Simple and Fail-safe Method to Transform Miniprep Escherichia coli Strain K12 Plasmid DNA Into Viable Agrobacterium tumefaciens EHA105 Cells for Plant Genetic Transformation
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...
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Bio-protocol LLC
2025-01-01
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| author | Beenzu Siamalube Emmanuel Ehinmitan Maina Ngotho Justus Onguso Steven Runo |
| author_facet | Beenzu Siamalube Emmanuel Ehinmitan Maina Ngotho Justus Onguso Steven Runo |
| author_sort | Beenzu Siamalube |
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| description | 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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| institution | Kabale University |
| issn | 2331-8325 |
| language | English |
| publishDate | 2025-01-01 |
| publisher | Bio-protocol LLC |
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| spelling | doaj-art-c48b0db0b21f4df998ac8449edb8f2172025-02-07T08:16:31ZengBio-protocol LLCBio-Protocol2331-83252025-01-0115110.21769/BioProtoc.5174Simple and Fail-safe Method to Transform Miniprep Escherichia coli Strain K12 Plasmid DNA Into Viable Agrobacterium tumefaciens EHA105 Cells for Plant Genetic TransformationBeenzu Siamalube0Emmanuel Ehinmitan1Maina Ngotho2Justus Onguso3Steven Runo4Department of Molecular Biology and Biotechnology, Pan African University Institute for Basic Sciences, Technology and Innovation, Nairobi, KenyaDepartment of Molecular Biology and Biotechnology, Pan African University Institute for Basic Sciences, Technology and Innovation, Nairobi, KenyaDepartment of Animal Science, Jomo Kenyatta University of Agriculture and Technology, Nairobi, KenyaInstitute for Biotechnology Research, Jomo Kenyatta University of Agriculture and Technology, Nairobi, KenyaDepartment of Biochemistry, Microbiology and Biotechnology, Kenyatta University, Nairobi, KenyaAgrobacterium-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.https://bio-protocol.org/en/bpdetail?id=5174&type=0 |
| spellingShingle | Beenzu Siamalube Emmanuel Ehinmitan Maina Ngotho Justus Onguso Steven Runo 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 |
| title | Simple and Fail-safe Method to Transform Miniprep Escherichia coli Strain K12 Plasmid DNA Into Viable Agrobacterium tumefaciens EHA105 Cells for Plant Genetic Transformation |
| title_full | Simple and Fail-safe Method to Transform Miniprep Escherichia coli Strain K12 Plasmid DNA Into Viable Agrobacterium tumefaciens EHA105 Cells for Plant Genetic Transformation |
| title_fullStr | Simple and Fail-safe Method to Transform Miniprep Escherichia coli Strain K12 Plasmid DNA Into Viable Agrobacterium tumefaciens EHA105 Cells for Plant Genetic Transformation |
| title_full_unstemmed | Simple and Fail-safe Method to Transform Miniprep Escherichia coli Strain K12 Plasmid DNA Into Viable Agrobacterium tumefaciens EHA105 Cells for Plant Genetic Transformation |
| title_short | Simple and Fail-safe Method to Transform Miniprep Escherichia coli Strain K12 Plasmid DNA Into Viable Agrobacterium tumefaciens EHA105 Cells for Plant Genetic Transformation |
| title_sort | simple and fail safe method to transform miniprep escherichia coli strain k12 plasmid dna into viable agrobacterium tumefaciens eha105 cells for plant genetic transformation |
| url | https://bio-protocol.org/en/bpdetail?id=5174&type=0 |
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