Controlling A. tumefaciens

[Alan McHughen]

While developing more efficient transformation efficiencies for flax, one 
of the parameters we investigated was length of co-culture period (flax 
hypocotyls + Agrobacterium *in vitro*). Traditionally, 2-3 days of such 
coculture is standard, largely due to the overgrowth of Agrobacterium 
observed with longer durations. We noted a better transformation 
efficiency in the hypocotyls cocultured for longer periods when the Agro 
did NOT overgrow the plant tissue. Because this non-overgrowth was a 
relatively rare event, the overall efficiency was low, however. 

Dr J-Z Dong, a postdoc in the lab at the time, started culturing the 
cocultivated hypocotyl + Agrobacterium on a piece of filter paper overlay 
on the culture medium; this dramatically reduced the Agrobacteria 
growth, enabling co-culture periods of up to 12 days. We eventually 
determined our most efficient co-culture period using this method for 
recovery of transgenic plants to be seven days. The use of the filter 
paper overlay to control Agrobacteria (and other aspects of this work) is 
recorded in Dong and McHughen; Plant Science 88:61-71 (1993).