Energy-dispersive x-Ray Analysis of Phosphorus, Potassium, Magnesium, and Calcium in Globoid Crystals in Protein Bodies from Different Regions of Cucurbita maxima Embryos

The seeds of Cucurbita maxima contain protein bodies with electrondense globoid crystals. Because of their density globoid crystals are ideal material for energy-dispersive x-ray (EDX) analysis studies of elemental composition. Fixation trials were carried out to test globoid crystal extraction during glutaraldehyde fixation, water washing, and ethanol dehydration. Glutaraldehyde fixation without subsequent washing or dehydration alone produced no significant changes in elemental composition of cotyledon globoid crystals. If glutaraldehyde fixation was followed by water washes or ethanol dehydration there was some loss of the major globoid crystal elements but the relative percentages of the elements P, K, Ca, and Mg remained relatively unchanged. In this paper results of a study of the P, K, Mg, and Ca content of globoid crystals in different tissues of squash embryos are presented. The globoid crystals in the radicle were found to be the least dense in the embryo. Globoid crystals from all embryo regions contained P, K, and Mg. In the various embryo regions P and Mg maintained relatively constant proportions of the globoid crystal composition while K and Ca varied. Of particular significance is the distribution of Ca which is generally an immobile element. Calcium was found in highest amounts in the globoid crystals of the radicle and stem regions while globoid crystals in much of the cotyledon contained little, if any, Ca. The Ca storage thus seems to be spatially arranged in a manner that would aid early growth of the root-shoot axis.     

X-ray Analysis Studies of Elements Stored in Protein Body Globoid Crystals of Triticum Grains

Energy-dispersive x-ray analysis was used to investigate the elemental storage within protein bodies, specifically the globoid crystals, in grains of wheat. Areas of the grain investigated included various parts of the embryo, the aleurone layer plus starchy endosperm near the embryo and the aleurone layer plus starchy endosperm farthest from the embryo. Variations did occur grain-to-grain, cell-to-cell and, in certain regions, intracellularly. No protein bodies with electron-dense globoid crystals were found in the starchy endosperm. Generally globoid crystals contained P, K, and Mg in all areas investigated. Globoid crystals from the aleurone layer farthest from the embryo on occasion contained Ca, whereas aleurone globoid crystals near the embryo sometimes contained Fe. In most of the embryo regions examined, a few globoid crystals contained Ca along with P, K, and Mg. No specific pattern to the Ca distribution could be found. Welldefined elemental distribution occurred with Mn. Manganese was found only in globoid crystals located in the base and midregions of the stele in the radicle. Thus, in wheat there is some specific distribution of minerals dependent upon cell type and/or position in the grain.     

Composition of globoid crystals from embryo protein bodies in five species of cucurbita

Previous energy-dispersive x-ray analysis studies of globoid crystal composition in seed protein bodies gave an indication that there might be a correlation between seed size and the type of elements stored in globoid crystals. This possibility was tested by conducting energy-dispersive x-ray analysis studies of P, K, Mg, and Ca levels in globoid crystals of four embryo regions (radicle, stem, cotyledon center palisade mesophyll, cotyledon center spongy mesophyII) in each of five different Cucurbita species (C. mixta, C. moschata, C. foetidissima, C. pepo, and C. andreana). The species were chosen to provide a range of seed size and weight. Globoid crystals from all embryo regions in all five species contained P, K, and Mg. Some variations in the levels of these elements did occur but there was no consistent pattern with regard to area of the seed or with regard to seed size. Calcium distribution showed significant variations. In species with large seeds (C. mixta, C. moschata) Ca was mainly found in globoid crystals in the radicle. Globoid crystals in species with small seeds (C. foetidissima, C. pepo, C. andreana) contained Ca in all embryo regions tested. The results of this study support the concept that Ca distribution in globoid crystals can be correlated with seed weight.     

玉米胚胎发育、萌发与胚的结构及子叶二型性

运用扫描电镜与半薄切片技术,观察了玉米(Zea mays L.)的胚发育过程,得到以下认识:第一、关于原胚.玉米合子细胞分裂形成的原胚分为胚柄、胚基与胚体三部分.胚柄短小,寿命短暂.胚基具有生长带,纵向伸长长度大,胚基的上部参与形成胚根鞘,其余部分干缩后附在胚根鞘末端.第二、玉米胚的背腹极性及二型子叶.原胚初期胚体出现背腹极性,腹面的细胞小,细胞质稠密,液泡较少;背面的细胞较大,细胞质稠密度略低,液泡较多.原胚后期胚体分化为腹部与背部,腹部从腹面的中央突起,背部在腹部的周围(从左至右侧)及整个胚体背面.进入幼胚时期,腹部分化为胚芽鞘、生长锥、胚轴、胚根和胚根鞘(大部分).期间,胚芽鞘原基和根原始细胞的分化都从胚体的中轴部位开始,然后向两侧和四周扩展,表现出胚体腹面形态的两侧对称性.原胚的背部形成盾片原基,盾片原基经历向左、右、上、下的迅速扩展和加厚的生长,将整个腹部所分化形成的构件藏于盾片的纵沟之中,最后盾片从纵沟的边缘长出的左、右侧鳞均向胚体的中轴线生长,完整显示出玉米胚腹面的两侧对称.玉米胚由腹部顶端形成胚芽鞘和生长锥的情况与水稻胚的胚芽鞘(顶生子叶)和生长锥的形成相同,玉米的胚芽鞘也是顶生子叶,盾片则是侧生子叶.玉米异型子叶的由来在于胚体的背腹极性.第三、玉米胚的真实形态结构及胚胎发育时期的划分.玉米胚是一个胚轴,其顶部是具胚芽鞘的胚芽,中部是具侧生子叶(盾片)的胚轴,下部是具胚根鞘的胚根.盾片从背面到腹面包住整个胚轴系统,在胚的腹面上可见从盾片边缘衍生的左、右侧鳞的边缘相迭,只在接缝线的上、下端留下蝌蚪状的小孔,使胚芽鞘和胚根鞘的末端稍露出.胚胎发育分为4个时期: 1.原胚期--从合子细胞分裂开始至分化背部与腹部为止;2.腹部迅速分化期;3.盾片快速生长期;4.侧鳞生长、胚套形成期.第四、获取垂直于胚腹面正中央纵切面是正确认识玉米胚形态的关键.     

玉米胚胎发育、萌发与胚的结构及子叶二型性

运用扫描电镜与半薄切片技术,观察了玉米(Zea mays L.)的胚发育过程,得到以下认识：第一、关于原胚。玉米合子细胞分裂形成的原胚分为胚柄、胚基与胚体三部分。胚柄短小,寿命短暂。胚基具有生长带,纵向伸长长度大,胚基的上部参与形成胚根鞘,其余部分干缩后附在胚根鞘末端。第二、玉米胚的背腹极性及二型子叶。原胚初期胚体出现背腹极性,腹面的细胞小,细胞质稠密,液泡较少;背面的细胞较大,细胞质稠密度略低,液泡较多。原胚后期胚体分化为腹部与背部,腹部从腹面的中央突起,背部在腹部的周围(从左至右侧)及整个胚体背面。进入幼胚时期,腹部分化为胚芽鞘、生长锥、胚轴、胚根和胚根鞘(大部分)。期间,胚芽鞘原基和根原始细胞的分化都从胚体的中轴部位开始,然后向两侧和四周扩展,表现出胚体腹面形态的两侧对称性。原胚的背部形成盾片原基,盾片原基经历向左、右、上、下的迅速扩展和加厚的生长,将整个腹部所分化形成的构件藏于盾片的纵沟之中,最后盾片从纵沟的边缘长出的左、右侧鳞均向胚体的中轴线生长,完整显示出玉米胚腹面的两侧对称。玉米胚由腹部顶端形成胚芽鞘和生长锥的情况与水稻胚的胚芽鞘(顶生子叶)和生长锥的形成相同,玉米的胚芽鞘也是顶生子叶,盾片则是侧生子叶。玉米异型子叶的由来在于胚体的背腹极性。第三、玉米胚的真实形态结构及胚胎发育时期的划分。玉米胚是一个胚轴,其顶部是具胚芽鞘的胚芽,中部是具侧生子叶(盾片)的胚轴,下部是具胚根鞘的胚根。盾片从背面到腹面包住整个胚轴系统,在胚的腹面上可见从盾片边缘衍生的左、右侧鳞的边缘相迭,只在接缝线的上、下端留下蝌蚪状的小孔,使胚芽鞘和胚根鞘的末端稍露出。胚胎发育分为4个时期：1．原胚期——从合子细胞分裂开始至分化背部与腹部为止;2．腹部迅速分化期;3．盾片快速生长期;4．侧鳞生长、胚套形成期。第四、获取垂盲于胚腹面正中央纵切面是正确认识玉米胚形态的关键。     

Mineral reserves in castor beans: the dry seed

Elemental composition and distribution of the mineral reserves in the endosperm and embryo tissues of Ricinus communis cultivars Hale and Zanzibarensis were investigated. Energy dispersive x-ray analysis was used to determine the elemental composition of the globoid crystals, while atomic absorption spectrometry allowed quantification of the elements, particularly Ca, in various seed regions. No major differences were found between the two cultivars with regard to the elemental distribution in globoid crystals. While the majority of globoid crystals contained P, K, and Mg, the occasional one also contained Ca. In extremely rare instances, Fe was detected in globoid crystals. Ca-containing globoid crystals were more common in provascular cell protein bodies in the stem and radicle. Polarized light microscopy, micro-incineration, and acid solubility tests demonstrated the presence of calcium oxalate crystals in the innermost testa which adheres to the endosperm and is often mistakenly identified as endosperm. Atomic absorption spectrometry revealed that most of the calcium present in castor bean seeds is localized in the testa. On a perseed-region basis, the much larger endosperm contains more Ca than does the embryo. However, on a unit-weight basis, the radicle-plus-stem regions contain considerably more Ca than does the cotyledon or endosperm, an observation that is consistent with the observed distribution pattern for Ca-containing globoid crystals.     

Protein bodies in seeds

Membrane bound protein bodies (aleurone grains) are thought to be the main subcellular location of protein and mineral storage in seeds. In addition to structurally homogeneous proteinaceous matrix, protein bodies may contain protein crystalloids, electron–dense globoid crystals, electron–transparent soft globoids, and crystals of calcium oxalate. Protein crystalloids vary in shape, size and number. For example, cotyledon mesophyll cell protein bodies in the Cucurbitaceae generally contain protein crystalloids whereas those of Compositae and Cruciferae do not. Globoid crystals, which are rich in phytin, vary greatly in size and number per protein body. Some species have numerous small globoid crystals per protein body whereas others have one or two large globoid crystals per protein body. Phosphorus and various cations (K, Mg, Ca, Fe, Ba, Mn) located in globoid crystals can be studied with an energy dispersive X–ray (EDX) analysis system mounted on a transmission electron microscope. In some cases, cations such as Ca, Mn and Fe are specifically localized in globoid crystals of certain tissues or embryo regions. Further investigations may allow elemental composition of globoid crystals to be used in studies of systematics. Biref–ringent crystals, sometimes in the form of single large crystals but frequently in the form of druses, are present in protein bodies of some species. At least some endosperm protein bodies of all Umbelliferous species examined contain druse crystals. While seed protein bodies of relatively few species have been studied with electron microscopy, there are indications that protein bodies could be a useful character for studies in plant systematics.     

Spatial X-ray fluorescence micro-imaging of minerals in grain tissues of wheat and related genotypes

Main conclusion

Wheat and its related genotypes show distinct distribution patterns for mineral nutrients in maternal and filial tissues in grains. X-ray-based imaging techniques are very informative to identify genotypes with contrasting tissue-specific localization of different elements. This can help in the selection of suitable genotypes for nutritional improvement of food grain crops.

Abstract

Understanding mineral localization in cereal grains is important for their nutritional improvement. Spatial distribution of mineral nutrients (Mg, P, S, K, Ca, Fe, Zn, Mn and Cu) was investigated between and within the maternal and filial tissues in grains of two wheat cultivars (Triticum aestivum Cv. WH291 and WL711), a landrace (T. aestivum L. IITR26) and a related wild species Aegilops kotschyi, using micro-proton-induced X-ray emission (µ-PIXE) and micro-X-ray fluorescence (µ-XRF). Aleurone and scutellum were major storage tissues for macro (P, K, Ca and Mg) as well as micro (Fe, Zn, Cu and Mn) nutrients. Distinct elemental distribution patterns were observed in each of the four genotypes. A. kotschyi, the wild relative of wheat and the landrace, T. aestivum L. IITR26, accumulated more Zn and Fe in scutellum and aleurone than the cultivated wheat varieties, WH291 and WL711. The landrace IITR26, accumulated far more S in grains, Mn in scutellum, aleurone and embryo region, Ca and Cu in aleurone and scutellum, and Mg, K and P in scutellum than the other genotypes. Unlike wheat, lower Mn and higher Fe, Cu and Zn concentrations were noticed in the pigment strand of A. kotschyi. Multivariate statistical analysis, performed on mineral distribution in major grain tissues (aleurone, scutellum, endosperm and embryo region) resolved the four genotypes into distinct clusters.     

应用电子探针对植物根际和根内营养元素微区分布的探讨

用电子探针可检测出玉米、大豆根际和根内含有Na,Mg,Al,Si,P,S,Cl,K,Ca,Ti,Fe,Cu和Zn 13种元素。这些元素在根际土壤、粘液层和根组织内的含量分布有一定的规律性。除Si,Al,Ca,Fe在根际土壤中峰值较高外,Ti仅在土壤中达到可检测量;S,Fe和Zn富集在粘液层,Mg,P,Cl只在根组织内才有较明显的峰。这些规律可作为区分根—土界面的参考指标。K含量在根内明显高于根际土壤,并由表皮层到中柱径向增加;Ca则与K不同,且受植物种类的影响。     

Radicle ofEchinocactus platyacanthus (Cactaceae)

Radicle of matureEchinocactus platyacanthus embryo is approximately 320 m long and represents less then 1/7 of the embryonal axis length. The radicle-hypocotyl boundary can be distinguished according to the striking difference in the size and shape of cells in protoderm and procambium, as well as discontinuity and different number of the cell files in the ground meristem. The root cap is small, consists of 4 layers of cells covering the apex of the radicle. The upper limit of the root cap is approximately 100 m closer towards the radicle tip than the radicle-hypocotyl boundary. Ultrastructure of radicle cells showed numerous lipid bodies as is typical for other oily seeds. Protein bodies of variable structure were also present together with other cell structures. Striking differences in protein body structure were found when protoderm and ground meristem were compared. Several small globoid crystals were present in each protein body of the protoderm, while protein bodies in the radicle ground meristem mostly contained one large globoid crystal. X-ray microanalysis revealed presence of P, K and Mg in all analyzed globoid crystals. Fe, Ca and Zn were detected in some of them.Abbreviations EDX microanalysis energy-dispersive X-ray microanalysis - GC(s) globoid crystals - ICP spectroscopy ion-coupled plasma spectroscopy - LM light microscopy - PB(s) protein bodies - SEM scanning electron microscopy - TEM transmission electron microscopy     

Immunodetection and immunolocalization of globulin storage proteins during zygotic and somatic embryo development in Zea mays

Globulins (GLB) are storage proteins that accumulate to high levels during zygotic embryo development of Zea mays L. We visualized the distribution of GLB during zygotic embryo development by immunolabelling of polyethylene glycol sections with a GLB-specific antiserum and a fluorescent secondary antibody. In sections of embryos at 10 days after pollimation (DAP), GLB were detected in the scutellar node only. Sections of embryos of 17 DAP showed, besides the presence of GLB in the scutellar node, the presence of a low amount of GLB in the coleoptile and the leaf primordia. In 30-DAP embryos GLB were localized in the root, the coleorhiza, the leaf primordia, the coleoptile and in all cells of the scutellum with the exception of the epidermis and the pro-vascular tissues. The subcellular location of GLB was visualized by immunolabelling of ultrathin sections with anti-GLB and a gold-conjugated secondary antibody. Scutellum cells and root cortex cells of 30-DAP embryos were packed with protein storage vacuoles (PSV), which differed in electron density. GLB were either evenly distributed throughout the PSV or were localized in electron-dense inclusions within the PSV. SDS-PAGE and immunoblot analysis of total protein extracts indicated the presence of a low amount of the GLB1 processing intermediate proGLB1'in globular as well as mature somatic embryos. After maturation on an ABA-containing medium, somatic embryos showed the additional presence of the next GLB1 processing intermediate GLB1. By immuno-electron microscopy it was possible to localize GLB in globular deposits in PSV in scutellum cells of these somatic embryos.     

Immunodetection and immunolocalization of globulin storage proteins during zygotic and somatic embryo development in Zea mays

Globulins (GLB) are storage proteins that accumulate to high levels during zygotic embryo development of Zea mays L. We visualized the distribution of GLB during zygotic embryo development by immunolabelling of polyethylene glycol sections with a GLB-specific antiserum and a fluorescent secondary antibody. In sections of embryos at 10 days after pollimation (DAP), GLB were detected in the scutellar node only. Sections of embryos of 17 DAP showed, besides the presence of GLB in the scutellar node, the presence of a low amount of GLB in the coleoptile and the leaf primordia. In 30-DAP embryos GLB were localized in the root, the coleorhiza, the leaf primordia, the coleoptile and in all cells of the scutellum with the exception of the epidermis and the pro-vascular tissues. The subcellular location of GLB was visualized by immunolabelling of ultrathin sections with anti-GLB and a gold-conjugated secondary antibody. Scutellum cells and root cortex cells of 30-DAP embryos were packed with protein storage vacuoles (PSV), which differed in electron density. GLB were either evenly distributed throughout the PSV or were localized in electron-dense inclusions within the PSV. SDS-PAGE and immunoblot analysis of total protein extracts indicated the presence of a low amount of the GLB1 processing intermediate proGLB1' in globular as well as mature somatic embryos. After maturation on an ABA-containing medium, somatic embryos showed the additional presence of the next GLB1 processing intermediate GLB1'. By immuno-electron microscopy it was possible to localize GLB in globular deposits in PSV in scutellum cells of these somatic embryos.     

Histochemical and ultrastructural studies on <Emphasis Type="Italic">Salix alba</Emphasis> and <Emphasis Type="Italic">S. matsudana</Emphasis> seeds

Mature seeds of Salix alba L. and Salix matsudana Koidz. are exendospermous and consist of an embryo and a seed coat. Ultrastructural studies show the presence of protein bodies, lipid bodies, chloroplasts, and a nucleus in the cells of most of the embryo tissues. Protein bodies always contain two or more globoid crystals. Energy-dispersive X-ray analysis of globoid crystals revealed the presence of P, K, Mg and Ca as the main constituents in all tissues. The chloroplasts present well-developed grana and, frequently, starch grains in the stroma. In cells of apical meristems, plastid endomembranes are non-organised in grana and deposits of phytoferritin are present in the stroma. Some cells of the subdermal layers of the cotyledons and hypocotyl-radicle axis present a large central vacuole and a narrow peripheral band of cytoplasm within which the protein bodies are scarce. Seeds of the two species studied here have recently been characterised as orthodox with short viability. The present study was carried out in an attempt to advance in the characterisation of these seeds as part of a comprehensive study of Salicaceae seeds.     

Calcium distribution in globoid crystals of cucurbita cotyledon protein bodies

Energy-dispersive x-ray analysis was used to investigate the location of globoid crystals with relatively high Ca levels within cotyledons of Cucurbita maxima, Cucurbita mixta, and Cucurbita andreana. The small globoid crystals in both upper and lower epidermal cells commonly contained Ca. Ca was present in globoid crystals of all provascular regions with the exception of the very small provascular regions of C. maxima. In C. maxima and C. mixta cotyledons, some cases were observed where Ca was found in the globoid crystals of the first layer of mesophyll cells surrounding the provascular region, but in general Ca was absent from globoid crystals of palisade and spongy mesophyll cells. In C. andreana, globoid crystals of palisade and spongy mesophyll cells commonly contained at least some Ca. Cell position and cell type are factors affecting the Ca content of globoid crystals in protein bodies.     

Spatial Distribution of Epigenetic Modifications in Brachypodium distachyon Embryos during Seed Maturation and Germination

Seed development involves a plethora of spatially and temporally synchronised genetic and epigenetic processes. Although it has been shown that epigenetic mechanisms, such as DNA methylation and chromatin remodelling, act on a large number of genes during seed development and germination, to date the global levels of histone modifications have not been studied in a tissue-specific manner in plant embryos. In this study we analysed the distribution of three epigenetic markers, i.e. H4K5ac, H3K4me2 and H3K4me1 in ‘matured’, ‘dry’ and ‘germinating’ embryos of a model grass, Brachypodium distachyon (Brachypodium). Our results indicate that the abundance of these modifications differs considerably in various organs and tissues of the three types of Brachypodium embryos. Embryos from matured seeds were characterised by the highest level of H4K5ac in RAM and epithelial cells of the scutellum, whereas this modification was not observed in the coleorhiza. In this type of embryos H3K4me2 was most evident in epithelial cells of the scutellum. In ‘dry’ embryos H4K5ac was highest in the coleorhiza but was not present in the nuclei of the scutellum. H3K4me1 was the most elevated in the coleoptile but absent from the coleorhiza, whereas H3K4me2 was the most prominent in leaf primordia and RAM. In embryos from germinating seeds H4K5ac was the most evident in the scutellum but not present in the coleoptile, similarly H3K4me1 was the highest in the scutellum and very low in the coleoptile, while the highest level of H3K4me2 was observed in the coleoptile and the lowest in the coleorhiza. The distinct patterns of epigenetic modifications that were observed may be involved in the switch of the gene expression profiles in specific organs of the developing embryo and may be linked with the physiological changes that accompany seed desiccation, imbibition and germination.     

Morphogenesis in in vitro Culture of Terminal Floral Apex of Banana

The technique by which plantlets was able to be regenerated via organogenesis or and somatic embryogenesis in fruit banana were developed in this experiment. Somatic embryogenesis was induced and the embryoid of banana possessed typlcal structure of embryo: scutellum, coleoptile and coleorhiza in monocotyledon embryo.     

High resolution structures of p-aminobenzamidine- and benzamidine-VIIa/soluble tissue factor: unpredicted conformation of the 192-193 peptide bond and mapping of Ca2+, Mg2+, Na+, and Zn2+ sites in factor VIIa

Factor VIIa (FVIIa) consists of a gamma-carboxyglutamic acid (Gla) domain, two epidermal growth factor-like domains, and a protease domain. FVIIa binds seven Ca(2+) ions in the Gla, one in the EGF1, and one in the protease domain. However, blood contains both Ca(2+) and Mg(2+), and the Ca(2+) sites in FVIIa that could be specifically occupied by Mg(2+) are unknown. Furthermore, FVIIa contains a Na(+) and two Zn(2+) sites, but ligands for these cations are undefined. We obtained p-aminobenzamidine-VIIa/soluble tissue factor (sTF) crystals under conditions containing Ca(2+), Mg(2+), Na(+), and Zn(2+). The crystal diffracted to 1.8A resolution, and the final structure has an R-factor of 19.8%. In this structure, the Gla domain has four Ca(2+) and three bound Mg(2+). The EGF1 domain contains one Ca(2+) site, and the protease domain contains one Ca(2+), one Na(+), and two Zn(2+) sites. (45)Ca(2+) binding in the presence/absence of Mg(2+) to FVIIa, Gla-domainless FVIIa, and prothrombin fragment 1 supports the crystal data. Furthermore, unlike in other serine proteases, the amide N of Gly(193) in FVIIa points away from the oxyanion hole in this structure. Importantly, the oxyanion hole is also absent in the benzamidine-FVIIa/sTF structure at 1.87A resolution. However, soaking benzamidine-FVIIa/sTF crystals with d-Phe-Pro-Arg-chloromethyl ketone results in benzamidine displacement, d-Phe-Pro-Arg incorporation, and oxyanion hole formation by a flip of the 192-193 peptide bond in FVIIa. Thus, it is the substrate and not the TF binding that induces oxyanion hole formation and functional active site geometry in FVIIa. Absence of oxyanion hole is unusual and has biologic implications for FVIIa macromolecular substrate specificity and catalysis.     

Cytology and Histology in Somatic Embryogenesis of Indica Rice

The somatic embryogenesis was established from mature dehulled seeds. The histological research showed that embryogenic calli were initiated first from absorbed cells of scutellum of mature seed. And then the embryoids derived from the surface of embryogenic callus. Having been the same structure like a zygotic embryo of rice, the embryoids possessed the major parts of scutellum, coleoptile and coleorhiza. In an embryoid, several developmental stages of pro-embryoid, including single embryogenic cells, two, four and multiple cell stage pro-embryeids and some abnormal embryoids were observed. It could be concluded from this experiment that the embryoid from somatic cell culture in Indica rice possessed an original form of a plant in structure like a zygotic did and derived from a single cell.     

Histology of somatic embryogenesis in cultured immature embryos of maize (Zea mays L.)

Summary The developmental histology of somatic embryo (=embryoid) formation in cultured immature embryos of hybrid maize cultivars (Zea mays L.) is described. Embryos cultured on media containing 2% sucrose formed distinct globular embryoids. These embryoids arose either directly by divisions confined to the epidermal and the subepidermal cells at the coleorhizal end of the scutellum or from a soft and friable embryogenic callus produced by them. On media containing 6% sucrose divisions were initiated in the cells adjacent to the procambium of the cultured embryos. Subsequently, zones of meristematic cells also were observed in the region of the node and in the basal portion of the scutellum. Mature, well organized somatic embryos as well as a compact nodular type of embryogenic callus were produced as a result of localized meristematic activity along the tip of the scutellum toward the coleorhiza. Some embryos formed only the compact type of callus, and shoot primordia were organized later in the surface layers of this callus.Abbreviations CH casein hydrolysate - MS Murashige and Skoog's nutrient medium - 2,4-D 2,4-dichlorophenoxyacetic acid     

Quantification of galactosylsphingosine in the twitcher mouse using electrospray ionization-tandem mass spectrometry.

Globoid cell leukodystrophy (Krabbe disease) is an autosomal recessive inherited neurodegenerative disorder caused by the deficiency of the lysosomal enzyme beta-galactosylceramidase. The pathogenesis of the disorder has been proposed to arise from the accumulation of the cytotoxic metabolite galactosylsphingosine (psychosine). The twitcher mouse is a naturally occurring murine model of globoid cell leukodystrophy. We have developed a rapid, sensitive, and specific mass spectrometric method for determining the galactosylsphingosine concentration in the tissues of twitcher mice. Galactosylsphingosine is extracted from the tissues in methanol, isolated using strong cation-exchange and C18 solid-phase extraction chromatography, and then directly analyzed using electrospray ionization-tandem mass spectrometry. A lactosylsphingosine internal standard has been employed for quantification. The assay demonstrated significant accumulation of galactosylsphingosine in the brain, spinal cord, and kidney of twitcher mice. It is anticipated that this method may be of use in the monitoring of experimental therapies for globoid cell leukodystrophy.