Figure 4

Two-dimensional pooling of the cotyledons in 96-well plates. Step 1: Twelve 8 × 8 grids were prepared (as depicted by numericals 1, 2, 3..... 12 and colored red, yellow, brown.... lavender, respectively,). Step 2: The eight seedlings arrayed in column 1 of the first grid were removed and arranged linearly. The right and the left cotyledons of the seedlings were excised and arranged in the same order as present in the column of the grid from which they were removed. Step 3: In two new deepwell plates, the cotyledons were placed in the wells as shown in the Figure. One set of cotyledons was placed in 1A-1H wells in the ROW plate whereas the other set of cotyledons was placed in 1A well of COLUMN plate. Step 4: Eight seedlings from 2A-2H of plate 1 (red colored) were removed and their cotyledons excised as in step 2. Step 5: One set of cotyledons was placed in 1A-1H of ROW plate whereas the other set was put in 1B well of COLUMN plate. Step 6: Similarly the seedlings were removed from columns 3, 4, 5, 6, 7 and 8 and pooled as described steps 2 and 3 in different wells. After pooling the seedlings of eight columns, both the ROW and COLUMN plate had eight cotyledons each in 1A-1H wells. Step 7: The whole procedure was repeated for the second, third....twelveth grid (as shown with yellow, brown ....lavender colors in Figure) to generate eight pooled ROW and COLUMN plates.