A relationship between seed development, Arabinogalactan-proteins (AGPs) and the AGP mediated promotion of somatic embryogenesis

Arabinogalactan-protein (AGP) epitopes are known to display developmentally regulated patterns of expression in several plant tissues. Therefore, AGPs have been suggested to play a role in plant development. Somatic embryogenesis is regulated by AGPs as well as by EP3 endochitinases. Using four different methods we have analysed the composition of AGPs in immature carrot seeds. The results obtained show that: (1) the native electrophoretic mobility of such AGPs changes during development; (2) AGP epitopes in immature seeds are developmentally regulated; (3) enzymatically released fragments of AGPs show that the composition of these molecules changes as a function of development; and (4) the biological activity of AGPs on the formation of somatic embryos changes depending on the age of the seeds. Our results suggest that degradation of maternally derived AGPs occurs after fertilization, while cellularization of the endosperm leads to synthesis of a new set of AGPs. The presence of an endochitinase cleavage site as well as the capacity to increase somatic embryogenesis only occurred in AGPs that were isolated from seeds in which the endosperm had been cellularized. Apparently, both EP3 endochitinases and somatic embryogenesis-promoting AGPs are developmentally regulated in immature carrot seeds.     

Arabinogalactan proteins are essential in somatic embryogenesis of Daucus carota L.

Daucus carota L. cell lines secrete a characteristic set of arabinogalactan proteins (AGPs) into the medium. The composition of this set of AGPs changes with the age of the culture, as can be determined by crossed electrophoresis with the specific AGP-binding agent, β-glucosyl Yariv reagent. Addition of AGPs isolated from the medium of a non-embryogenic cell line to an expiant culture initiated the development of the culture to a non-embryogenic cell line. Without addition of AGPs or with addition of carrot-seed AGPs an embryogenic cell line was established. Three-month-old embryogenic cell lines usually contain less than 30% of dense, highly cytoplasmic cells, i.e. the embryogenic cells, but when carrot-seed AGPs were added this percentage increased to 80%. Addition of carrot-seed AGPs to a two-year-old, non-embryogenic cell line resulted in the re-induction of embryogenic potential. These results show that specific AGPs are essential in somatic embryogenesis and are able to direct development of cells.     

Developmental pathways of somatic embryogenesis

Somatic embryogenesis is defined as a process in which a bipolar structure, resembling a zygotic embryo, develops from a non-zygotic cell without vascular connection with the original tissue. Somatic embryos are used for studying regulation of embryo development, but also as a tool for large scale vegetative propagation. Somatic embryogenesis is a multi-step regeneration process starting with formation of proembryogenic masses, followed by somatic embryo formation, maturation, desiccation and plant regeneration. Although great progress has been made in improving the protocols used, it has been revealed that some treatments, coinciding with increased yield of somatic embryos, can cause adverse effects on the embryo quality, thereby impairing germination and ex vitro growth of somatic embryo plants. Accordingly, ex vitro growth of somatic embryo plants is under a cumulative influence of the treatments provided during the in vitro phase. In order to efficiently regulate the formation of plants via somatic embryogenesis it is important to understand how somatic embryos develop and how the development is influenced by different physical and chemical treatments. Such knowledge can be gained through the construction of fate maps representing an adequate number of morphological and molecular markers, specifying critical developmental stages. Based on this fate map, it is possible to make a model of the process. The mechanisms that control cell differentiation during somatic embryogenesis are far from clear. However, secreted, soluble signal molecules play an important role. It has long been observed that conditioned medium from embryogenic cultures can promote embryogenesis. Active components in the conditioned medium include endochitinases, arabinogalactan proteins and lipochitooligosaccharides.     

Fertile wheat (Triticum aestivum L.) regenerants from protoplasts of embryogenic suspension culture

We report regeneration of fertile, green plants from wheat (Triticum aestivum L. cv. Aura) protoplasts isolated from an embryogenic suspension initiated from somatic early-embryogenic callus. The present approach combines the optimization of protoplast culture conditions with screening for responsive genotypes. In addition to the dominant effect of the culture media, the increase in fresh mass and the embryogenic potential of somatic callus cultures varied considerably between the various genotypes tested. Establishment of suspension cultures with the required characters for protoplast isolation was improved by reduction of the ratio between cells and medium and by less frequent (monthly) transfer into fresh medium. A new washing solution was introduced to avoid the aggregation of protoplasts. However, the influence of the culture medium on cell division was variable in the different genotypes. We could identify cultures from cultivar Aura that showed approximately a 9% cell division frequency and morphogenic response. The protoplast-derived microcolonies formed both early and late-embryogenic callus on regeneration medium and green fertile plants were obtained through somatic embryogenesis. The reproducibility of plant regeneration from protoplast culture based on the cultivar Aura was demonstrated by several independent experiments. The maintenance of regeneration potential in Aura suspension cultures required establishment of new cultures within a 9-month period.     

Arabinogalactan-proteins in Cichorium somatic embryogenesis: effect of β-glucosyl Yariv reagent and epitope localisation during embryo development

 Direct somatic embryogenesis was induced in root tissues of the Cichorium hybrid `474' (C. intybus L. var. sativum×C. endivia L. var. latifolia). Addition of β-d-glucosyl Yariv reagent (βGlcY), a synthetic phenylglycoside that specifically binds arabinogalactan-proteins (AGPs), to the culture medium blocked somatic embryogenesis in a concentration-dependent manner with complete inhibition of induction occurring at 250 μM βGlcY. The AGP-unreactive α-d-galactosyl Yariv reagent had no biological activity in this system. Upon transfer of 250 μM βGlcY-treated roots to control conditions, somatic embryogenesis was recovered with a time course similar to that of control roots. The βGlcY penetrated roots and bound abundantly to developing somatic embryos, to the root epidermis and the stele. Immunofluorescence and immunogold labelling using monoclonal antibodies (JIM13, JIM16 and LM2) revealed that AGPs were localised in the outer cell walls peripheral cells of the globular embryo. A spatio-temporal expression of AGPs appeared to be associated with differentiation events in the somatic embryo during the transition from the globular stage to the torpedo stage. To verify βGlcY specificity, molecules that bound βGlcY were extracted from treated conditioned medium and identified as AGPs by using the same monoclonal antibodies. In addition, AGPs were found to be abundantly present in the medium during embryogenic culture. All of these results establish the implication of AGPs in embryo development, and their putative role in somatic embryogenesis is discussed. Received: 26 August 1999 / Accepted: 28 January 2000     

Involvement of arabinogalactan proteins in the regeneration process of cultured protoplasts of Marchantia polymorpha

Protoplasts of Marchantia polymorpha L. (liverwort) regenerated new cell walls in initial culture. However, the survival rate of regenerated cells decreased rapidly after this stage. The decrease in survival rate was suppressed by the β-glucosyl Yariv reagent (βglcY), which binds to arabinogalactan proteins (AGPs), only when it was added to culture medium during the period of incipient cell wall regeneration. The addition of βglcY after the period of incipient cell wall regeneration had no effect on the survival rate. These results suggested the involvement of AGPs in the cell wall regeneration process. After cell wall regeneration, the regenerated cells started to divide actively after being transferred to a medium with 1% activated charcoal (AC). Protoplasts that had been cultured with βglcY during the period of incipient cell wall regeneration and then transferred to the AC medium divided vigorously, and the cell division rate was remarkably increased (>80%). However, without transfer to the AC medium, βglcY at concentrations higher than 20 μg ml⁻¹ inhibited cell division. No effect on cell survival nor cell division was observed with the α-galactosyl Yariv reagent. Staining of β-1,3-glucan (callose) with aniline blue (AB) showed that a large amount of β-1,3-glucan was deposited in the regenerated cell walls of the protoplasts cultured without βglcY, while little or no β-1,3-glucan was stained by AB in protoplasts cultured with βglcY. These results suggest that AGPs and β-1,3-glucan play important roles in the survival and subsequent cell division of regenerated cells of M. polymorpha protoplast cultures.     

Characterization of proteins secreted during maize microspore culture: arabinogalactan proteins (AGPs) stimulate embryo development

To study molecules secreted from cultured plant cells that promote development, maize microspores were transferred into culture and the conditioned media were collected over time and analysed. Electrophoresis indicated that both non-glycosylated and glycosylated proteins including arabinogalactan proteins (AGPs) appeared in the medium and their concentration increased during the time of culture. The development of embryos was correlated with the presence of specific extracellular proteins, using an experimental system based on a tunicamycin inhibition test. In addition, a precise protein analysis was conducted using MALDI-TOF and ESI-MS-MS techniques. These approaches have allowed the identification of 5 other types of proteins: a cell wall invertase, two thaumatin isoforms, one 1-3 beta-glucanase and two chitinase isoforms. Altogether these experiments and results open ways for research aimed at understanding which molecules stimulate embryo formation. Moreover, AGPs may be used to stimulate the development of microspores (pollen embryogenesis) prepared from non-responsive genotypes.     

Tobacco zygotic embryogenesis in vitro: the original cell wall of the zygote is essential for maintenance of cell polarity, the apical-basal axis and typical suspensor formation

We have developed a reliable in vitro zygotic embryogenesis system in tobacco. A single zygote of a dicotyledonous plant was able to develop into a fertile plant via direct embryogenesis with the aid of a co-culture system in which fertilized ovules were employed as feeders. The results confirmed that a tobacco zygote could divide in vitro following the basic embryogenic pattern of the Solanad type. The zygote cell wall and directional expansion are two critical points in maintaining apical-basal polarity and determining the developmental fate of the zygote. Only those isolated zygotes with an almost intact original cell wall could continue limited directional expansion in vitro, and only these directionally expanded zygotes could divide into typical apical and basal cells and finally develop into a typical embryo with a suspensor. In contrast, isolated zygote protoplasts deprived of cell walls could enlarge but could not directionally elongate, as in vivo zygotes do before cell division, even when the cell wall was regenerated during in vitro culture. The zygote protoplasts could also undergo asymmetrical division to form one smaller and one larger daughter cell, which could develop into an embryonic callus or a globular embryo without a suspensor. Even cell walls that hung loosely around the protoplasts appeared to function, and were closely correlated with the orientation of the first zygotic division and the apical-basal axis, further indicating the essential role of the original zygotic cell wall in maintaining apical-basal polarity and cell-division orientation, as well as subsequent cell differentiation during early embryo development in vitro.     

Cellular and molecular changes associated with somatic embryogenesis induction in Agave tequilana

In spite of the importance of somatic embryogenesis for basic research in plant embryology as well as for crop improvement and plant propagation, it is still unclear which mechanisms and cell signals are involved in acquiring embryogenic competence by a somatic cell. The aim of this work was to study cellular and molecular changes involved in the induction stage in calli of Agave tequilana Weber cultivar azul in order to gain more information on the initial stages of somatic embryogenesis in this species. Cytochemical and immunocytochemical techniques were used to identify differences between embryogenic and non-embryogenic cells from several genotypes. Presence of granular structures was detected after somatic embryogenesis induction in embryogenic cells; composition of these structures as well as changes in protein and polysaccharide distribution was studied using Coomassie brilliant blue and Periodic Acid-Schiff stains. Distribution of arabinogalactan proteins (AGPs) and pectins was investigated in embryogenic and non-embryogenic cells by immunolabelling using anti-AGP monoclonal antibodies (JIM4, JIM8 and JIM13) as well as an anti-methyl-esterified pectin-antibody (JIM7), in order to evaluate major modifications in cell wall composition in the initial stages of somatic embryogenesis. Our observations pointed out that induction of somatic embryogenesis produced accumulation of proteins and polysaccharides in embryogenic cells. Presence of JIM8, JIM13 and JIM7 epitopes were detected exclusively in embryogenic cells, which supports the idea that specific changes in cell wall are involved in the acquisition of embryogenic competence of A. tequilana.     

Somatic hybridization in Citrus: navel orange (C. sinensis Osb.) and grapefruit (C. paradisi Macf.)

Summary Protoplasts of navel orange, isolated from embryogenic nucellar cell suspension culture, were fused with protoplasts of grapefruit isolated from leaf tissue. The fusion products were cultured in the hormone-free medium containing 0.6 M sucrose. Under the culture conditions, somatic embryogenesis of navel orange protoplasts was suppressed, while cell division of grapefruit mesophyll protoplasts was not induced. Six embryoids were obtained and three lines regenerated to complete plants through embryogenesis. Two of the regenerated lines exhibited intermediate morphological characteristics of the parents in the leaf shape. Chromosome counts showed that these regenerated plants had expected 36 chromosomes (2n=2x=18 for each parent). The rDNA analysis using biotin-labeled rRNA probes confirmed the presence of genomes from both parents in these plants. This somatic hybridization system would be useful for the practical Citrus breeding.     

Arabinogalactan protein profiles and distribution patterns during microspore embryogenesis and pollen development in Brassica napus

Arabinogalactan proteins (AGPs), present in cell walls, plasma membranes and extracellular secretions, are massively glycosylated hydroxyproline-rich proteins that play a key role in several plant developmental processes. After stress treatment, microspores cultured in vitro can reprogramme and change their gametophytic developmental pathways towards embryogenesis, thereby producing embryos which can further give rise to haploid and double haploid plants, important biotechnological tools in plant breeding. Microspore embryogenesis constitutes a convenient system for studying the mechanisms underlying cell reprogramming and embryo formation. In this work, the dynamics of both AGP presence and distribution were studied during pollen development and microspore embryogenesis in Brassica napus, by employing a multidisciplinary approach using monoclonal antibodies for AGPs (LM2, LM6, JIM13, JIM14, MAC207) and analysing the expression pattern of the BnAGP Sta 39–4 gene. Results showed the developmental regulation and defined localization of the studied AGP epitopes during the two microspore developmental pathways, revealing different distribution patterns for AGPs with different antigenic reactivity. AGPs recognized by JIM13, JIM14 and MAC207 antibodies were related to pollen maturation, whereas AGPs labelled by LM2 and LM6 were associated with embryo development. Interestingly, the AGPs labelled by JIM13 and JIM14 were induced with the change of microspore fate. Increases in the expression of the Sta 39–4 gene, JIM13 and JIM14 epitopes found specifically in 2–4 cell stage embryo cell walls, suggested that AGPs are early molecular markers of microspore embryogenesis. Later, LM2 and LM6 antigens increased progressively with embryo development and localized on cell walls and cytoplasmic spots, suggesting an active production and secretion of AGPs during in vitro embryo formation. These results give new insights into the involvement of AGPs as potential regulating/signalling molecules in microspore reprogramming and embryogenesis.     

The role of arabinogalactan proteins binding to Yariv reagents in the initiation, cell developmental fate, and maintenance of microspore embryogenesis in Brassica napus L. cv. Topas

Arabinogalactan proteins (AGPs) are extracellular proteoglycans involved in plant growth and development. The addition of beta-D-glucosyl Yariv reagent (betaGlcY), a synthetic phenylglycoside that specifically reacts with AGPs, to the culture medium notably disturbed microspore embryogenesis in a concentration-dependent manner. The initiation of microspore embryogenesis was clearly inhibited by 30 microM betaGlcY and completely inhibited by 50 microM betaGlcY. The transfer of microspore-derived embryos at different developmental stages into NLN6 medium containing 50 microM betaGlcY prohibited their normal development, as approximately 21.24, 43.99, and 59.73%, respectively, of the treated globular-, heart-, and torpedo-stage embryos exhibited numerous root hair-like structures. Both heart-stage and torpedo-stage embryos showed a rapid growth of roots with a large number of clustered root hairs. Some root hair-like structures were also observed on the apical portions of embryos. Microscopy of the treated embryos revealed that the basic patterns of cells at both the radial and apical-basal axes were greatly altered, such that the cells lost their ability to carry out programmed embryogenesis. These results show that the betaGlcY-AGP interaction modulates the developmental fate of embryonic cells, especially epidermal cells, and thereby strongly affects root generation and development. Immunofluorescence microscopy revealed that both JIM8 and JIM13 binding to AGP co-localize with betaGlcY-binding sites. Thus, AGPs binding to betaGlcY, co-localized with Jim8- and Jim13-binding protein, appear to play a crucial role in the initiation of Brassica microspore embryogenesis and the maintenance of cell differentiation during embryonic development. In addition, these proteins may also be involved in the regulation of root generation.     

阿拉伯半乳糖蛋白在被子植物中的功能

阿拉伯半乳糖蛋白(arabinogalactan proteins,AGPs)是一类富含羟脯氨酸/脯氨酸的高度糖基化的蛋白分子,在高等植物的细胞壁、质膜和胞外基质中广泛存在。AGPs是一类重要的糖蛋白,它在被子植物营养生长和生殖发育的各个环节都可能发挥作用,涉及体细胞胚胎发生、细胞增殖、细胞膨大、细胞程序性死亡、损伤防御、根形态建成、花粉管生长以及植物激素信号传导等。植物结构基因组学及功能基因组学的快速发展,使得人们对AGPs的表达模式和功能特点有了更深入的认识。本文首先就AGPs的分子结构和分类,然后重点就利用基因组序列信息分析以及正、反向遗传学等手段进行的AGPs在植物营养生长、生殖发育、细胞程序性死亡,以及分子互作和信号传导等方面的作用的研究进行了综述。     

Plant regeneration from highly embryogenic callus,cell suspension and protoplast cultures of Trifolium fragiferum

Using various media, tissue and protoplast cultures plant regeneration systems were developed for Trifolium fragiferum (2n=16). (L.). The best media for induction of embryogenic cultures were based on Kao (1977) or Kao and Michayluk (1975). Somatic embryogenesis was observed in cultures derived from green leaf mesophyll protoplasts of branching plants, somatic embryo protoplasts and cell suspension protoplasts, leaflets and various explants of immature zygotic embryos. The process of somatic embryogenesis was maintained for over two years on Murashige and Skoog's (1962) medium supplemented with 0.5 mg l^-1 benzyladenine and 0.05 mg l^-1 naphthaleneacetic acid. These long term cultures were capable of regenerating plants that were fertile and produced seeds. These results were compared with those from protoplast, tissue and organ culture of other species of the Trifolium genus. This revised version was published online in June 2006 with corrections to the Cover Date.     

Ontogeny of embryogenic callus in Medicago truncatula: the fate of the pluripotent and totipotent stem cells

Background and Aims

Understanding the fate and dynamics of cells during callus formation is essential to understanding totipotency and the mechanisms of somatic embryogenesis. Here, the fate of leaf explant cells during the development of embryogenic callus was investigated in the model legume Medicago truncatula.

Methods

Callus development was examined from cultured leaf explants of the highly regenerable genotype Jemalong 2HA (2HA) and from mesophyll protoplasts of 2HA and wild-type Jemalong. Callus development was studied by histology, manipulation of the culture system, detection of early production of reactive oxygen species and visualization of SERK1 (SOMATIC EMBRYO RECEPTOR KINASE1) gene expression.

Key Results

Callus formation in leaf explants initiates at the cut surface and within veins of the explant. The ontogeny of callus development is dominated by the division and differentiation of cells derived from pluripotent procambial cells and from dedifferentiated mesophyll cells. Procambium-derived cells differentiated into vascular tissue and rarely formed somatic embryos, whereas dedifferentiated mesophyll cells were competent to form somatic embryos. Interestingly, explants incubated adaxial-side down had substantially less cell proliferation associated with veins yet produced similar numbers of somatic embryos to explants incubated abaxial-side down. Somatic embryos mostly formed on the explant surface originally in contact with the medium, while in protoplast microcalli, somatic embryos only fully developed once at the surface of the callus. Mesophyll protoplasts of 2HA formed embryogenic callus while Jemalong mesophyll protoplasts produced callus rich in vasculature.

Conclusions

The ontogeny of embryogenic callus in M. truncatula relates to explant orientation and is driven by the dynamics of pluripotent procambial cells, which proliferate and differentiate into vasculature. The ontogeny is also related to de-differentiated mesophyll cells that acquire totipotency and form the majority of embryos. This contrasts with other species where totipotent embryo-forming initials mostly originate from procambial cells.Key words: Callus, dedifferentiation, leaf veins, Medicago truncatula, pluripotency, procambium, protoplasts, reactive oxygen species, SERK, somatic embryogenesis, stem cells, totipotency     

Somatic embryogenesis of Cyclamen persicum in liquid medium

A method is described for the production of somatic embryos of Cyclamen persicum Mill. in liquid medium. Five steps are involved; initiation of embryogenic cell lines, proliferation of pro-embryogenic masses (PEMs) on auxin-containing medium, development of somatic embryos on hormone-free medium with high osmolarity, germination and subsequent plantlet formation. Cell lines were initiated by culturing the explant, the seedling tuber, directly in liquid medium. Three parameters were important for obtaining embryogenic cell lines; explant density, hormone concentrations and subculture regime. The rate of uptake of the hormones 2,4-dichlorophenoxyacetic acid (2,4-D) and kinetin influenced the formation of PEMs. Highly embryogenic cell lines were obtained only when PEMs had formed within 5–7 weeks. PEMs were proliferated for at least 24 months and could be isolated from each subculture for the production of somatic embryos. A high sucrose content (175 m M ) in the development medium without hormones ensured efficient embryo development from PEMs. A subsequent subculture in low sucrose concentration (58 m M ) induced the formation of a tuber, thus promoting germination. Arabinogalactan-proteins (AGPs) from carrot seeds and AGPs bound by the monoclonal antibody ZUM 18 increased the number of PEMs in a culture, showing that the activity of AGPs is not species specific.     

Plant regeneration from embryogenic cell suspensions and protoplasts of dessert banana cv. ‘Da Jiao’ (<Emphasis Type="Italic">Musa paradisiacal</Emphasis> ABB Linn.) via somatic embryogenesis

The ‘Da Jiao’ cultivar of banana (Musa paradisiacal ABB Linn.) is an ideal germplasm to produce new banana varieties resistant to Fusarium oxysporum f. sp. cubense (FOC) race 4, for this cultivar is not only a popular dessert banana in south China, but also bears high resistance to FOC race 4. In this study, we established a homogeneous embryogenic cell suspension (ECS) of ‘Da Jiao’ and obtained regenerated plants from ECS-derived protoplasts via somatic embryogenesis. The ECS was initiated from yellow friable callus induced from immature male inflorescence on M1 medium. A pre-culture was used to select ECS in M2 medium without 2,4-dichlorophenoxyacetic acid for 10 d. Addition of 1.0 mg L⁻¹ abscisic acid to M3 medium could enhance the frequency of somatic embryogenesis by about 2.6-fold. Protoplasts, with a yield range of 5–6 × 10⁶ per milliliter, were isolated from the ECS. About 0.35% of the protoplasts formed microcallus, which contained about 100 cells, after 1 mo of feeder layer culture with ECS of Musa acuminate cv. Mas (AA) as nurse cells. Healthy plantlets (0.14%) were regenerated from the microcallus through somatic embryogenesis.     

Somatic embryogenesis from cell suspension and protoplast cultures ofCapsella bursa-pastoris (L.) Medic

Summary Rapidly growing cell suspension cultures of shepherd’s purse (Capsella bursa-pastoris L. Medic.) were established from leaf-derived calli. These suspensions remained unorganized in the presence of 2,4-D, but underwent extensive root organogenesis in a growth regulator-free liquid medium. Attempts to induce direct embryogenesis in liquid cultures were unsuccessful, but numerous embryos were obtained from cells plated onto growth-regulator-free solid medium. These embryos were frequently abnormal, and secondary embryogenesis was problematic for plant recovery but fertile plants were recovered. Viable protoplasts could readily be isolated from these cell suspensions. After 1 wk of culture, protoplast viability was 62%, and 7% of the cells had divided. Embryogenesis was observed from protoplast-derived microcolonies, plated on growth-regulator-free medium. Although these somatic embryos were difficult to root, plants were recovered. New cell suspensions were more recently established, which were only 4 to 6 mo. old when plant regeneration was attempted. Numerous shoots were obtained when these cells were plated onto growth-regulator-free solid media. However, these shoots differed from the embryos previously obtained in that they readily rooted and rapidly developed into plantlets. This system may allow the use of shepherd’s purse as a gene source for introgression of agronomically interesting traits intoBrassica crop species through protoplast manipulation and somatic hybridization.     

The histodifferentiation events involved during the acquisition and development of somatic embryogenesis in oil palm (Elaeis guineensis Jacq.)

Understanding the fate and dynamics of cells during callus formation is essential to understanding totipotency and the somatic embryogenesis (SE) mechanisms. In the present study, the histodifferentiation events involved during the acquisition and development of somatic embryogenesis in oil palm (Elaeis guineensis Jacq.) was investigated. Zygotic embryos were inoculated on SE induction medium, and at 14 days the first divisions of the procambial and perivascular cells were observed. This region progressed to the formation of meristematic masses at 21 days, indicating their procambial and perivascular origin. Primary calli emerged at 45 days of culture, followed by progression to embryogenic calli at 90 days. The formation of proembryos (PE) from the meristematic cells occurred at 135 days of cultivation. The PE were isolated from the tissue of origin by the slight thickening of the cell wall, indicating their unicellular origin. When transferred to the maturation phase, differentiation of the somatic embryos at different developmental stages (globular and torpedo) was observed. The differentiated somatic embryos presented protoderm, procambial strands and plumules. Afterwards, they were transferred to culture medium without growth regulators in which conversion of the somatic embryos from torpedo stage into plants was observed. These results enable a greater understanding of the SE process and plantlet formation in E. guineensis.