Leaf explant age

Leaf explants at different ages may have different responses to Agrobacterium virulence. To determining the best explant age for Agrobacterium inoculation, we tested 14-, 21-, and 28-day old leaf explants cut off from plantlets cultured in thidiazuron (TDZ) pretreatment medium for their transient expression efficiency of a reporter gene after cocultivation with Agrobacterium. The result shows that there was no significant difference for transient expression of the GUS gene at the three different ages we tested.

Agrobacterium cell density

Agrobacterium cell density plays an important role in transformation efficiency. While higher cell density usually makes more transformation events occur, excessive Agrobacterium growth can be a serious problem for the further selection culture and shoot growth even if antibiotic is applied. Agrobacterium cell density at OD600 3.0, 2.1, 1.5, and 0.5 were tested to see their effect on GUS gene transient expression and Agrobacterium elimination in the selection culture. We found that cell density at OD600 3.0 and 2.1 had significantly higher transient expression than those at OD600 1.5 and 0.5. Agrobacterium overgrowth was found only in OD600 3.0 treatment.