Agrobacterium-Mediated Genetic Transformation: A Method to Genetically Transform the Rice Genome via Genetically Engineered Agrobacterium tumefaciens

Agrobacterium tumefaciens, a plant pathogenic bacterium, can be genetically engineered by introducing an artificially prepared Ti plasmid, carrying a gene of interest in its transfer DNA or T-DNA region, and a helper plasmid carrying the virulence or Vir genes for transferring the T-DNA into plant cells.

For Agrobacterium-mediated genetic transformation, begin with a young rice inflorescence and cut it into small pieces. Transfer the cuttings to a growth medium and incubate to facilitate the growth of the cuttings into an undifferentiated cell mass called a callus.

Now, transfer the callus to an Agrobacterium infiltration medium containing acetosyringone - a chemoattractant for Agrobacterium. Next, add a culture of genetically engineered Agrobacterium to the medium and incubate briefly.

The acetosyringone molecules activate the cell receptor protein - VirA - on the Agrobacterium membrane, which further mediates the phosphorylation of a transcriptional activator protein - VirG. Phosphorylated VirG moves toward the helper plasmid and activates the transcription of a cascade of virulence genes, including channel proteins and endonucleases - VirD1 and VirD2.

The channel proteins form a transport channel connecting the bacterial and plant cell, whereas the endonucleases bind to the border sequences in the Ti plasmid and cleave the T-DNA with VirD2, bound at one end. Thereafter, VirD2 directs the entry of T-DNA via the transport channel into the callus cells and facilitates its integration into the rice genome.

Wipe each inflorescence with a 70% alcohol swab, and let it dry before cutting. Bring the inflorescence to a sterile lab bench, and cut it into small pieces with sterilized scissors. Then, transfer the cuttings to a Petri plate containing NBD2 medium.

Incubate the plate in the dark at 26 degrees Celsius, for 10 to 14 days to induce callus. To perform transformation, transfer a single colony from the YEB plate with selective antibiotics to 5 milliliters of liquid YEB medium containing the same antibiotics, in a 50-milliliter conical sterile test tube. Shake the tube on an orbital shaker at 250 x g and 25 to 28-degree Celsius until bacteria grow to an OD₆₀₀ of 0.5.

Add 1 milliliter of bacterial suspension to 100 milliliters of YEB medium with the same selective antibiotics, in a 250-milliliter conical flask, and shake the flask on an orbital shaker at 250 x g, and 28-degree Celsius for four hours. Centrifuge the culture at 4,000 x g for 10 minutes at room temperature, to collect the bacteria. Discard the supernatant, and resuspend the pellet with AAM-AS medium, and dilute the suspension to OD₆₀₀ of 0.4.

After the incubation, collect around 150 healthy, light-yellow, embryogenic calli into a 150-milliliter sterile flask. Add 50 to 75 milliliters of the bacterial cell suspension into the flask, and then add 10 to 25 milliliters of fresh AAM-AS medium, to immerse the calli for 10 to 20 minutes, shaking occasionally.

Pour the bacterial suspension out of the flask carefully, and dry the calli with sterile filter paper. Then, place them on a Petri dish with NBD-AS medium, and cover them with filter paper. Incubate the calli at 25 to 28 degree Celsius in the dark for three days, checking them for bacterial overgrowth.

After three days of co-cultivation, transfer the calli to a sterile Petri dish using filter paper, then, air-dry them for two hours on a clean bench. Ensure that the calli are not adhered to the filter paper, and transfer them to the primary selection medium NBD2.

After two weeks, transfer the calli evenly to a new plate containing fresh selection medium. Then, move the calli to fresh MS medium for differentiation, and the shoot buds to MS medium (with 10 mg/L Hygromycin) to proliferate more shoots.