## 4 Discussion  
Phase-contrast tomography provided by synchrotron radiation is a relevant approach for exploring the three-dimensional organization of highly hydrated plant tissues, without the destructive cut and the use of any contrasting agent. In particular, it was possible to identify a large number of biological structures within whole wheat grains at various stages of development without cutting artefacts. Low-resolution setup in synchrotron provided high-quality 3D images of the whole grains, with an enhanced level of contrast compared to that obtained from classical laboratory microtomography. However, some reconstruction defaults like rings artefact could be still noticed. They were thin enough to allow identification of the structures of interest. Image processing allowed for segmentation of the grains, and visualization of their 3D shapes. This information is of interest for the study of the development of the grain. When using a higher resolution, a larger number of details can be revealed from 3D images: more tissue layers can be identified, cells can be better individualized, and dense molecular structures (starch grains) can be observed within cells. However, there are several drawbacks using synchrotron-based tomography. The synchrotron facilities are restricted in access and the planification should be organized months in advance. Therefore, one should carefully plan the plant production (especially when it takes a rather long time to get plants at the proper stage; in our case 5 months), experimental setup, and anticipate possible difficulties. For instance, our settings were designed to fix the grains in a vertical position in order to scan several grains without further manipulations and prevent dehydration of tissues. However, the tape we used produced a strong signal and was too tightly attached to the grains at some positions. For some applications, such as studying the shape or dimensions of organs, lab-based microtomography remains a relevant tool. Details can be compared with that obtained from microscopy: microscopy provides finer anatomical details, as well as information about the local chemical composition. However, microscopy imaging requires physical cross-sections of the sample. This results in usual 2D information, with approximate location and orientation, and the presence of artifacts caused by the preparation steps of the slice (sectioning, drying, embedding, staining, etc. In literature, 3D reconstruction of entirely sectioned and imaged grain samples is scarce because it is complicated and time-consuming [15]. The three-dimensional nature of the images resulting from synchrotron tomography lets us envision the investigation of the 3D architecture of the tissues, their organization within the grain, as well as the 3D morphology of the cells within specific tissues. Segmentations of some tissues and cells are feasible; however, systematic segmentation of tissues and cells for a comprehensive study remains impossible so far with the experimental setups and processing methods used. The exploration and interpretation of dense 3D data have however shown to be difficult. Generating and interpreting 3D sliced with arbitrary orientation requires orientation skills, the opacity of the tissues makes 3D rendering difficult, and the size of the data sets (several GB) requires powerful hardware and/or software. Further exploration of dense 3D data will require smart integration of 3D image processing algorithms together with an interactive selection of regions or adjustment of algorithm parameters. Comparison with optical microscopy images illustrates the complementarity of the imaging approaches. Promising future works would be to combine the detailed information obtained from 2D microscopy with the global 3D anatomical description of the grain. This would require efficient algorithms for 2D to 3D registration of images from different modalities, as well as a more exhaustive sampling of the wheat grain using microscopy.