A rapid, direct observation method to isolate mutants with defects in starch grain morphology in rice

Starch forms transparent grains, referred to as starch grains (SGs), in amyloplasts. Despite the simple glucose polymer composition of starch, SGs exhibit different morphologies depending on plant species, especially in the endosperm of the Poaceae family. This study reports a novel method for preparing thin sections of endosperm without chemical fixation or resin embedding that allowed us to visualize subcellular SGs clearly. Using this method, we observed the SG morphologies of >5,000 mutagenized rice seeds and were able to isolate mutants in which SGs were morphologically altered. In five mutants, named ssg (substandard starch grain), increased numbers of small SGs (ssg1-ssg3), enlarged SGs (ssg4) and abnormal interior structures of SGs (ssg5) were observed. Amylopectin chain length distribution analysis and identification of the mutated gene suggested a possible allelic relationship between ssg1, ssg2, ssg3 and the previously isolated amylose-extender (ae) mutants, while ssg4 and ssg5 seemed to be novel mutants. Compared with conventional observation methods, the methods developed here are more effective for obtaining fine images of subcellular SGs and are suitable for the observation of a large number of samples.