GUS assay
After 3 days of co-cultivation, some calli
were washed to remove excess Agrobacterium then
incubated in X-Gluc solution at 37 oC for 1 hour to
overnight (Jefferson, 1987). The transient GUS
expression in the calli was examined and compared
with that in the untransformed calli. For the
transformed plants, leaf segments were cut and
immersed in X-Gluc solution followed by incubation
at 37 oC for 1 hour to overnight. After staining, the
leaf segments were bleached out in 95 % ethanol to
remove chlorophyll and then GUS expression was
investigated.