFP) and constitutive system (A. tumefaciens C58C1 with pYL102, pYL101c::virG-N54D, and pE35S::sfGFP), respectively, during the transient transformation of Nicotiana benthamiana. Conclusion, significance and impact of study: The constructed IPTG-inducible virulence (vir) binary vector system shows potential for enhancing Agrobacterium-mediated plant transformation efficiency through the regulation of virulence gene expressions. This study also provides valuable insights for potential future enhancements in the Agrobacterium vector system.

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