Embryo production through somatic embryogenesis can be used to study cell differentiation in plants

Somatic embryogenesis is the process by which somatic cells, under induction conditions, generate embryogenic cells, which go through a series of morphological and biochemical changes that result in the formation of a somatic embryo. Somatic embryogenesis differs from zygotic embryogenesis in that it is observable, its various culture conditions can be controlled, and a lack of material is not a limiting factor for experimentation. These characteristics have converted somatic embryogenesis into a model system for the study of morphological, physiological, molecular and biochemical events occurring during the onset and development of embryogenesis in higher plants; it also has potential biotechnological applications. The focus of this review is on embryo development through somatic embryogenesis and especially the factors affecting cell and embryo differentiation.     

Degradation of yolk platelets in the early amphibian embryo is regulated by fusion with late endosomes

The eggs of many animal species contain a large store of yolk platelets, lipid droplets and glycogen granules; these are consumed during early embryogenesis. However, the mechanisms by which degradation of these stored materials occurs during early embryogenesis are not clearly understood. The mechanisms underlying yolk degradation in amphibian (newt) embryos were investigated. Electron microscopy using an anion marker, cationic ferritin, revealed that yolk platelets were degraded after fusion with late endosomes containing primary lysosomes. Electron microscopy and the results of experiments using a number of reagents with selective effects on intracellular transport suggested that yolk degradation activity in early amphibian embryos may be regulated at the point of fusion between late endosomes and yolk platelets.     

Action of 1,25-dihydroxyvitamin D3 and parathyroid hormone on 45calcium uptake by the yolk sac membrane of chick embryos

During development, the chick embryo mobilizes the calcium it needs from two extraembryonic sources, initially from the yolk and later from the eggshell. Calcium may be hormonally regulated during avian embryogenesis, but details of this regulation are lacking. We investigated the effects of 1,25-dihydroxycholecalciferol [1,25(OH)2D3], bovine parathyroid hormone [bPTH], and vehicle [ethanol or saline] on blood calcium values and incorporation of 45Ca into the yolk sac membrane of 9, 12, and 15 day chick embryos. Control data were also collected from uninjected 6 day embryos. Solutions were injected directly into the yolk sac compartment 48 and 24 hours prior to the experiment. Exogenous 1,25(OH)2D3 induced hypercalcemia in all age groups examined, while exogenous PTH induced hypercalcemia in day 12 and 15 embryos. Small disks of yolk sac membrane were incubated in medium to which 45Ca was added and assayed for 45Ca content at various intervals after start of incubation. In control yolk sac tissue, the uptake of 45Ca was greatest in younger embryos with decreasing uptake at developmentally more advanced ages; 1,25(OH)2D3 treatment significantly enhanced the uptake of 45Ca into yolk sac tissue in all groups (9, 12, and 15 day embryos). PTH treatment caused a significant elevation in 45Ca uptake in the day 12 and 15 embryos.     

Antioxidants and total oxyradical scavenging capacity during grass shrimp, Palaemonetes pugio, embryogenesis

During embryogenesis in grass shrimp the capacity to scavenge oxyradicals increased as measured by the Total Oxyradical Scavenging Capacity (TOSC) assay. The increase in TOSC during embryogenesis was associated with increasing concentrations of a number of antioxidants, including coenzyme Q (ubiquinone), alpha-tocopherol and reduced glutathione. Glutathione concentrations ranged from 0.004 to 0.005 nmol/embryo in early embryo stages and reached concentrations between 0.16 to 0.23 nmol/embryo in late embryo stages. Ascorbate remained essentially constant (0.16-0.20 nmol/embryo) throughout embryogenesis and may provide the preponderance of TOSC during early embryo development. Carotenoids were associated with yolk lipovitellin and these antioxidants decreased as yolk was absorbed during embryogenesis. Astaxanthin and beta-carotene were identified in embryos with astaxanthin always the principal carotenoid. In early embryo stages there are maternally derived antioxidants but as embryogenesis proceeds there is an assembly of a complex antioxidant system by newly formed cells and tissues.     

Morphogenesis of extraembryonic membranes and placentation in Mabuya mabouya (Squamata, Scincidae)

Topological and histological analyses of Mabuya mabouya embryos at different developmental stages showed an extraembryonic membrane sequence as follows: a bilaminar omphalopleure and progressive mesodermal expansion around the whole yolk sac at gastrula stages; mesodermal split and formation of an exocoelom in the entire embryonic chamber at neurula stages; beginning of the expansion of the allantois into the exocoelom to form a chorioallantoic membrane at pharyngula stages; complete extension of the allantois into the exocoelom between limb-bud to preparturition stages. Thus, a placental sequence could be enumerated: bilaminar yolk sac placenta; chorioplacenta; allantoplacenta. All placentas are highly specialized for nutrient absorption from early developmental stages. The bistratified extraembryonic ectoderm possesses an external layer with cuboidal cells and a microvillar surface around the whole yolk sac, which absorbs uterine secretions during development of the bilaminar yolk sac placenta and chorioplacenta. During gastrulation, with mesodermal expansion a dorsal absorptive plaque forms above the embryo and several smaller absorptive plaques develop antimesometrially. Both structures are similar histologically and are active in histotrophic transfer from gastrula stages until the end of development. The dorsal absorptive plaque will constitute the placentome and paraplacentome during allantoplacental development. At late gastrula-early neurula stages some absorptive plaques form chorionic concavities or chorionic bags that are penetrated by a long uterine fold and seem to have a specialized histotrophic and/or metabolic role. The extraembryonic mesoderm does not ingress into the yolk sac and neither an isolated yolk mass nor a yolk cleft are formed. This derived pattern of development may be related to the drastic reduction of the egg size and obligatory placentotrophy from early developmental stages. Our results show new specialized placentotrophic structures and a novel arrangement of extraembryonic membrane morphogenesis for Squamata.     

Ontogeny of the mouse hematopoietic system

In vertebrates the extraembryonic mesoderm of the yolk sac (YS) is the first site during embryogenesis where morphologically discernible hematopoiesis may be found. Later hematopoiesis shifts into the embryo proper, first to the liver, the major fetal hematopoietic site, then to definitive hematopoietic territories, the spleen and bone marrow. It is widely accepted that in the mouse this picture reflects the migration of pluripotent hematopoietic stem cells (HSC) from the YS accompanied by subsequent colonization of the hematopoietic tissues during embryogenesis. However, there is no conclusive evidence showing unequivocally the initiating role of the YS in murine adult hematopoiesis. Recently, we have demonstrated the important role of embryo body tissues in the development of CFU-S before the establishment of definitive hematopoiesis in the fetal liver. This finding suggests that the early development of the hematopoietic system in the mouse is more complex than has been previously proposed and we consider here the early hematopoietic events in the developing mouse embryo.     

Embryo lipoproteins and yolk lipovitellin consumption during embryogenesis in Macrobrachium borellii (Crustacea: Palaemonidae)

The prawn Macrobrachium borellii has lecithotrophic eggs with highly-abbreviated development. The major yolk component is lipovitellin (LV), a lipoprotein with 30% lipids (by weight). LV consumption during embryogenesis was followed by ELISA and Western blot analysis using an anti-LV polyclonal antibody. No cross-reacting proteins were observed and LV-like lipoproteins were strongly recognized by the antibody in hemolymph (vitellogenin), yolk (LV) and embryos (LVe), as determined by Western Blot analysis. LV decreased significantly along development from 9.4 to 1.1 microg/mg egg. Consumption rate of LV was slow in early embryogenesis, followed by a rapid utilization in late embryonic stages. Significant LVe amounts were still present at hatching. LV apolipoproteins were selectively degraded during embryo development, being the highest molecular weight subunit the most affected. Comparison among in vitro, in vivo and theoretical proteolysis suggested that trypsin may be involved in LV degradation during late embryogenesis. Embryo lipoprotein (HDLe) synthesis was first detected at stage 6. HDLe shared the same density, MW and subunit composition as adult hemolymph HDL(1) and did not cross-react with LV-like lipoproteins. Though expressed at low concentration, it fulfilled embryo needs for lipid transport among organs.     

Fatty acid esterification in the yolk sac membrane of the avian embryo

The transfer of lipid from the yolk to the avian embryo is mediated by the yolk sac membrane (YSM). Some, but not all, of the published morphological evidence supports the view that the lipid undergoes a cycle of hydrolysis and re-esterification during translocation across the YSM. The present study aims to test this view by investigating the capacity of the YSM to perform esterification of free fatty acids to form acyl-lipids. YSM pieces (area vasculosa), obtained from the chicken embryo at day 10 of development, were incubated in vitro in medium containing [¹⁴C]-palmitic acid. Radioactivity was rapidly incorporated into the tissue lipid indicating a high capacity for esterification. The incorporation was linear with time during the 1-h incubation. Approximately 84% of the incorporated label was recovered in triacylglycerol, 12% was incorporated into phospholipid and less than 1% was detected in cholesteryl ester. [¹⁴C]-palmitic acid was incorporated primarily at the sn-1/3 positions in the triacylglycerol molecule and at the sn-1 position of phospholipid. The incorporation of label into tissue pieces obtained from the non-vascularized peripheral region of the YSM (area vitellina) was much more limited than that observed for the area vasculosa. The results support the hypothesis that yolk lipid is hydrolyzed and re-esterified during transfer across the YSM.Abbreviations YSM yolk sac membrane - VLDL very-low density lipoprotein Communicated by G. Heldmaier     

Developmental fate of the yolk protein lipovitellin in embryos and larvae of winter flounder, Pleuronectes americanus.

The developmental fate of the vitellogenin-derived yolk protein, lipovitellin (Lv), was investigated in winter flounder embryos and yolk-sac larvae. Since Lv is present as only one major polypeptide in ovulated winter flounder eggs, unlike the multiple yolk polypeptides found in the mature eggs of most teleosts, this system is presented as a simpler model of yolk protein structure and utilization during teleostean development. Winter flounder Lv is cleaved during embryogenesis from a 94 kD polypeptide at fertilization to 67 kD and 26 kD polypeptides at hatching. The rate of this proteolytic processing is slow during early embryonic development, but enters a more rapid phase between days 8 and 12 post-fertilization in embryos reared at 4-5 degrees C, and approaches 50% completion at day 10. Lv processing is essentially complete 3 days before hatching; nevertheless, major degradation of the Lv peptide by the developing winter flounder does not occur until after hatching. The Stokes radius of Lv changes only moderately following processing, from 4.50 nm in unfertilized eggs to 4.19 nm in late embryos and newly hatched larvae, whereas the processed Lv retains its heat stability relative to other yolk polypeptides. Nearly 50% of its lipid content, however, is released from the Lv particle during embryogenesis, concomitant with cleavage of the Lv 94 kD polypeptide. Lv processing may thus render a portion of the yolk protein-associated lipid more accessible to the developing embryo, whereas other yolk components are retained for later use by the winter flounder larva. Alternately, removal of lipid may lead to proteolytic vulnerability of the Lv polypeptide. In either case, only a portion of the lipid moiety of the Lv particle appears to play a significant nutritive role for the embryo, whereas its protein component is reserved for larval use. J. Exp. Zool. 284:686-695, 1999.     

Protein synthesis and differentiation in a clonal line of teratocarcinoma and in preimplantation mouse embryo.

Undifferentiated cells of a clonal line of teratocarcinoma can differentiate in vitro into embryoid bodies with morphological and biochemical features of early mouse embryo. During the first step of differentiation protein synthesis has been analysed by 2 dimensional gel electrophoresis. While new proteins are synthesized, the synthesis of others turned off with the appearance of endodermal cells in embryoid bodies. We have compared protein synthesis during teratocarcinoma differentiation and during early mouse embryogenesis at three stages of mouse preimplantation embryo. The results demonstrate that only the late blastocyst protein synthesis pattern shows most of the polypeptides identified in the differentiated protein synthesis pattern of teratocarcinoma. In contrast, protein synthesis during the early stages of mouse embryonic development is very different from protein synthesis in undifferentiated teratocarcinoma.     

Aspects of the reproductive biology of the bass, Dicentrarchus labrax L. I. An histological and histochemical study of oocyte development

Histological and histochemical studies of oocyte development in the bass, Dicentrarchus labrax L., showed that three types of inclusions are formed during vitellogenesis. Lipid yolk accumulates first as lipid droplets, followed by protein yolk in the form of discrete protein yolk granules. The third type of inclusion are the small cortical alveoli (intravesicular yolk/yolk vesicles, i.e.'carbohydrate yolk') which form in the peripheral cytoplasm after both the lipid and protein yolk have started to accumulate. While the protein yolk granules maintain their structural integrity through to maturation, forming a densely packed zone in the mid-outer cortex, the lipid yolk droplets continually coalesce and migrate centripetally, forming a prominent zone of large lipid droplets in the inner-mid cortex. From the histological study of oocyte development, a number of distinct developmental stages are delineated, while gross examination of the paired ovary revealed that, depending on its stage of development, it can be placed into one of seven maturity stages.     

Morphologic characteristics of definitive and provisional structures of normal 17-day-old human embryos]

The structure of the presomite human embryo was investigated at embryogenesis. The embryonic shield is a three-layer gastrula 810 mkm long in the anteroposterior direction and 855 mkm wide (at the level of the primitive nodule). The primitive streak is 200 mkm long; the primitive nodule is well pronounced. All three germ layers are separately followed only in the cranial end of the embryo. The chordo-mesodermal process, 80 mkm long, is seen and is situated anterior to the primitive nodule, between ecto- and endoderm; in its zone, as well as in the area of the primary nodule and the primary streak, along the middle line, the germ layers are in close contact with each other. In the caudal end the mesoderm grows thin, and the external and internal layers come into contact forming the cloacal membrane. Extraembryonic formations are described: amniotic vesicle, yolk sac, amniotic peduncle, allantois and chorionic membrane wall. Together with the extraembryonic ecto- and endoderm, exocoelomic mesoderm participates in the formation of walls of the primitive germ vesicles. The yolk sac wall contains blood islets. Primary blood vessels are detected in the connective tissue matrix of the chorionic layer and in the amniotic peduncle. According to the anamnesis, morphological data and comparing to the data of the literature on presomitic human embryos, the age of the embryo "Krym" is determined as old as 17 days.     

Differential gene expression during early murine yolk sac development

The visceral yolk sac (VYS), composed of extraembryonic mesoderm and visceral endoderm, is the initial site of blood cell development and serves important nutritive and absorptive functions. In the mouse, the visceral endoderm becomes a morphologically distinct tissue at the time of implantation (E4.5), while the extraembryonic mesoderm arises during gastrulation (E6.5–8.5). To isolate genes differentially expressed in the developing yolk sac, polymerase chain reaction (PCR) methods were used to construct cDNA from late primitive streak to neural plate stage (E7.5) murine VYS mesoderm and VYS endoderm tissues. Differential screening led to the identification of six VYS mesoderm-enriched clones: ribosomal protein L13a, the heat shock proteins hsc 70 and hsp 86, guanine-nucleotide binding protein-related gene, cellular nucleic acid binding protein, and ã-enolase. One VYS endoderm-specific cDNA was identified as apolipoprotein C2. In situ hybridization studies confirmed the differential expression of these genes in E7.5 yolk sac tissues. These results indicate that representative cDNA populations can be obtained from small numbers of cells and that PCR methodologies permit the study of gene expression during early mammalian postimplantation development. While all of the mesoderm-enriched genes were ubiquitously expressed in the embryo proper, apolipoprotein C2 expression was confined to the visceral endoderm. These results are consistent with the hypothesis that at E7.5, the yolk sac endoderm provides differentiated liver-like functions, while the newly developing extraembryonic mesoderm is still a largely undifferentiated tissue. © 1995 wiley-Liss, Inc.     

Germ layer specification and axial patterning in the embryonic development of the freshwater planarian Schmidtea polychroa

Although patterning during regeneration in adult planarians has been studied extensively, very little is known about how the initial planarian body plan arises during embryogenesis. Herein, we analyze the process of embryo patterning in the species Schmidtea polychroa by comparing the expression of genes involved in the establishment of the metazoan body plan. Planarians present a derived ectolecithic spiralian development characterized by dispersed cleavage within a yolk syncytium and an early transient embryo capable of feeding on the maternally supplied yolk cells. During this stage of development, we only found evidence of canonical Wnt pathway, mostly associated with the development of its transient pharynx. At these stages, genes involved in gastrulation (snail) and germ layer determination (foxA and twist) are specifically expressed in migrating blastomeres and those giving rise to the temporary gut and pharyngeal muscle. After yolk ingestion, the embryo expresses core components of the canonical Wnt pathway and the BMP pathway, suggesting that the definitive axial identities are established late. These data support the division of planarian development into two separate morphogenetic stages: a highly divergent gastrulation stage, which segregates the three germ layers and establishes the primary organization of the feeding embryo; and subsequent metamorphosis, based on totipotent blastomeres, which establishes the definitive adult body plan using mechanisms that are similar to those used during regeneration and homeostasis in the adult.     

Ultrastructure of embryonic envelopes and integument ofOncopeltus fasciatus dallas (Insecta,Heteroptera)

Summary The embryo ofOncopeltus fasciatus forms a typical secondary dorsal organ (SDO). It develops after katatrepsis from the contracting serosa, the cells of which decrease in diameter but increase considerably in height. After 66 h, the SDO represents a protrusion of the serosal epithelium above the head and is then reduced to a disc-shaped formation, which sinks into the yolk, where it disintegrates after 80 h.During its typical expression, between 66 and 78 h, the SDO shows a zonal arrangement of its cell organelles. The nucleus, which is located in the basal cell region, has a very irregular outline and includes several nucleoli and globular inclusion bodies. Rough and smooth ER are well developed around the nucleus and suggest the involvement of the organ in protein secretion as well as in lipid metabolism. Electron-lucent vacuoles and electron-dense granules, sometimes enclosed in the vacuoles, accumulate in the apical cell region, and are obviously extruded into the peripheral (extraembryonic) space. The formation of intercellular clefts and delicate cytoplasmic extensions facing the yolk and microvilli facing the periphery evidence a transporting function of the epithelium. Blisters intercalated in extended junctional complexes between apical cell regions point to the transport of solutes.Because of the similarities of the processes observed in the SDO and in Malpighian tubules of larvae, an excretory function of the SDO is suggested. Final products of yolk and embryo are apparently transported to the extraembryonic space, where they accumulate during embryogenesis.Phylogeny, relationship, and function of the different embryonic glands in Arthropoda (primary and secondary DO and pleuropodia) are discussed.Dedicated to Prof. Dr. B. Scharrer on the occasion of her birthdaySupported by the Deutsche ForschungsgemeinschaftI am grateful to Miss K. Schmidtke and Mrs. M. Ullmann for technical assistance     

Identification of an enhancer that controls up-regulation of fibronectin during differentiation of embryonic stem cells into extraembryonic endoderm

The extraembryonic endoderm is derived from inner cell mass cells of the blastocyst during early mouse embryogenesis. Formation of the extraembryonic endoderm, which later contributes to the yolk sac, appears to be a prerequisite for subsequent differentiation of the inner cell mass. While embryonic stem cells can be induced to differentiate into extraembryonic endoderm cells in vitro, the molecular mechanisms underlying this process are poorly understood. We used a promoter trap approach to search for genes that are expressed in embryonic stem cells and are highly up-regulated during differentiation to the extraembryonic endoderm fate. We showed that fibronectin fits this expression profile. Moreover we identified an enhancer in the 12th intron of the fibronectin locus that recapitulated the endogenous pattern of fibronectin expression. This enhancer carries Sox protein-binding sequences, and our analysis demonstrated that Sox7 and Sox17, which are highly expressed in the extraembryonic endoderm, were involved in enhancer activity.     

Metabolic fates of yolk lipid and individual fatty acids during embryonic development of the coot and moorhen

There is currently little information regarding the metabolic fates of yolk lipid and individual fatty acids during embryonic development of free-living avian species. Here we report the pattern of lipid utilization during embryonic development of the coot (Fulica atra) and the moorhen (Gallinula chloropus), two related species producing precocial offspring from eggs with a distinctive fatty acid composition and with an incubation period similar to that of the chicken. By the time of hatching, the proportions of the initial yolk lipid that had been transferred to the embryo were 88.2% and 79.8% for the coot and moorhen respectively. During the whole incubation period, 42.9% and 40.0% of the initial yolk lipid of the coot and moorhen respectively were lost from the system due to oxidation for energy, equating to 47.8% and 50.0% respectively of the actual amount of lipid transferred over this time. Thus, the lipid received by the embryos of both species is partitioned almost equally between the alternative fates of energy metabolism and incorporation into tissue lipids. In the coot, this 50:50 split between oxidation and tissue formation was maintained during the hatching process. The proportions of arachidonic (20:4n-6) and docosahexaenoic (22:6n-3) in the yolk lipids of these species were 2.5-3.5 times higher than in eggs of domestic poultry. In contrast to the situation in the chicken, there was no preferential uptake of 22:6n-3 from the yolk during coot and moorhen development. The fatty acid compositions of the whole body lipids of the coot and moorhen hatchlings were almost identical to those of the initial yolks indicating that, unlike the chicken, these species display relatively little overall biomagnification of 20:4n-6 and 22:6n-6 during development. It is suggested that the yolk fatty acid profiles of the coot and moorhen are particularly well matched to the requirements of the embryo, reducing the need for selective uptake of 22:6n-3 and for the overall biomagnification of 22:6n-3 and 20:4n-6.     

Antioxidative signalling pathways regulate the level of reactive oxygen species at the endometrial-extraembryonic membranes interface during early pregnancy

Conceptus (embryo and associated extraembryonic membranes) implantation and development require a reciprocal biochemical and physical interactions between the extraembryonic membranes and the endometrium. However, the enzymatic antioxidative pathways controlling reactive oxygen species production at the endometrial-extraembryonic membrane interface early in pregnancy are not known. We aimed therefore to determine the content of malondialdehyde, as biomarkers of lipid peroxidation, and the activities of the major antioxidant enzymes, copper-zinc containing and manganese containing superoxide dismutases, catalase and glutathione peroxidase, in sheep extraembryonic membranes, caruncular and intercaruncular endometrium zones sampled at specific stages of pregnancy corresponding to the conceptus implantation (day 16) and the early post-implantation period (day 21). Malondialdehyde content in caruncular, intercaruncular and extraembryonic tissues was not different between stages of the pregnancy. Extraembryonic membranes demonstrated increased manganese containing superoxide dismutase and glutathione peroxidase activities, whereas catalase activity in these tissues decreased from day 16 to day 21. Caruncular tissues demonstrated increased manganese containing superoxide dismutase activity from day 16 to day 21. Intercaruncular tissues demonstrated increased copper-zinc containing superoxide dismutase, manganese containing superoxide dismutase and catalase activities from day 16 to day 21. The ovine extraembryonic membranes exhibit dynamic changes in enzymatic antioxidative pathways different from those of endometrial tissues during the transition from implantation to post-implantation period. This biochemical data provides novel insights into the developmental changes in antioxidative pathways of extraembryonic membranes and endometrium during early conceptus development.     

Analysis of extraembryonic mesodermal structure formation in the absence of morphological primitive streak

During mouse gastrulation, the primitive streak is formed on the posterior side of the embryo. Cells migrate out of the primitive streak to form the future mesoderm and endoderm. Fate mapping studies revealed a group of cell migrate through the proximal end of the primitive streak and give rise to the extraembryonic mesoderm tissues such as the yolk sac blood islands and allantois. However, it is not clear whether the formation of a morphological primitive streak is required for the development of these extraembryonic mesodermal tissues. Loss of the Cripto gene in mice dramatically reduces, but does not completely abolish, Nodal activity leading to the absence of a morphological primitive streak. However, embryonic erythrocytes are still formed and assembled into the blood islands. In addition, Cripto mutant embryos form allantoic buds. However, Drap1 mutant embryos have excessive Nodal activity in the epiblast cells before gastrulation and form an expanded primitive streak, but no yolk sac blood islands or allantoic bud formation. Lefty2 embryos also have elevated levels of Nodal activity in the primitive streak during gastrulation, and undergo normal blood island and allantois formation. We therefore speculate that low level of Nodal activity disrupts the formation of morphological primitive streak on the posterior side, but still allows the formation of primitive streak cells on the proximal side, which give rise to the extraembryonic mesodermal tissues formation. Excessive Nodal activity in the epiblast at pre‐gastrulation stage, but not in the primitive streak cells during gastrulation, disrupts extraembryonic mesoderm development.     

An integrated overview of seed development in Arabidopsis thaliana ecotype WS

This work is part of a research program aiming at identifying and studying genes involved in Arabidopsis thaliana seed maturation. We focused here on the Wassilewskija ecotype seed development and linked physiological and biochemical data, including protein, oil, soluble sugars, starch and free amino acid measurements, to embryo development, to obtain a complete and thorough reference data set. A. thaliana seed development can be divided into three stages. During early embryogenesis (i.e. morphogenesis), seed weight and lipid content were low whereas important amounts of starch were transiently accumulated. In the second stage, or maturation phase, a rapid increase in seed dry weight was observed and storage oils and proteins were accumulated in large quantities, accounting for approximately 40% of dry matter each at the end of this stage. During the third and last stage (late maturation including acquisition of desiccation tolerance), seed dry weight remained constant while an acute loss of water took place in the seed. Storage compound synthesis ended concomitantly with sucrose, stachyose and raffinose accumulation. This study revealed the occurrence of metabolic activities such as protein synthesis, in the final phase of embryo desiccation. A striking correlation between peaks in hexose to sucrose ratio and transition phases during embryogenesis was observed.