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