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Background  The visualization of internal 3D-structure of tissues at micron resolutions without staining by contrast reagents is desirable in plant researches, and it can be achieved by an X-ray computed tomography (CT) with a phase-retrieval technique. Recently, a laboratory-based X-ray microscope adopting the phase contrast CT was developed as a powerful tool for the observation of weakly absorbing biological samples. Here we report the observation of unstained pansy seeds using the laboratory-based X-ray phase-contrast CT.    Results  A live pansy seed within 2 mm in size was simply mounted inside a plastic tube and irradiated by in-house X-rays to collect projection images using a laboratory-based X-ray microscope. The phase-retrieval technique was applied to enhance contrasts in the projection images. In addition to a dry seed, wet seeds on germination with the poorer contrasts were tried. The phase-retrieved tomograms from both the dry and the wet seeds revealed a cellular level of spatial resolutions that were enough to resolve cells in the seeds, and provided enough contrasts to delineate the boundary of embryos manually. The manual segmentation allowed a 3D rendering of embryos at three different stages in the germination, which visualized an overall morphological change of the embryo upon germination as well as a spatial arrangement of cells inside the embryo.    Conclusions  Our results confirmed an availability of the laboratory-based X-ray phase-contrast CT for a 3D-structural study on the development of small seeds. The present method may provide a unique way to observe live plant tissues at micron resolutions without structural perturbations due to the sample preparation.

Visualization of internal 3D structure of small live seed on germination by laboratory-based X-ray microscopy with phase contrast computed tomography