DNA construct instability in bacteria used for Agrobacterium mediated plant transformation

The use of plasmid in the production of genetically modified (GM) crops is highly essential in research and in commercial production of GM plants. However plasmid instability constitutes a major problem in the use of recombined microorganisms in the production of GM crops. In this study we evaluated the stability of p8114 carrying a gene coding for a transcription factor (TFIIIA) driven by Cassava Vein Mosaic Virus (CsVMV) promoter and an nptII selectable marker driven by 35S promoter in the T-DNA. The plasmid was amplified in E.coliDH5α strain on Luria Broth (LB)agar supplemented with 100 µg/ml kanamycin. The colonies were confirmed by Restriction Fragment Length Analysis (RFLA) and by DNA sequencing. The confirmed colonies were stored as glycerol stock at -80 0 C and as DNA extracts in TE buffer at 4 0 C. Agrobacterium strains LBA4404, EHA 105 and AGL1 were also transformed with DNA from the confirmed colonies. Plasmid stability was evaluated after 3 months. Sixteen to hundred percent level of instability was observed in E.colicolonies stored at -80 0 C and 50% level of instability in plasmid transformed into Agrobacterium strain LBA4404. Agrobacterium strain LBA4404 showed a higher level of stability 75% compared to EHA 105 (0%) and AGL1 (50%).

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