Binary Bacterial Artificial Chromosome Vector Based Gene Transformation in Arabidopsis Plants: A Technique to Generate Transgenic Plants With Single-Copy Insertion
Transcrição
Begin with Arabidopsis plants bearing immature flower heads. Dip the flower heads in Agrobacterium cell suspension carrying T-DNA cloned with binary bacterial artificial chromosome or BIBAC, a vector replicating in both E. coli and Agrobacterium systems. This T-DNA binary vector can deliver large transgenes such as herbicide resistance gene and fluorescent selectable markers along a seed-specific promoter.
While the flower heads are still immersed, gently agitate the plants to enhance their contact with Agrobacterium. With its inherent ability to infect plants, Agrobacterium transfers the transgene into the floral cell. As the transgene enters the cell, it moves to the nucleus and fuses with the plant genome.
Now, wrap the plant in cling film and incubate in the dark to retain humidity for improved transformation. Remove the film and grow plants under physiological conditions until they produce seeds.
Next, harvest the seeds and observe under fluorescence microscope to select transformants. Now, grow the transformed seeds in suitable conditions until germination. Spray the plants with herbicide, and incubate.
Plants with multiple transgene insertion experience gene silencing and wither, whereas plants with a single-copy insertion express an active herbicide-metabolizing enzyme and survive the treatment. Extract genomic DNA from the surviving transformants and perform PCR to calculate efficiency of single-copy insertion.
