Binary Bacterial Artificial Chromosome Vector Based Gene Transformation in Arabidopsis Plants: A Technique to Generate Transgenic Plants With Single-Copy Insertion

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.

To prepare the Arabidopsis plants for transformation, grow Arabidopsis plants in a greenhouse or climate-controlled growth chamber, until they are flowering. Clip the first bolts to allow more secondary bolts to emerge. Plants are ready for dipping four to six days after clipping, when the plants have many immature flower heads, and not many fertilized siliques.

To perform floral dipping, dip inflorescences for 5 to 10 seconds in Agrobacterium suspension. Use gentle agitation. Wrap the above-ground parts of the plants in cling film to keep the humidity high, and cover the plant pots with a box to keep the plants in the dark.

Incubate the plants for two days in a greenhouse or growth chamber. After two days, remove the box and the cling film, and grow the plants to maturity in a greenhouse or growth chamber. To increase the efficiency of transformation, the same plants can be re-dipped seven days after the first dipping.

Next, when the transformed plants are mature and dry, harvest the seeds. Pool and analyze the seeds of plants transformed with the same construct as a single set. In case plants are transformed with a BIBAC-RFP-Gateway plasmid derivative, identify transgenic plants by analyzing the seeds using fluorescence microscopy.

To detect DsRed expression in seed coats, image the seeds at an excitation of 560 nanometers, and emission of 600 to 650 nanometers. Separate the fluorescent seeds from non-fluorescent counterparts using forceps.

To screen for transgenic plants transformed with a BIBAC-BAR-Gateway plasmid derivative, sow the seeds in trays filled with soil. Ensure an even spreading of seeds over trays, by suspending seeds in 0.1% agar in 0.5X MS medium, and spread the seeds using a 1-milliliter pipette.

Stimulate the seeds to germinate in a synchronous manner by incubating the seeds for at least two days at 4 degrees Celsius. This can be done before or after sowing the seeds. Two and three weeks after sowing the seeds, spray the seedlings with 0.5% Glufosinate-ammonium solution. Use 500 milliliters of Glufosinate-ammonium solution per 1 square meter. Transfer surviving seedlings to pots. Analyze the Glufosinate-ammonium-resistant plants, by PCR for the presence of the construct of interest.