Multiscale 3d Reconstruction Of Ginkgo Biloba Embryo And Arabidopsis Thaliana Seed

As the unique reproductive organs of plants,seeds are not only the source of nutrition for seedling growth,but also the food source for human and animals.They have the important function of continuation and transmission of species development.Three-dimensional reconstruction technology can capture the small probability events in cells and tissues.Analysis for the differences of three-dimensional structure and the differences of three-dimensional structure in a variety of specific areas from the organizational level can deeply understand their mechanism.Therefore,the use of 3D reconstruction technology is of great significance for the study of internal structure characteristics and spatial distribution.In this study,Ginkgo biloba embryos and Arabidopsis thaliana wild-type seeds were used as the research materials.Three kinds of three-dimensional reconstruction techniques,including micro-CT（computed tomography）,LSFM（light sheet fluorescence microscopy）and automatic collector of ultrathin sections scanning electron microscopy（Auto CUTS-SEM）,were used to generate multi-scale structural data,which were processed with software Image J,CTan and Imaris for functional analysis.The three-dimensional structure and cell arrangement of seeds were deeply explored.The main results are as follows:（1）Compared with osmium tetroxide（Os O₄）,cesium iodide（Ce I）treatment of Ginkgo biloba embryos showed better contras and clearer background in micro-CT image,which would be more suitable for micro-CT samples.The area and volume of the embryos were smaller in the primary development stage,while increased in the late mature stage.（2）The porosity of analysis in Ginkgo biloba embryos showed that the mature embryo in the early stage had the highest porosity,while the late mature embryo had the lowest.In addition,the porosity of cotyledon was higher than that of radicle.（3）The number of secretory cavities of Ginkgo biloba embryo was the least at the early stage of mature embryo and the volume and surface area were also small.With the number of secretory cavities increased,the volume and surface area gradually increased at the late stage of embryo maturation.（4）The micro-CT images of Arabidopsis thaliana seeds showed that the seeds contained cotyledons,hypocotyls,seed coat,aleurone layer and other structures.In addition,the three-dimensional analysis of micro-CT image showed that the surface area,volume and porosity of Col-0 were the largest,while the surface area,volume and porosity of Ws were the smallest.（5）Spatial topological analysis showed that the degree,closeness and BC of Ler are relatively larger than those of the other two materials,while the eccentricity is the smallest.（6）The method of chemical fixation combined with en-bloc staining is suitable for the collection of serial ultrathin sections of Arabidopsis thaliana.The serial ultrathin section library of Arabidopsis thaliana embryo was established by using Auto CUTS-SEM technology,and the three-dimensional reconstruction of tissue proved that the technology is suitable for large plant tissue samples.（7）The structure of vascular tissue in each ultrathin section was extracted,and processed with three-dimensional reconstruction showed that vascular tissue was distributed in the whole Arabidopsis thaliana embryo like branches.The ultrastructure of Arabidopsis thaliana embryo showed that the inner structure of Arabidopsis thaliana embryo cells contained abundant organelles.In conclusion,this study demonstrated the role of three techniques in the analysis of the three-dimensional development and evolution process of Ginkgo biloba embryo and secretory cavity,as well as in the analysis of the tissue cell arrangement of Arabidopsis thaliana seed from different scales,providing technical support for exploring plant seed development and three-dimensional structure.