Developmental Stages Of Somatic Embryos Research Articles

Inhibited polar auxin transport results in aberrant embryo development in Norway spruce

Current hypotheses concerning the role of polar auxin transport in embryo development are entirely based on studies of angiosperms, while little is known about how auxin regulates pattern formation in gymnosperms. In this study, different developmental stages of somatic embryos of Norway spruce (Picea abies) were treated with the polar auxin transport inhibitor 1-N-naphtylphthalamic acid (NPA). Effects of the treatments on auxin content, embryo differentiation and programmed cell death (PCD) were analysed. During early embryo development, NPA-treatment led to increased indole-3-acetic acid (IAA) content, abnormal cell divisions and decreased PCD, resulting in aberrant development of embryonal tube cells and suspensors. Mature embryos that had been treated with NPA showed both apical and basal abnormalities. Typically the embryos had abnormal cotyledon formation and irregular cell divisions in the area of the root meristem. Our results show that polar auxin transport is essential for the correct patterning of both apical and basal parts of conifer embryos throughout the whole developmental process. Furthermore, the aberrant morhologies of NPA-treated spruce embryos are comparable with several auxin response and transport mutants in Arabidopsis. This suggests that the role of polar auxin transport is conserved between angiosperms and gymnosperms.

MASS PROPAGATION OF COFFEE TRANSPLANTS UNDER SCALED-UP PHOTOAUTOTROPHIC MICROPROPAGATION SYSTEM

Somatic embryos were obtained from leaf discs of coffee plants cultured in vitro. The main objective of our study was to develop a scaled-up micropropagation system under photoautotrophic conditions (PA) using Coffea arabusta somatic embryos as a model plant. At different developmental stages of somatic embryos (torpedo, precotyledonary, cotyledonary and germinated), the physiological variables in relation to the photosynthetic ability were investigated. Results revealed that cotyledonary and germinated stage embryos were physiologically capable to grow photoautotrophically. However, high photosynthetic photon flux (PPF: 100 μmol m 2 s -1 ) treatment for 14 days increased the photosynthetic efficiency of the somatic embryos and possibly make them more suitable to grow under PA conditions. To confirm these findings the somatic embryos were cultured under PA conditions (sugar-free medium with CO 2 enrichment in the culture vessel environment and high PPF). From the results it was concluded that cotyledonary stage is the earliest stage somatic embryo, which can be successfully grown photoautotrophically for conversion into plantlets. To scale up the plantlet conversion process of coffee somatic embryos under PA conditions a bioreactor was especially designed. The growth and conversion percentage of cotyledonary stage embryos cultured in the newly developed bioreactor were compared with those of commercially available systems such as RITA temporary immersion bioreactor and Magenta box. Results revealed that the growth, percent conversion and ex vitro survival of plants were highest in the newly developed bioreactor.

Monoclonal antibody against a cell wall marker protein for embryogenic potential of Dactylis glomerata L. suspension cultures

We identified and isolated a monoclonal antibody (MAb 3G2) raised against extracellular proteins from microcluster cells of orchard grass (Dactylis glomerata L.) embryogenic suspension culture. MAb 3G2 recognized with high specificity an antigen ionically bound within the primary cell wall and in the culture medium of microcluster cells. Two-dimensional polyacrylamide gel analysis and blotting of proteins on PVDF membrane showed that MAb 3G2 detected a single polypeptide of apparent molecular mass of 48 kDa and an isoelectric point (pI) of 5.2, designated EP48. A transient expression during somatic embryogenesis was observed for EP48. Indirect immunofluorescence showed that this protein highly accumulated in the cell walls of some single cells, microclusters and partly in proembryogenic masses (PEMs), but not in globular embryos of the embryogenic cell line and microclusters from the non-embryogenic cell line. Signal intensity varied between individual cells of the same population and in successive stages of somatic embryo development. Screening of several D. glomerata L. embryogenic and non-embryogenic cell lines with MAb 3G2 indicated the presence of ECP48 in only embryogenic suspension cultures at early stages of embryo development long before morphological changes have taken place and thus it could serve as an early marker for embryogenic potential in D. glomerata L. suspension cultures.

CDNA-AFLP study of gene expression during somatic embryogenesis in Fraxinus angustifolia Vhal.

SummaryTranscriptional changes during somatic embryo development in Fraxinus angustifolia have been analysed by the cDNA-AFLP technique. Polymorphic Transcript Derived Fragments (TDFs), potentially useful for molecular physiological characterisation of somatic embryogenesis in F. angustifolia, were observed. Of 143 TDFs, 15 were sequenced and some showed high similarity with known gene sequences. In particular, TDFs of 1-aminocyclopropane-1-carboxylic acid synthase, trehalose-6-phosphate synthase, leuco-anthocyanidin dioxygenase and three retrotransposon-like sequences were expressed differentially during the four stages of somatic embryo development. The patterns of expression of these genes, and the potential role(s) of their products in controlling somatic embryogenesis are discussed.

Somatic embryogenesis from callus cultures of Terminalia chebula Retz.: an important medicinal tree

Somatic embryogenesis was obtained from cotyledon and mature zygotic embryo callus cultures of Terminalia chebula Retz. Callus cultures of cotyledon and mature zygotic embryo were initiated on induction medium containing Murashige and Skoog (MS) nutrients with 1.0 mg/l 2,4-dichlorophenoxyacetic acid (2,4-D) either 0.01 or 0.1 mg/l Kinetin and 30 g/l sucrose. Induction of somatic embryogenesis, proliferation and development was obtained through different culture passages. Embryogenic cotyledon callus with globular somatic embryos was obtained on MS basal medium supplemented with 50 g/l sucrose. Globular somatic embryos were observed from mature zygotic embryo callus on induction medium. Different stages of somatic embryo development from cotyledon and mature zygotic embryo calluses were observed on MS basal medium supplemented with 50 g/l sucrose after 4 weeks of culture. Histological studies have revealed the developmental stages of somatic embryos. A maximum of 40.3±1.45 cotyledonary somatic embryos/callus was obtained from mature zygotic embryo compared to 7.70±0.37 cotyledonary somatic embryos/callus initiated from cotyledons. Germination of somatic embryos and conversion to plants were achieved. Highest frequency of germination (46.66±0.88) of somatic embryos was obtained on MS basal medium containing benzyladenine (0.5 mg/l) with 30 g/l sucrose.

Origin of Somatic Embryogenesis is Proliferating Root Primordia in Seed Derived Oat Callus

The ontogeny of somatic embryos and proliferating root primordia in tissue cultures of mature seeds of oats is described. Callus formation was initiated on developing embryos of mature oat seeds maintained on Murashige and Skoog's medium containing 2mgl-1 2,4-dichlorophenoxyacetic acid. The root primordia on subculture medium did not elongate but multiplied by forming new primordia directly from root meristem, after callusing or by cleaving pre-existing root primordia. In the root meristem region somatic embryogenesis could also occur. The first somatic embryos were observed at the end of the first subculture. Different developmental stages of somatic embryos were histologically identified in the culture. Occasionally shoot buds were formed directly from root primordia. Among 5 tested cultivars Risto had the best response on callus culture medium. 200 shoots/plantlets per gram of a 10-month culture were obtained on MS germination medium containing 0.01 mgl-1 ABA and 6% sucrose.

Somatic embryogenesis from leaf explants of Australian fan flower, Scaevola aemula R. Br.

Somatic embryogenesis from leaf explants of Scaevola aemula R. Br. was achieved. Somatic embryos were induced from explants cultured on MS medium supplemented with 0.2 mg/ 2,4-dichlorophenoxyacetic acid and 0.2-0.5 mg/l 6-benzylaminopurine (BAP). Various developmental stages of somatic embryos were found on this medium-from globular embryos to germinated embryos. The transfer of globular embryos to MS medium containing 0.5 mg/l BAP resulted in a high frequency of shoot regeneration. Leaf explants cultured on MS medium containing different combinations of BAP and alpha-naphthaleneacetic acid formed adventitious shoots and roots. Histological examination confirmed the process of somatic embryogenesis. Induction of somatic embryogenesis in Scaevola provides a system for studying embryogenesis in Australian native plants and will facilitate the improvement of these plants using genetic transformation techniques.

Somatic embryogenesis and plant regeneration from cotyledon explants of a timber-yielding leguminous tree, Dalbergia sissoo Roxb.

Efficient plant regeneration through somatic embryogenesis was achieved from callus cultures derived from semi-mature cotyledon explants of Dalbergia sissoo Roxb., a timber-yielding leguminous tree. Somatic embryos developed over the surface of embryogenic callus and occasionally, directly from cotyledon explants without intervening callus phase. Callus cultures were initiated from cotyledon pieces of D. sissoo on Murashige and Skoog (1962) medium supplemented with 4.52, 9.04, 13.57, and 18.09 mumol/L 2,4-dichlorophenoxyacetic acid and 0.46 mumol/L Kinetin. Maximum percentage response for callus formation was 89% on MS medium supplemented with 9.04 mumol/L 2,4-D' and 0.46 mumol/L Kn. Somatic embryogenesis was achieved after transfer of embryogenic callus clumps to 1/2-MS medium without plant growth regulators (1/2-MSO). Average numbers of somatic embryos per callus clump was 26.5 on 1/2-MSO medium after 15 weeks of culture. Addition of 0.68 mmol/L L-glutamine to 1/2-MSO medium enhanced somatic embryogenesis frequency from 55% to 66% and the number of somatic embryos per callus clump from 26.5 to 31.1. Histological studies were carried out to observe various developmental stages of somatic embryos. About 50% of somatic embryos converted into plantlets on 1/2-MSO medium containing 2% sucrose, after 20 days of culture. Transfer of somatic embryos to 1/29-MSO medium containing 10% sucrose for 15 days prior to transfer on 1/2-MS medium with 2% sucrose enhanced the conversion of somatic embryos into plantlets from 50 to 75%. The plantlets with shoots and roots were transferred to 1/2 and 1/4-liquid MS medium, each for 10 days, and then to plastic pots containing autoclaved peat moss and compost mixture (1:1). 70% of the plantiets survived after 10 weeks of transfer to pots. 120 regenerated plantlets out of 150 were successfully acclimatised. After successful acclimatisation, plants were transferred to earthen pots.

Sucrose metabolism during somatic and zygotic embryogeneses in Norway spruce: content of soluble saccharides and localisation of key enzyme activities

Changes in soluble carbohydrate compounds and their amounts were analysed during the somatic embryo maturation and seed development of Norway spruce (Picea abies (L.) Karst.). The data were correlated to the localisation of key enzymes of carbohydrate metabolism (invertase, EC 3.2.1.26; sucrose synthase, EC 2.4.1.13; phosphoglucomutase, EC 5.4.2.2). The decrease in total carbohydrate content as well as the accumulation of sucrose in later stages was a common feature in both systems. At the beginning of somatic embryo maturation the activity of sucrose synthase was low, and it gradually increased during development. At the final cotyledonary stage the SuSy localisation in somatic embryos resembled the pattern observed in cotyledonary stage zygotic embryos. Activity of invertase (pH 6.0) was detected in the early stages of somatic embryo development. Afterwards the activity decreased to almost undetectable levels in mature cotyledonary embryos. Very low invertase activity was detected during the period of seed development studied. The activity of phosphoglucomutase was high throughout the whole process of maturation in both zygotic and somatic embryos. The results are discussed in respect to the transition of developing embryos from metabolic to storage sink and the possible signalling potential of the changes.

Somatic embryogenesis and plantlet regeneration from cotyledon explants isolated from emblings and seedlings of hybrid firs

Summary Embryogenic tissues have been initiated on cotyledon explants dissected from seedlings or emblings of hybrid firs. Cotyledons of seedling origin ( Abies alba x A. cephalonica ) gave a relatively low initiation frequency (1.94 percnt;). In embling-derived cotyledons ( Abies alba x A. cephalonica, Abies alba x A. numidica ), the initiation was cell-line dependent and reached values between 1.25 and 24.28 percnt;. The established embryogenic cell lines are being maintained in long-term cultures. The origin and development of the somatic embryos have been traced histologically. The early stages of somatic embryo development have been characterised by cell division activity (predominantly periclinal) in the epidermal and subepidermal layers of cotyledons and subsequently by development of nodular structures. Further differentiation led to the formation and emergence of somatic embryos on the surface of cotyledons. Somatic embryo development and plantlet regeneration occurred from proliferating tissues initiated from cotyledons of embling as well as seedling origin.

Pyrimidine metabolism during somatic embryo development in white spruce ( Picea glauca)

Summary Pyrimidine metabolism was investigated at various stages ofsomatic embryo development of white spruce ( Picea glauca ). The contribution of the de novo and the salvage pathways of pyrimidine biosynthesis to nucleotide and nucleic acid formation and the catabolism of pyrimidine was estimated by the exogenously supplied [6- 14 C]orotic acid, an intermediate of the de novo pathway, and with [2- 14 C]uridine and [2- 14 C]uracil, substrates of the salvage pathways. The de novo pathway was very active throughout embryo development. More than 80 percnt; of [6- 14 C]orotic acid taken up by the tissue was utilized for nucleotide and nucleic acid synthesis in all stages of this process. The salvage pathways of uridine and uracil were also operative. Relatively high nucleic acid biosynthesis from uridine was observed, whereas the contribution of uracil salvage to the pyrimidine nucleotide and nucleic acid synthesis was extremely limited. A large proportion of uracil was degraded as 14 CO 2 , probably via β-ureidopropionate. Among the enzymes of pyrimidine metabolism, orotate phosphoribosyltransferase was high during the initial phases of embryo development, after which it gradually declined. Uridine kinase, responsible for the salvage of uridine, showed an opposite pattern, since its activity increased as embryos developed. Low activities of uracil phosphoribosyltransferase and non-specific nucleoside phosphotransferase were also detected throughout the developmental period. These results suggest that the flux of the de novo and salvage pathways of pyrimidine nucleotide biosynthesis in vivo is roughly controlled by the amount of these enzymes. However, changing patterns of enzyme activity during embryo development that were measured in vitro did not exactly correlate with the flux estimated by the radioactive precursors. Therefore, other fine control mechanisms, such as the fluctuation of levels of substrates and/or effectors may also participate to the real control of pyrimidine metabolism during white spruce somatic embryo development.

Induction of somatic embryogenesis in cashewnut (Anacardium occidentale L.)

Somatic embryogenesis was induced in callus cultures derived from nucellar tissue of cashewnut (Anacardium occidentale L.). Callus was obtained from nucellar tissue after 3 wk of culture on semisolid Murashige and Skoog (MS) basal medium supplemented with 2,4-dichlorophenoxyacetic acid (2,4-D, 5 μM)+gibberellic acid (GA3, 15 μM)+N6-benzyladenine (BA, 5 μM). This callus gave rise to an embryogenic mass after 9 wk on maintenance medium containing 2,4-D (10 μM)+GA3 (15 μM)+4% sucrose +0.5% activated charcoal +10% coconut water (CW) +0.05% casein hydrolysate (CH). The embryogenic mass, after transfer to medium supplemented with 2,4-D (5 μM)+GA3 (30 μM)+4% sucrose +0.5% activated charcoal +10% CW +0.05% CH, gave rise to somatic embryos. The developmental stages of somatic embryos were observed using light and stereo microscopes. Histological study of somatic embryo development was also carried out. The present study would be useful for clonal propagation, and variety improvement in cashewnut, which is essential due to its increasing demand and export potential.

Two-dimensional electrophoretic analysis of proteins associated with somatic embryogenesis development in Cupressus sempervirens L.

Two-dimensional gel electrophoretic analysis and histological studies were performed on somatic embryos in cypress. Embryogenic cultures were obtained from in vitro culture of immature seeds. On a modified Murashige and Skoog (MS) medium they showed an intense and repetitive cleavage polyembryogenesis phenomenon which maintained them in a continuous proliferating status instead of undergoing a complete embryogenic development. Only the addition of bovine serum albumin to the culture allowed somatic embryo development and maturation. Major histological differences were noticed between developing and nondeveloping embryogenic cultures. Attempts to find proteins that could be associated with developmental stages of somatic embryos have been achieved. Proteins were extracted and analyzed by two-dimensional electrophoresis from nondeveloping embryogenic cultures (S0) and from embryogenic cultures at three different stages of somatic embryo development: small size and rounded shape embryos (S1), increased size embryos with a well-developed suspensor (S2) and embryos with two well-separated cotyledons (S3). The results revealed some qualitative and quantitative protein variations between the two cultures. Some could be connected with the induction of pro-embryo differentiation whereas others should be more related to the mechanisms involved in somatic embryo development and maturation. Specific polypeptides associated with the presence of bovine serum albumin (BSA) in the medium have been detected.

Biosynthesis of pregnane derivatives in somatic embryos of Digitalis lanata

Activities of the enzymes cholesterol monooxygenase (side chain cleaving) (SCCE) Δ 5-3β- hydroxysteroiddehydrogenase Δ 5-Δ 4- ketosteroid isomerase, progesterone 5β-reductase, 3β-hydroxysteroid 5β-oxidoreductase, 3α-hydroxysteroid 5β-oxidoreductase, progesterone 5α-reductase and 3β-hydroxysteroid 5α-oxidoreductase which are involved in biosynthesis and transformation of pregnane derivatives were determined in different developmental stages of somatic embryos of Digitalis lanata. All enzymes were found to be present in proembryogenic masses (PEMs) as well as in globular and bipolar embryos. Most SCCE activity was found in the mitochondria fraction. Cholesterol, sitosterol and stigmasterol, precursors of the pregnanes, occurred in somatic embryos in amounts of about 1 μg mg −1 protein. Pregnenolone was found in traces only (about 20 ng mg −1 protein). Feeding of progesterone caused an increase of the contents of 5α-pand 5β-pregnandione, progesterone, 5β-pregnane-3α-ol,20-one and 5α-pregnane-3β-ol,-20-one. In contrast, administration of cholesterol caused a small increase of pregnenolone only. These results indicate that the rate limiting step in pregnane (and probably in cardenolide biosynthesis) is compartmentation of CSSE in the mitochondria of the somatic embryos of D. lanata.

Ultrastructural Changes in Cotyledons of Pineapple Guava (Myrtaceae) During Somatic Embryogenesis

Ultrastructural studies (SEM and TEM) were performed on cotyledons of pineapple guava ( Feijoa sellowianaBerg, Myrtaceae) induced to form embryos on medium containing 1.0 mg l −1(4.5μ M2,4-D) and 0.3 Msucrose. At the time of culture, the cells were filled with protein and lipid bodies. Microbodies and poorly differentiated organelles could also be seen. In contrast to germinating cotyledons, where lipid and protein reserves were quickly metabolized, cells of the embryogenically induced cotyledons showed evidence of reserve consumption only after 5 d of culture. Subepidermal cells of the upper cotyledonary surface underwent several divisions giving rise to a meristematic layer of several cells thickness from which somatic embryos developed. Embryos could also be formed directly by successive divisions of epidermal cells. Cells involved in somatic embryo formation contained a large nucleus with a conspicuous nucleolus and dense cytoplasm where numerous ribosomes, mitochondria, plastids with starch and short profiles of rough endoplasmic reticulum were present. Plasmodesmata were present both in cell walls of the meristematic cells and in few celled embryos whereas in degenerating embryos or in more advanced stages of somatic embryo development no plasmodesmata could be found. Although oil bodies were not observed in the meristematic cells they were identified in very young embryos, being the first reserve compounds to appear. Cells not involved in somatic embryo differentiation were characterized by the presence of several microbodies containing a crystalloid inclusion and elongated mitochondria.

Total protein pattern expression during induction and development of carrot somatic embryos

Differential gene expression revealed by the analysis of total protein patterns was investigated in somatic embryogenesis of Daucus carota L. The aim of this work was to identify markers, and to assess the relationships between induction and different developmental stages of somatic embryogenesis. Proteins extracted from an embryogenic cell suspension and five developmental stages of somatic embryos were separated by two-dimensional gel electrophoresis. Both quantitative and qualitative changes were detected, and the relationships between the protein patterns were visualized using an unrooted tree. The different organs of somatic plantlets, namely root, hypocotyl and cotyledon, were analyzed separately for spatial differences in protein distribution. A comparison of six protein patterns revealed that the variable proteins were modulated according to five profiles, three of which are most relevant. Among the variable proteins, ten were specific to the cell suspension stage and might be assigned either to callogenesis or to embryogenesis induction. Six proteins found only in somatic embryos were shown to fluctuate at different developmental stages and represent morphogenesis markers. Proteins absent during the early stages of embryo development but expressed in the cell suspension stage as well as in the later stages, might be correlated with the presence of 2,4-D. In fact, the unrooted tree revealed that the cell suspension protein pattern was closer to that of somatic plantlets than to earlier stages of embryogenesis. Furthermore, the CS pattern was closer to that of roots, which are auxin inducible, than to the other organ of the plantlet. Therefore, possible effects of exogenous and endogenous auxins on protein synthesis are discussed.

Histology of somatic embryo initiation and development in chickpea ( Cicer arietinum L.)

The histology of somatic embryo initiation and development in chickpea ( Cicer arietinum L.) from different explants was studied. Immature cotyledonary segments, immature embryo axes and mature embryo axes of cultivar PG12 were cultured on Murashige and Skoog's (MS) basal medium supplemented with 3.0 mg/l 2,4,5-trichlorophenoxyacetic acid (2,4,5-T) with and without a gelling agent. In addition, young leaflets of cultivar C235 and immature embryo axes of cultivar PG5 were also cultured on MS basal medium supplemented with 1.25 mg/l 2,4-dichlorophenoxyacetic acid (2,4-D) + 0.25 mg/l kinetin and 3.0 mg/l 2,4-D +0.1 mg/l 6-benzylaminopurine (BAP). Explants with different developmental stages of somatic embryos were fixed for histological examination, cut to 10 μm thick sections, stained with hematoxylin-eosin and observed microscopically. Histological studies revealed that somatic embryos arose indirectly with an intervening callus phase from immature cotyledons and immature embryo axes, directly from mature embryo axes and by both routes from young leaflets.

Somatic embryo development in carrot is associated with an increase in levels of S‐adenosylmethionine, S‐adenosylhomocysteine and DNA methylation

Almost homogeneous populations representing different developmental stages of somatic embryos (globular, torpedo‐shaped, plantlets) and vacuolated cells were obtained from a cell suspension culture of carrot. The concentrations of S‐adenosylmethionine (SAM), S‐adenosylhomocysteine (SAH) and methylated DNA were determined in embryos at different developmental stages and were found to increase during somatic embryogenesis. The highest increase during embryogenesis was a 5‐fold increase in the level of SAM. A considerable increase in the methylation index (SAM/SAH ratio) was also found. We propose that the levels of SAM and SAH may be involved in the control of somatic embryogenesis by affecting the level of DNA methylation, which in turn might cause differential changes in gene activation. An increase in the level of SAM may be a prerequisite for progression of embryogenesis and the development of complete embryos.

Induction of somatic embryogenesis inCynara cardunculusL. (Compositae)

SUMMARY This report describes procedures for the production of somatic embryos from cotyledons and first leaves of in-vitro grown seedlings of Cynara cardunculus L. (Compositae). Embryogenic calli were obtained after 4–5 weeks of culture on solid B5 medium containing selected combinations and concentrations of 2,4-dichlorophenoxyacetic acid (2,4-D) and kinetin (KIN) or 6-benzylaminopurine (BAP). In the presence of 2,4-D at 1 mg litre−1 and KIN at 0–1 mg litre−1, initial stages of somatic embryo development were formed on the surface of the embryogenic calli. Cotyledon explants presented a higher percentage of embryogenic induction than leaf explants. The transfer of these cultures to B5liquid medium containing 2,4-D (0–1, 0–5, 1 mg litre−1) and zeatin (ZEA) at 1 mg litre−1 allowed induction of more embryos and their further development. 2,4-D at the optimum concentration of 0.1 mg litre−1 was necessary for embryo development. Some embryos presented abnormalities that were not prevented by the addition of abscisic acid (ABA) to the culture medium. Mature embryos when transferred to the same liquid medium lacking growth regulators showed root apex growth and greening of cotyledons. However, the germination of the embryos was still only occasional and plantlets obtained showed arrest of growth before development of leaves.

Pyrolysis mass spectrometry of developmental stages of maize somatic embryos

SUMMARY The diversity in biopolymers such as polysaccharides, (poly)phenols, nucleic acids and lipids in the developmental stages of two types of histologically different somatic embryos of maize was investigated, using in-source pyrolysis low voltage electron impact mass spectrometry (PyMS) and the data were compared with those obtained for zygotic embryos. The first type of somatic embryo is attached to callus tissue and shows the same histo-differentiation characteristics as zygotic embryos. The second type grows from clusters of cells in liquid culture and its shoot meristem is blocked in development (Emons & Kieft 1991). In-source pyrolysis low voltage electron impact mass spectrometry shows that during callus initiation from young zygotic embryos the relative abundances of di- and triglycerides decrease, but increase again after establishment of the embryogenic callus. During callus initiation mass peaks for ferulic acid appear. The embryogenic cell clusters contain more pentosans and hexosans than the non-embryogenic elongated cells. In the globular stage, the hexosans increase. At the ovoid stage, when the first cells with vascular thickenings are observed in the sections, which are brightly fluorescent and can be stained with phloroglucinol, mass peaks for lignin are observed in the mass spectrum. The mass spectra of the developmental stages of somatic embryos grown attached to callus are quite comparable to those of zygotic embryos. Both structures contain far less phenolics, no lignin-like polyphenols, and a much higher amount of polysaccharides than the single somatic embryos.