Simple and Fail-safe Method to Transform Miniprep Escherichia coli Strain K12 Plasmid DNA Into Viable Agrobacterium tumefaciens EHA105 Cells for Plant Genetic Transformation
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Date
2025
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bio-protocol
Abstract
Agrobacterium-mediated gene transformation method is a vital molecular biology technique employed to develop transgenic
plants. Plants are genetically engineered to develop disease-free varieties, knock out unsettling traits for crop improvement,
or incorporate an antigenic protein to make the plant a green factory for edible vaccines. The method’s robustness was
validated through successful transformations, demonstrating its effectiveness as a standard approach for researchers working
in plant biotechnology. It enables the introduction of foreign DNA into plant genomes. Conventionally, plant genetic
transformation has relied on time-consuming, costly, and technically demanding procedures, such as electroporation and
chimeric viruses or biolistic methods, which usually yield variable transformation efficiencies. This study presents a simple
and fail-safe protocol that involves a modified freeze-thaw and heat-shock concoction method. This approach involves a
streamlined plasmid miniprep procedure to isolate high-quality plasmid DNA from Escherichia coli K12 strain, followed
by a target-specific transfer into A. tumefaciens EHA105 strain. The optimized method minimizes DNA degradation and
maximizes uptake by Agrobacterium cells, making it a reproducible and accessible protocol for various genetic engineering
applications. The transformation efficiency is consistently high, enhancing plasmid uptake while maintaining cell viability,
requiring minimal specialized equipment and reagents. The proposed protocol offers significant advantages, including
simplicity, reliability, and cost-effectiveness, positioning it as a valuable alternative to traditional techniques in the field of
plant biotechnology.
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Siamalube, B. et al. (2025). Simple and Fail-safe Method to Transform Miniprep Escherichia coli Strain K12 Plasmid DNA Into Viable Agrobacterium tumefaciens EHA105 Cells for Plant Genetic Transformation. Bio-protocol 15(1): e5174. DOI: 10.21769/BioProtoc.5174