3,3-diaminobenzidine (DAB) staining
Second leaves of three-week-old tomato plants were detached, petioles
were wrapped in wet tissue paper and leaves were placed on petri dishes
in trays filled with water with only the petiole touching the water.
After 24 h in an incubator (24°C, 16:8 light/dark), each leaflet was
treated by applying three 20 µl droplets of 300 µM PA solution, three 20
µl droplets of mock solution or wounding with a scalpel (positive
control). Leaflets were sampled 1, 3, 6 and 24 h post treatment and
DAB-stained as in Daudi & O’Brien (2012). Four leaves (16 leaflets)
were used per treatment and time point, and the experiment was repeated
twice.