The protocol was adapted from (67). Purified root starch (5 mg) was suspended in 165 μl of sterile water, and 10 μl was transferred to a 96-well, flat-bottom plate containing 190 μl of water. Starch granules were detected using a flow cytometer. Moist starch granules have a refractive index of 1.52 (68). Here, we use a CytoFlex [settings: forward scatter (FSC) gain, 20; threshold FSC, 6000; max, 3000 events s−1; λ = 488 nm] to measure 50,000 granules per sample. The diameter was derived from the FSC value based on polymethyl methacrylate sizing microbeads (refractive index: 1.49; ø: 1, 4, 8, 20, and 30 μm) to determine the correlation of diameter to FSC value. Size gates were selected on the basis of standardization and WT biological data.

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