Development and Application of a Plate Method for Visualizing Aleurone Layers in Mature Rice Grains

Rice bran oil, a valuable ingredient of rice bran (composed mainly of embryo and aleurone), is gaining increasing attention for its abundance and benefits for human health. To increase production, breeders have selected for enlarged embryos and thickened aleurone layers. However, breeding for the latter is impeded by the time-consuming and labor-intensive process of observation of aleurone traits. Here, we established a new method for visualizing aleurone layers comprising embedding of mature grains in a plate, dichromatic staining of half grains, and computer-assisted image analysis. With this 'plate method', a batch of up to 100 grains fixed on the plate can be handled and examined more efficiently than the standard cryomicrotome method, which only processes the grains individually. In addition, the results obtained from the plate method were highly correlated with that of the cryomicrotome method in terms of aleurone area (r = 0.92) and mean aleurone thickness (r = 0.93). This new method allowed us to rapidly assess the aleurone phenotypes of more than 22,000 mutagenized grains of 'Mizuhochikara', with 700-1000 grains per day. As a result, one mutant line with thickened aleurone layer was successfully isolated.