In situ Imaging of Pea Starch in Seeds

A method has been developed for the in situ imaging of starch in dry seeds by exploiting the tight packing of the starch and protein storage reserves within the cells of the embryo. The method can be adapted to prepare seed samples which are suitable for light microscopy (birefringence and iodine staining), scanning electron microscopy and atomic force microscopy. Its potential for imaging the internal structure of starch granules without any prior isolation process is demonstrated for round smooth peas. Using a standard ultramicrotome, thin sections were cut directly from selected regions of dry pea seeds and examined by light microscopy before and after hydration. The sectioning procedure left a planed surface with the internal structure of the starch granules exposed. This material was examined by scanning electron microscopy and atomic force microscopy directly or after controlled hydration. In the hydrated pea samples, the growth ring structure and blocklet sub-structure of individual starch granules within the seed were visualised directly by atomic force microscopy. Furthermore, the effects of hydration and staining were monitored and have been used to introduce contrast into the images. The observations have revealed new information on the blocklet distribution within pea starch granules and the physical origins of the growth ring structure of the granules: the blocklet distribution suggests that the granules contain alternating bands with different levels of crystallinity, rather than alternating amorphous and crystalline growth rings.