2.2 Cutting process
After 12 hours dipping time, 5 geminated seeds in each group were incubated in 1 mM nitro-blue tetrazolium (NBT) in 10mM Tris-HCl buffer (pH 7.34) at room temperature for 30 minutes. Then all the stained seeds were washed with ultrapure water and frozened below -100 oC. In order to compare the amounts of ROS in the same location of seeds dipped in the water with and without NBs. All the seeds were cut vertically into 100μm slices continuously from the tip of sprout part with a cutting machine (Leica CM1950, Leica Biosystems Co. Ltd., Japan). The section areas for the first sliced sample and second sliced sample were about 0.1μm2and 0.25 μm2, respectively.