Section Methods For Cereal Mature Whole Kernel And Their Application In Investigation Of Starch In Maize Caryopsis

Cereal mature kernels play an important role in human diet,livestock farming,and industrial production.The endosperm is the main part of the kernel and full of storage starch and protein.The morphology,quantity and spatial distribution of endosperm cell,starch and protein in the kernel affect the yield and quality of the kernel.More and more studies have shown that the endosperm cell,starch and protein in different regions of mature kernel are heterogeneous,especially in the high-amylose cereal endosperm.Therefore,there is a growing interest in investigation of endosperm cell,starch and protein in different regions of mature kernel.However,the section of mature whole kernel cannot be prepared using the existing section methods,leading to that the in situ studies of different regions in kernel are difficult.In the present study,the novel resin section method and non-embedded section method were established to prepare the sections of cereal mature whole kernels.A novel method was also established to observe and quantitatively analyze endosperm cells.The spatiotemporal accumulation and characteristics of starch in developing maize caryopsis and the heterogeneity of starch granules in high-amylose maize endosperm were investigated using the above established methods.This study could provide technical methods for in situ obseryation and analysis of different regions of cereal mature kernels,and reveal the heterogeneity of endosperm cell,starch and protein in the kernel.In addition,it could also enrich the knowledge of spatiotemporal accumulation and characteristics of starch in developing maize caryopsis,and provide some important information for the quality breeding of cereal crops and their processing and utilization.The main results are as follows:1.The establishment and applications of novel resin section method for preparing the sections of cereal mature whole kernels.After the cereal mature kernels were softened and fixed using a fixation solution,the whole kernels were cut transversely or longitudinally with a sharp razor blade to prepare the sample blocks.The samples were then gradually dehydrated with gradient ethanol and infiltrated with LR White resin.Finally,the samples were embedded in pure LR White resin to obtain the resin embedding blocks.The resin embedding blocks were clamped firmly in the sample holder of an ultramicrotome,trimmed using a razor blade and glass knife,and dry-sectioned with a glass knife.The sections were controlled using a copper hook to prevent the sections from curling or sticking to the glass knife or the block,and transferred to the slides.The prepared resin sections of cereal whole kernels were stained with Fluorescent brightener 28,iodine solution and Coomassie brilliant blue R250 to clearly display the endosperm cell wall,starch and storage protein,respectively,which could be used to analyze the morphology,number and spatial distribution of endosperm cell,starch and protein in the kernels.The sections of mature rice,maize and wheat whole kernels showed that there were significant differences in the morphology of endosperm cells and starch granules in different regions of endosperm,and the storage protein was mainly distributed in the sub-aleurone layer.The established method for the sections of cereal mature whole kernels is also suitable for sections of the developing,germinated and cooked whole kernels.2.The establishment and application of novel non-embedded section method for preparing the section of cereal mature whole kernels.The mature kernel or the resin block with the kernel sample was firstly clamped in the sample holder of the ultramicrotome.The sample surface was flattened with a sharp razor blade,and further polished into the smooth surface with a glass knife.The vitreous or transparent kernels could be directly sectioned under the microtome with a glass knife,but the floury kernels needed to be treated with nail polish before sectioning.The transparent nail polish was added to the floury region of the kernel,and waited for a short time to make it dry and solidify.After that,the sample was sectioned.During the sectioning process,the sections were controlled with copper hooks to prevent the slices from curling and sticking to the glass knife or the block.Finally,the sections were transferred to a glass slide.The sections of rice,maize,wheat and barley whole kernels prepared by the established method could clearly show the morphology of starch stained with iodine solution.The starch granules in different regions of cereal endosperm were heterogeneous in morphology and quantity.The shape and size of starch granules in the transparent(or vitreous)and floury regions of endosperm were completely different.The sections of high-amylose rice and maize whole kernels exhibited that the starch in different regions of the endosperm not only had different shapes and sizes,but also had significant differences in starch properties.The non-embedded sections could be used to not only observe the morphology of the starch,but also in situ analyze the characteristics of the starch.In addition,it could also be used to rapidly isolate mutants with altered starch morphology from a large number of cereal mutants due to the simple and rapid preparation of kernel section.3,The establishment and application of a new processing method for images of endosperm cells.The image of endosperm cells was firstly opened in the Photoshop software,and the interest region of endosperm cells was copied and pasted onto a new canvas with black background.The "Pen tool" in Photoshop software was used to accurately outline the endosperm cells and saved as "Paths".After that,the "Brush tool" was used to stroke the "Paths" to clearly display the endosperm cell walls on the image background.After the black background layer was duplicated and moved over the image layer,the "Paths”was stroked again on the "Background layer copy" to highlight the endosperm cell wall on the black background.Finally,the processed image was saved.The images,which were photographed from resin sections of mature rice,maize and wheat kernels stained with iodine solution,both safranin O and methyl violet,and fluorescent brightener 28,respectively,were processed using the established method.The processed images could not only clearly and visually display the morphology of endosperm cells,but also be used to directly and quantitatively analyze the morphological parameters of endosperm cells including area,length,width and circularity(roundness)using various morphological analysis softwares.After the resin sections of whole kernels were processed using this image processing method,they could be used to not only observe and quantitatively analyze the morphology of endosperm cells in different regions of developing and cereal mature kernels,and but also analyze the formation of endosperm tissue during caryopsis development.The processed image was more accurate and efficient for analyzing morphological parameters using image analysis software than the unprocessed image.4.The application of section method on spatiotemporal accumulation of starch in developing maize whole caryopsis.The sections of developing normal and waxy maize whole caryopses were prepared using the established section method of cereal whole kernel.The sections were stained with iodine solution to clearly exhibit the spatiotemporal accumulation of starch in developing caryopses.The results showed that pericarp starch took the form of compound granules,was distributed in the bottom of caryopses,and degraded from the top to the bottom.Embryo starch mostly took the form of simple granules and accumulated in the scutellum beginning approximately 10 days after pollination.In the endosperm,starch accumulated longitudinally from the top to the bottom and transversely from the center to the periphery with caryopsis development.The peripheral endosperm cells synthesized starch faster than did the inner ones.Simple and compound starches were both observed,but the compound starch granules were distributed in the central region of the endosperm.At a late stage of development,compound starch was only observed in the bottom central portion of the endosperm.The starches were isolated from the endosperm,embryo and pericarp at 20 days after pollination,their amylose contents and crystalline structure were investigated.The pericarp and embryo starch had the highest and lowest amylose content,respectively,in normal maize.The amylose contents of waxy maize pericarp and embryo starches were similar to that of normal maize embryo,but amylose was hardly detected in the endosperm of waxy maize.The starches from the endosperm,embryo and pericarp of normal and waxy maize all had A-type crystallinity.Expression analysis of genes related to starch synthesis indicated that the granule-bound starch synthase ? and ? involved in amylose synthesis were expressed only in endosperm and embryo,respectively,but both expressed in the pericarp.Soluble starch synthase,starch branching enzymes and starch debranching enzymes involved in amylopectin synthesis were all detected in the endosperm,embryo and pericarp,but their expression levels were significantly different.5.The application of whole kernel section method on heterogeneous starch granules of high-amylose maize endosperm.The heterogeneity of endosperm starch granules was investigated in maize inbreds with 19.2%,23.0%,36.4%,41.8%and 62.5%amylose contents.The individual,biphasic,aggregate,,and elongated starch granules were detected in maize endosperm.The individual granules existed mainly in maize inbreds with 19.2%and 23.0%amylose content,the biphasic granules were observed mainly in maize inbreds with 36.4%and 41.8%amylose content,and the aggregate and elongated granules were detected mainly in maize inbred with 62.5%amylose content.The resin section of whole kernel showed that the starch granules in the outer and bottom of endosperm gradually became small,and the aggregate and elongated granules gradually formed with the increase of amylose content.The individual,biphasic,aggregate and elongated starch granules existed mainly in the central endosperm.The amylose content of starch in the center endosperm was lower than that in the outer and bottom of endosperm among maize inbreds with amylose contents from 19.2%to 41.8%.The difference in amylose content became more significant with the increase of amylose content.The crystalline types of starches had no difference among different regions of endosperm for the same maize inbred.The biphasic starch granules were detected in the central region of developing endosperm in maize inbred with 36.4%amylose content,and gradually distributed in the upper region of central endosperm with the development of maize caryopsis.