Zygotic Embryo Development Research Articles

Dehydrin genes and their expression in recalcitrant oak (Quercus robur) embryos

In this work, three dehydrin genes, QrDhn1, QrDhn2, QrDhn3, were isolated from recalcitrant oak (Quercus robur). Their expression pattern was analyzed in both zygotic and somatic embryos as well as in vegetative tissues exposed to different kinds of abiotic stresses including desiccation, osmotic stress, and chilling. The QrDhn1 gene encoding for Y(n)SK(n) type dehydrin was expressed during later stages of zygotic embryo development but in somatic embryos only when exposed to osmotic or desiccation stress. In contrast, the other two oak dehydrin genes encoding for putative K(n) type dehydrins were expressed only in somatic embryos (both not-treated and osmotically stressed) and leaves of oak seedlings exposed to desiccation. Behavior of these genes suggests that different dehydrins are involved in processes of seed maturation and response to altered osmotic (water status) conditions in somatic embryos. Revealing further members of dehydrin gene family in recalcitrant oak might contribute to clarify non-orthodox seed behavior as well as identify mechanisms contributing to desiccation tolerance in plants.

Proteomic analysis of the development and germination of date palm (Phoenix dactylifera L.) zygotic embryos

By using a comparative proteomic approach (2-DE coupled to MS/MS), the development, maturation, and germination of date palm zygotic embryos, have been studied. Proteins were trichloroacetic acid (TCA)-acetone-phenol extracted and resolved by 2-DE in the 5-8 pH range. The total protein content and the number of spots resolved increased from early (12 weeks after pollination (WAP); 68.96 mg/g DW: 207 spots) to late (17 WAP; 240.85 mg/g DW: 261 spots) stages, decreasing upon germination (from 120.8 mg/g DW: 273 spots in mature embryos to 26.35 mg/g DW: 87 spots in 15 days after germination). Up to 194 spots showed qualitative or quantitative differences between stages. Statistical analysis of spot variation was performed by PCA, obtaining a more accurate grouping of the samples and determining the most discriminant spots. Samples were also clustered based on Pearson distance and Ward's minimum distance. Sixty-five variable spots were subjected to MS analysis, resulting in 21 identifications. The identified proteins belong to the following functional categories: enzymes of glycolysis, tricarboxylic acid cycle, and carbohydrate biosynthesis, protein translation, storage (glutelin), and stress-related proteins. The evolution pattern of the functional groups was examined and discussed in terms of metabolism adaptation to the different embryogenic and germination stages.

Histological comparison between wheat embryos developing in vitro from isolated zygotes and those developing in vivo

There is currently great interest shown in understanding the process of embryogenesis and, due to the relative inaccessibility of these structures in planta, extended studies are carried out in various in vitro systems. The culture of isolated zygotes in particular provides an excellent platform to study the process of in planta embryogenesis. However, very few comparisons have been made between zygotic embryos grown entirely in cultures and those grown in vivo. The present study analyses the differences and similarities between the in vitro and in vivo development of wheat zygotic embryos at the level of morphology and histology. The study was possible thanks to an efficient culture system and an appropriate method of preparing isolated wheat zygotes for microscopy. The in vitro embryos were fixed, embedded and sectioned in the two-celled, globular, club-shaped and fully differentiated stages. Embryos developing in vitro closely followed the morphology of their in planta counterparts and their cell types and tissues were also similar, demonstrating the applicability of the present culture system for studying the process of zygotic embryogenesis. However, some important differences were also detected in the case of in vitro development: the disturbance of or lack of initial polarity led to changes in the division symmetry of the zygotes and subsequently to the formation of uniform cells in the globular structures. Presumably, differences between the in vitro and in planta environments resulted in a lower level of differentiation and maturation in in vitro embryos and in abundant starch and protein accumulation in the scutellum.

Isolation and characterization of an embryo-specific Em-like gene of pedunculate oak (Quercus robur L.) and its temporal and spatial expression patterns during somatic and zygotic embryo development

The oak Em gene, QrEm, was isolated from Quercus robur L. zygotic embryos in RT-PCR using degenerate primers. The full-length cDNA was obtained by RACE. Sequence analyses revealed a high degree of similarity to previously identified Em-like genes from both monocots and dicots. Northern blot analyses showed that QrEm gene is expressed in zygotic embryos during the mid-maturation phase, with a decline following towards the end of seed development. In somatic embryos, abundant transcript accumulation was observed after treatment with 6% sorbitol or after partial desiccation. In situ hybridization experiments displayed similar tissue-specific expression patterns in both zygotic and somatic embryos, predominantly in the meristematic regions of root and shoot apices as well as provascular tissues, whereas almost no hybridization signal was found in the cotyledons. Wounding, drought and cold all failed to induce expression of the QrEm gene in vegetative tissue, suggesting that the QrEm gene is embryo-specific and its expression is developmentally regulated.

Characteristics of High α‐Linolenic Acid Accumulation in Seed Oils

Modern diets are often deficient in omega-3 fatty acids and additional dietary sources of omega-3 fatty acids are useful. In order to investigate the molecular basis of the high accumulation of the omega-3 fatty acid, alpha-linolenic acid (18:3), in three different plants, flax (Linum usitatissimum), Dracocephalum moldavica, and Perilla frutescens omega-3 desaturase activity, transcript levels, and 18:3 in-vivo synthesis were examined. The 18:3 content was found to be higher at the later developmental stage of D. moldavica (68%) compared with P. frutescens (59%) and flax (45%) cotyledons. The 18:3 and 18:2 contents in both PC and TAG were determined during various stages of seed development for all three plants in addition to soybean (Glycine max). Northern blot analysis data of three different stages of D. moldavica, flax, and P. frutescens compared with moderately low 18:3 producers, soybean (Glycine max), and Arabidopsis thaliana and Brassica napus, (8-10% 18:3) at a stage of zygotic embryo development of high triglyceride synthesis showed that omega-3 desaturase mRNA levels were higher in all three high 18:3 producers, flax, D. moldavica and P. frutescens. This indicates that the high level of alpha-linolenic acid in TAG may be largely controlled by the level of omega-3 desaturase gene expression. However, the PC versus TAG fatty acid composition data suggested that along with omega-3 desaturase other enzymes also play a role in 18:3 accumulation in TAG, and the high accumulators have a selective transfer of alpha-linolenic acid into TAG.

Buthionine sulfoximine (BSO)-mediated improvement in cultured embryo quality in vitro entails changes in ascorbate metabolism, meristem development and embryo maturation

Applications of buthionine sulfoximine (BSO), an inhibitor of GSH (reduced glutathione), which switches the cellular glutathione pool towards the oxidized form GSSG, positively influences embryo quality by improving the structure of the shoot apical meristem and promoting embryo maturation, both of which improve the post-embryonic performance of the embryos. To investigate the mechanisms underlying BSO-mediated improvement in embryo quality the transcript profiles of developing Brassica napus microspore-derived embryos cultured in the absence (control) or presence of BSO were analyzed using a 15,000-element B. napus oligo microarray. BSO applications induced major changes in transcript accumulation patterns, especially during the late phases of embryogenesis. BSO affected the transcription and activities of key enzymes involved in ascorbate metabolism, which resulted in major fluctuations in cellular ascorbate levels. These changes were related to morphological characteristics of the embryos and their post-embryonic performance. BSO applications also activated many genes controlling meristem formation and function, including ZWILLE, SHOOTMERISTEMLESS, and ARGONAUTE 1. Increased expression of these genes may contribute to the improved structural quality of the shoot poles observed in the presence of BSO. Compared to their control counterparts, middle- and late-stage BSO-treated embryos also showed increased accumulation of transcripts associated with the maturation phase of zygotic embryo development, including genes encoding ABA-responsive proteins and storage- and late-embryogenic abundant (LEA) proteins. Overall these transcriptional changes support the observation that the BSO-induced oxidized glutathione redox state allows cultured embryos to reach both morphological and physiological maturity, which in turn guarantees successful regeneration and enhanced post-embryonic growth.

Regeneration of zygotic-like microspore-derived embryos suggests an important role for the suspensor in early embryo patterning

The inaccessibility of the zygote and proembryos of angiosperms within the surrounding maternal and filial tissues has hampered studies on early plant embryogenesis. Somatic and gametophytic embryo cultures are often used as alternative systems for molecular and biochemical studies on early embryogenesis, but are not widely used in developmental studies due to differences in the early cell division patterns with seed embryos. A new Brassica napus microspore embryo culture system, wherein embryogenesis highly mimics zygotic embryo development, is reported here. In this new system, the donor microspore first divides transversely to form a filamentous structure, from which the distal cell forms the embryo proper, while the lower part resembles the suspensor. In conventional microspore embryogenesis, the microspore divides randomly to form an embryonic mass that after a while establishes a protoderm and subsequently shows delayed histodifferentiation. In contrast, the embryo proper of filament-bearing microspore-derived embryos undergoes the same ordered pattern of cell division and early histodifferentiation as in the zygotic embryo. This observation suggests an important role for the suspensor in early zygotic embryo patterning and histodifferentiation. This is the first in vitro system wherein single differentiated cells in culture can efficiently regenerate embryos that are morphologically comparable to zygotic embryos. The system provides a powerful in vitro tool for studying the diverse developmental processes that take place during the early stages of plant embryogenesis.

A carrot G-box binding factor-type basic region/leucine zipper factor DcBZ1 is involved in abscisic acid signal transduction in somatic embryogenesis

Carrot (Daucus carota) somatic embryogenesis has been extensively used as an experimental system for studying embryogenesis. In maturing zygotic embryos, abscisic acid (ABA) is involved in acquisition of desiccation tolerance and dormancy. On the other hand, somatic embryos contain low levels of endogenous ABA and show desiccation intolerance and lack dormancy, but tolerance and dormancy can be induced by exogenous application of ABA. In ABA-treated carrot embryos, some ABA-inducible genes are expressed. We isolated the Daucus carota bZIP1 (DcBZ1) gene encoding a G-box binding factor-type basic region/leucine zipper (GBF-type bZIP) factor from carrot somatic embryos. The expression of DcBZ1 was detected in embryogenic cells, non-embryogenic cells, somatic embryos, developing seeds, seedlings, and true leaves. Notably, higher expression was detected in embryogenic cells, true leaves, and seedlings. The expression of DcBZ1 increased in seedlings and true leaves after ABA treatment, whereas expression was not affected by differences in light conditions. During the development of zygotic and somatic embryos, increased expression of DcBZ1 was commonly detected in the later phase of development. The recombinant DcBZ1 protein showed specific binding activity to the two ABA-responsive element-like motifs (motif X and motif Y) in the promoter region of the carrot ABA-inducible gene according to results from an electrophoretic mobility shift assay. Our findings suggest that the carrot GBF-type bZIP factor, DcBZ1, is involved in ABA signal transduction in embryogenesis and other vegetative tissues.

Proliferation and differentiation from endosperms of Carthamus tinctorius

The endosperms of Carthamus tinctorius cv. HUS-305, excised at globular to heart-shaped stages of zygotic embryo development, were cultured on Murashige and Skoog's medium (MS) supplemented with different concentrations of 6-benzylaminopurine (BAP), kinetin, thidiazuron (TDZ), 2,4-dichlorophenoxyacetic acid (2,4-D) or α-naphthalene-acetic acid (NAA). The highest incidence of callusing was on 2,4-D supplemented media. However, embryos differentiated only from the calli developed on media supplemented with BAP, kinetin or TDZ with the last eliciting maximum embryogenic response. The addition of a reduced nitrogen source, casein hydrolysate to MS medium supplemented with BAP and/or NAA, did not stimulate the response. However, adenine sulphate (100 mg dm-3) promoted the induction of somatic embryos. Upon transfer to MS basal medium or the same supplemented with 0.61 µM gibberellic acid (GA3), plumular poles of few embryos elongated resulting in the development of shoots.

PpRab1, a Rab GTPase from maritime pine is differentially expressed during embryogenesis

Rab-related small GTP-binding proteins are known to be involved in the regulation of the vesicular transport system in eukaryotic cells. We report the characterization of a previously isolated full-length cDNA PpRab1 from Pinus pinaster. Amino acid sequence analysis revealed the presence of G1-G5 conserved domains of the GTPase Ras superfamily and a double cysteine motif in the C-terminal, characteristic of Rab proteins. The PpRab1 protein shows high sequence similarity to several Rab1 GTP-binding proteins in plants. Phylogenetic analysis showed that, within the Ras superfamily, PpRab1 is more closely related to the Rab family and within this, PpRab1 protein was found to cluster with Arabidopsis subfamily AtRABE, whose members are known to regulate ER-to-Golgi membrane trafficking steps. PpRab1 transcripts were expressed at constitutively high levels for the initial stages of zygotic embryo development, and then their relative abundance decreased as embryo matures. The PpRab1 transcript is not embryo-specific as it was found in roots, cotyledons and hypocotyls. An increase in PpRab1 expression level was observed when seeds are germinated and collected at successive time points of development. In situ RT-PCR analysis revealed an expression signal in early zygotic embryos. In view of the proposed roles of Rab1 GTP-binding protein, the possible function of the protein encoded by PpRab1 in embryogenesis is discussed.

Changes in levels of gibberellins, their putative conjugates, and ABA in zygotic embryos during late maturation and dry seed stages of Brassica napus cv Topaz

Two late stages [days 35 and 40 after pollination (DAP)] in zygotic embryo (ZE) development of Brassica napus were utilized to quantify, by the stable isotope-labeled dilution method, levels of “free” and “aglycone” gibberellins (GAs), as well as abscisic acid (ABA), during the programmed dehydration of the seed. GAs from both the early 13 hydroxylation and early non-hydroxylation pathways were present in these ZEs of B napus. Between 35 and 40 DAP endogenous ABA dropped precipitously (almost 30-fold) and this drop in ABA was accompanied by a significant reduction in levels of GA1 and even in levels of the inactive GA catabolites, GA8 and GA29. Levels of GA4 and putative GA85 also dropped appreciably, though not significantly. In contrast, the levels of GA20 and GA9 (the immediate precursors of GA1 and GA4, respectively) did not change in the ZEs during this transition. A fungal-derived cellulase was used to hydrolyze the highly water-soluble fraction, which will contain GA conjugates. Relatively high levels of several GAs (GA9, GA20) were thus quantified after hydrolysis as the aglycones, e.g., 56 and 25 ng/g DW of GA20 and 23 and 5 ng/g DW, of GA9, respectively at DAP 35 and DAP 40. Other GAs found after hydrolysis of the highly water-soluble fraction remained relatively constant between 35 and 40 DAP. An exception was the putative GA85 aglycone, which increased sixfold (free GA85 decreased by ca. half). The transition to the dry seed stage for ZEs of B. napus is thus accompanied not only by the expected reduction in ABA, but also by reduced levels of many “free” GAs, especially the bioactive, 3β-hydroxylated GAs. In contrast, levels of 3-deoxy GAs remain relatively high, implying a partial block in the 3β-hydroxylation “activation” step of GA biosynthesis.

IAA, ABA, polyamines and free amino acids associated with zygotic embryo development of Ocotea catharinensis

The aim of this work was to study morphological and biochemical aspects during zygotic embryogenesis in O. catharinensis, by measuring changes in the endogenous concentrations of proteins, amino acids, polyamines (PAs), indole-3-acetic acid (IAA) and abscisic acid (ABA). Buffer-soluble and insoluble protein contents were determined by spectrometry, and amino acids, PAs, IAA and ABA concentrations were determined by high performance liquid chromatography. Total amino acid accumulation, predominantly asparagine, occurred when the embryo showed completely developed cotyledons, with posterior reduction in the mature embryo. This decrease in total amino acid concentration in the mature embryo may result from their use in storage␣as well as for LEA protein synthesis. Free putrescine (Put) concentration decreased, while free spermine (Spm) increased during embryo development. This suggest a role for Put in the initial phases of embryogenesis when high rates of cell division occur, while elevated concentration of Spm are essential from the middle to the end of embryo development, when growth is mainly due to cell elongation. An IAA peak in zygotic embryos occurred during initial development, suggesting a link between growth and cellular division as well as with the establishment of bilateral symmetry. ABA concentration declined during initial stages of development then increased at the mature embryo stage, suggesting a possible relationship with dormancy and recalcitrance characteristics. Our results show that changes in the phytohormones (IAA, ABA and PAs) concentrations in combination with amino acids are likely important factors determining the developmental stages of O.␣catharinensis zygotic embryos.

Localization of an arabinogalactan protein epitope and the effects of Yariv phenylglycoside during zygotic embryo development of Arabidopsis thaliana

Arabinogalactan proteins (AGPs) are a class of highly glycosylated proteins widely distributed in higher plants and thought to be involved in plant growth and development. In the present paper, Western blotting with the monoclonal antibodies JIM4, JIM13, and LM2 showed that JIM13 reacted best with total protein extracts from flowers and siliques of Arabidopsis thaliana. This monoclonal antibody was therefore used as a probe to localize the AGP epitope in zygotic embryos at different developmental stages. Immunofluorescent labeling with JIM13 showed that AGPs were mainly distributed in the embryo proper and the top 1 to 2 cells and basal part of suspensors. The results of immunogold labeling confirmed the JIM13 epitope distribution in the different cells of the suspensor. AGP immunofluorescence was also observed at the shoot apex meristem during transition from the globular to the heart embryo stage, but this gradually disappeared after the torpedo stage. After (beta-D-Glc)(3) Yariv phenylglycoside (betaGlcY), a synthetic reagent that specifically binds to AGPs, was added to A. thaliana ovule culture medium, the survival rate and frequency of development of ovules at the zygote stage decreased in a concentration-dependent manner, with complete inhibition at 100 microM. The frequency of embryo differentiation from the globular stage to heart or later stages also decreased sharply. When betaGlcY was removed 24 h after inoculation, the inhibitory effects were reversible in a concentration-dependent and time-dependent manner. The results show that betaGlcY can inhibit embryo development and differentiation in A. thaliana, and the inhibitory effects are concentration dependent and reversible, indicating that AGPs are involved in embryo differentiation and shoot meristem formation. The possible roles of AGPs in A. thaliana zygotic embryo development are also discussed.

Improved somatic embryo maturation in loblolly pine by monitoring ABA-responsive gene expression

During loblolly pine zygotic embryo development, increases in mRNAs for three ABA-responsive LEA-like genes coincided with the two developmental stage-specific peaks of endogenous ABA accumulation (Kapik et al. 1995). These ABA concentration profiles from zygotic embryo development were used to develop several tissue culture approaches that altered the exposure of somatic embryos to exogenous ABA. Elevating exogenous ABA at a time corresponding to mid-maturation improved the germination and resulted in more zygotic-like expression of selected genes in somatic embryos. Extending the time on maturation medium for a fourth month increased embryo yield, dry weight, and germination in high-and low-yield genotypes. Optimizing the amounts of embryogenic suspension, plated and exogenous ABA concentration increased from 22 to 66% in the early-stage bipolar embryos that developed to the cotyledonary stage.

Comparative analysis of cell cycle events in zygotic and somatic embryos of Cyclamen persicum indicates strong resemblance of somatic embryos to recalcitrant seeds

Embryo development and germination of Cyclamen persicum have been comparatively characterized for zygotic and somatic embryos with regard to mitotic activity and morphology in order to identify developmental abnormalities in somatic embryogenesis. Zygotic embryo development proved to be highly synchronous with distinct periods of cell division, cell elongation and embryo maturation within a total period of 17 weeks of seed development. Somatic embryo development was accomplished within only 3 weeks, resulting in a mixture of morphologically highly variable embryos. No distinct developmental periods could be identified and no reduction of the mitotic activity was discovered for non-desiccated somatic embryos. Controlled desiccation of somatic embryos severely reduced their germination rate, demonstrating resemblance of somatic embryos to recalcitrant seeds, whereas zygotic Cyclamen seeds could be characterized as typically orthodox.

Histological aspects of avocado embryo development and effect of developmental stages on germination

Anatomical differentiation and storage product accumulation were studied during avocado (Persea americana Mill.) zygotic embryo development using histological techniques. The results were correlated with the germination capacity under in vitro conditions. The histodifferentiation phase, characterized by active cell division and differentiation, was accomplished by 100 d after pollination (DAP), when embryos reached 16–18 mm in length. After a phase in which there were no morphological changes, the maturation phase commenced at 125 DAP in embryos 24–26 mm in length. Accumulation of starch granules and the appearance of protein bodies occurred at this time. Germination was significantly influenced by the embryo developmental stage. Different parameters used as indicators of germination quality (germination percentage, germination speed and recovery of complete plantlets) showed a positive correlation with the age of the embryos. However, germination in vitro was not achieved fully until the end of zygotic embryo development, when the embryo reached physiological maturity.

The effect of osmoticum on ascorbate and glutathione metabolism during white spruce ( Picea glauca) somatic embryo development

Water stress is an important factor which regulates organized development of both zygotic and somatic embryos. Somatic embryos of white spruce were cultured in the presence of polyethylene glycol (PEG), a non-plasmolyzing agent which increases embryo quality and number, and mannitol, a plasmolyzing agent. The effects of these two compounds on both ascorbate and glutathione metabolism were investigated at different stages of embryo development. Compared to control and mannitol-treated embryos, embryos treated with PEG accumulated higher levels of endogenous ascorbate (ASC) in its reduced form, especially during the first half of the maturation period. This increase, also observed in immature seeds, was mainly the result of two different processes: activation of the de novo ASC machinery, and recycling of ASC from ascorbate free radicals (AFR) which was modulated by the activity of ascorbate free radical reductase (AFRR, EC. 1.6.5.4). The activity of this enzyme increased during the early phases of development in both PEG-treated somatic embryos and seeds. Compared to control somatic embryos, mannitol and PEG were shown to change the levels of reduced (GSH) and oxidized glutathione (GSSG). In particular, a constant decline in the GSH/GSSG ratio was observed in the presence of PEG. This pattern was also observed in maturing white spruce seeds. Overall, these data indicate that applications of non-plasmolyzing agents in the culture medium of spruce somatic embryos result in seed-like fluctuations of the ascorbate–glutathione metabolism, which may have a positive effect on embryo yield.

Diurnal O2 and carbohydrate levels in wheat kernels during embryony

In vitro zygotic and somatic embryogenesis procedures for wheat have been improved by simulating in ovulo nutritional, hormonal and dissolved oxygen (dO2) conditions. However, diurnal fluctuations in these conditions during early embryony are not well characterized. In this study, dO2 and water-soluble carbohydrate levels in wheat kernels were determined after 8h of light and 8h of dark at approximately 6, 12 and 18 day post anthesis (DPA). Clark style O2 microelectrodes, having a tip diameter of approximately 115μm, were inserted into intact kernels immediately distil to the developing embryo, and dO2 levels were recorded at 50μm intervals into the center of kernels. High-performance anion exchange chromatography with pulsed amperometric detection was used to quantify carbohydrate levels in endosperm sap. dO2 levels in the chlorophyllous layer of the pericarp reached 190mmolm−3 during the day, which probably represents, because of photosynthesis, a supersaturated O2 condition relative to the external environment (21% O2). At the embryo surface, dO2 levels at 6DPA ranged from 135 to 170mmolm−3. At 12 and 18DPA, dO2 levels at the embryo axis ranged from 100 to 150mmolm−3. At all three stages, dO2 levels in the center of the endosperm were below 13mmolm−3. Extreme fluctuations in carbohydrate levels were observed diurnally during rapid seed fill (12DPA). Levels of sucrose and short-chain fructans were much higher during the day than during the night. In contrast, fructose, glucose, and myo-inositol levels were much higher during the night than during the day. By 18DPA (hard dough stage), carbohydrate levels tended to be similar during the day and night. These dynamic fluctuations may assist in regulating embryony in ovulo, and their simulation might improve the development of somatic and zygotic embryos in vitro.

Isolation of the gene encoding Carrot leafy cotyledon1 and expression analysis during somatic and zygotic embryogenesis

The Arabidopsis thaliana LEC1 gene regulates embryo morphology and seed maturation. For a better understanding of its function, we isolated a carrot ( Daucus carota L. cv. US-Harumakigosun) counterpart of this gene, C-LEC1, from a cDNA library of carrot somatic embryos, since carrot is a better model plant for preparing large quantities of somatic embryos at the same developmental stage. The predicted amino acid sequence of C-LEC1 is similar to that of LEC1 and contains regions that are conserved in the heme-activated protein 3 (HAP3) subunit of plants, animals and microorganisms. C-LEC1 expression was detected in embryogenic cells, somatic embryos, and developing seeds. In situ hybridization analysis revealed C-LEC1 expression in the peripheral region of the embryos but not in the endosperm. Expression of C-LEC1 driven by Arabidopsis LEC1 promoter was able to complement the defects of the Arabidopsis lec1-1 mutant. These results suggest that C-LEC1 is a functional homolog of Arabidopsis LEC1, an important regulator of zygotic and somatic embryo development.

An in vitro Culture System used to Investigate Possible Interactions between the Embryo Proper and the Suspensor in Embryogenesis in Japanese Larch (Larix leptolepis GORDON)

It has been proposed that the suspensor has important roles in early embryogenesis in seed plants. However, the roles of the suspensor are not well understood, because the development of zygotic embryos normally occurs deep within both the endosperm and the maternal cells. In this paper, we report the development of an in vitro culture system to investigate the roles of the suspensor in the development of the embryo proper, using a somatic embryogenesis system with Japanese larch (Larix leptolepis GORDON). Our results indicate that the suspensor is essential for the normal development of somatic embryos of this species. This method provides a useful experimental system to investigate the interactions between the embryo proper and the suspensor.