Figure 2. DsRed accumulation in the pools of multiple leaf discs.

Leaf discs taken from all leaves of an eight-week-old N. tabacum plant were infiltrated with Agrobacterium containing a plasmid encoding DsRed. After a five-day incubation period (25°C, 16/8 h light/dark cycle, 7 klux), the TSP was extracted from 88 randomly selected leaf discs (Figure 2A). The DsRed accumulation was determined in each individual leaf disc extract by measuring its fluorescence, and this result was related to the TSP, which was determined via the Bradford assay. All values were randomly combined to generate virtual pools of 2, 3, 4 and so on, up to 20 leaf discs. The whole procedure was repeated twice for each pool. The outcome of this virtual pooling is shown in a scatter plot (Figure 2B) wherein each symbol represents one pool. To find the optimal pool size to minimize variation and effort, two constraints were set at 50% deviation (dotted lines) around the average fluorescence of all values (solid line). The selected pool size for all further assays (nine leaf discs) is indicated in red.