Non-embryogenic Cells Research Articles

Comparative quantitative proteomic analysis of embryogenic and non-embryogenic calli in maize suggests the role of oxylipins in plant totipotency

Totipotency, the ability of somatic plant cell to generate whole plant through somatic embryogenesis, is still not well understood. In this study, maize immature zygotic embryos were used to generate embryogenic (EC) and non-embryogenic (NEC) calli. In order to compare proteomes of EC and NEC, two-dimensional electrophoresis (2-DE) in combination with mass spectrometry was used. This approach resulted into 361 quantified 2-DE spots out of which 44 were found statistically significantly differentially abundant between EC and NEC. Mass spectrometry provided the identity for 23 proteins that were classified into 8 metabolic categories. The most abundant were proteins associated with energy followed by proteins associated with disease and defense. Based on the abundances of identified proteins in this and other studies, working model for plant totipotency was proposed. One aspect of this working model suggests that increased abundances of proteins associated with pyruvate biosynthesis and suppression of embryogenic genes might be responsible for differences between EC and NEC cells. Furthermore we speculate that the increased abundance of lipoxygenase in the NEC cells results in changes in the equilibrium levels of one or more signaling molecules and is at least partly responsible for somatic cell reprogramming during totipotency. Totipotency, the ability of somatic plant cell to generate whole plant through somatic embryogenesis, is still not well understood. In order to further advance understanding of this biological phenomenon, proteomes of embryogenic and non-embryogenic callus, derived from immature zygotic embryos of inbred maize line A19, were compared using 2-DE based proteomic technology. Based on the abundances of identified proteins in this and other studies, working model for plant totipotency was proposed. One aspect of this working model suggests that increased abundances of proteins associated with pyruvate biosynthesis and suppression of embryogenic genes might be responsible for differences between EC and NEC cells. Furthermore we speculate that the increased abundance of lipoxygenase in the NEC cells results in changes in the equilibrium levels of one or more signaling molecules and is at least partly responsible for somatic cell reprogramming during totipotency. This article is part of a Special Issue entitled: Environmental and structural proteomics.

A systematic comparison of embryogenic and non-embryogenic cells of banana (Musa spp. AAA): Ultrastructural, biochemical and cell wall component analyses

Plant regeneration via somatic embryogenesis is essential for improvement of vegetatively propagated banana (Musa spp. AAA) through biotechnological techniques. A systematic comparison of embryogenic cells (ECs) and non-embryogenic cells (NECs) of banana was performed in the present study. Immuno-dot blotting, immunoflourescence labeling, biochemical and ultrastructural analyses were employed to study differences between ECs and NECs.Both immuno-dot analysis and immunofluorescence labeling revealed a higher abundance of fucosylated xyloglucans in cell walls of ECs. Arabinogalactan-protein (AGP) epitopes recognized by LM2 and NH4.3E5 antibodies were more abundant in NECs. On the other hand, AGPs recognized by JIM16, MAC204 and MAC207 antibodies were more abundant in ECs. Biochemical analyses revealed that the content of total soluble sugars and reducing sugars, as well as levels of indole-3-acetic acid, abscisic acid and zeatin riboside were significantly higher in ECs in comparison to NECs. In contrast, NECs showed significantly higher content of gibberellic acid. In addition, NECs were highly vacuolated and contained lipid bodies, prominent mitochondria and plastids, however, endoplasmic reticulum and Golgi bodies were not so abundant when compared to ECs.This study revealed that cell walls of ECs are rich in fucosylated xyloglucan and in AGP epitopes labeled by JIM16, MAC204 and MAC207 antibodies, indicating that these cell wall components together with higher levels of auxin, abscisic acid and zeatin riboside are contributing to the embryogenic competence of banana. In contrast, NECs showed more abundant non-fucosylated and non-XXXG xyloglucans in their cell walls, and higher level of gibberellic acid. This study provides new and useful information about hormonal and cell wall regulation of banana embryogenic competence which contributes to better understanding of this process, and might lead to practical applications in the near future.

Accumulation pattern of dehydrins during sugarcane (var. SP80.3280) somatic embryogenesis

The objective of the present study was to determine dehydrin protein levels in sugarcane var. SP80-3280 during somatic embryogenesis. Dehydrins from embryogenic and non-embryogenic cell cultures were analyzed using western blot and in situ immunolocalization microscopy. Both techniques employ antibodies raised against a highly conserved lysine-rich 15-amino acid sequence termed the K-domain, which is extensively used to recognize proteins immunologically related to the dehydrin family. In embryogenic cultures, western blot analysis of the heat-stable protein fraction revealed eleven major bands ranging from 52 to 17 kDa. They were already visible on the first days, gradually increasing until reaching peak values around day 14, when organogenesis begins, to later decrease in concurrence with the appearance of green plantlets (around day 28). These fluctuations indicate that this pattern of accumulation is under developmental control. Dehydrins were mainly immunolocalized in the nuclei. A phosphatase treatment of protein extracts caused a mobility shift of the 52, 49, and 43 kDa dehydrin bands suggesting a putative modulation mechanism based on protein phosphorylation. In sugarcane embryogenic cultures, presence of dehydrins is a novel finding. Dehydrins were absent in non-embryogenic cultures. The novel findings regarding accumulation, nuclear localization, and phosphorylation of dehydrins provide a starting point for further research on the role of these proteins in the induction and/or maintenance of embryogenesis. The novel findings regarding accumulation, nuclear localization, and phosphorylation of dehydrins provide a starting point for further research on the role of these proteins in the induction and/or maintenance of embryogenesis.

Histological changes and differences in activities of some antioxidant enzymes and hydrogen peroxide content during somatic embryogenesis of Musa AAA cv. Yueyoukang 1

To determine the possible involvement of oxidative stress in somatic embryogenesis of banana (Musa spp.), and provide a means to overcome the recalcitrance in embryogenesis, changes in histology, hydrogen peroxide (H2O2) content and antioxidant enzyme activities during banana somatic embryogenesis were investigated. From embryogenic cells (ECs) to early globular embryos, nearly all cells showed typical meristematic characteristics with a high nucleus/cytoplasm ratio. Cells started to lose their meristematic characteristic during later globular stage. Starch granules in cells showed spatial and temporal changes during somatic embryogenesis. POD (E.C.1.11.1.7) activity decreased significantly after transfer of ECs to the embryo regeneration medium which was followed by a significant increase and peaked at the late stages of embryogenesis. CAT (E.C.1.11.1.6) activity showed similar pattern, however its highest activity appeared in the ECs instead of the later embryos. On the contrary, a gradual increase in SOD (E.C.1.15.1.1) activity was observed after inoculation of ECs on embryo regeneration medium until the globular stage. Similarly to SOD activity, the hydrogen peroxide (H2O2) content also increased after inoculation of ECs on embryo regeneration medium and it peaked in proembryos, followed by a significant decrease in later embryogenic stages. The H2O2 content as well as activities of all tested antioxidant enzymes were significantly higher in ECs than in non-embryogenic cells. These results suggested that high levels of antioxidant enzyme activity and high H2O2 content were necessary for embryogenic competence of ECs while high H2O2 content played an important role in early development of somatic embryogenesis of banana.

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.

Regeneration of Centella asiatica plants from non-embryogenic cell lines and evaluation of antibacterial and antifungal properties of regenerated calli and plants

BackgroundThe threatened plant Centella asiatica L. is traditionallyused for a number of remedies. In vitro plant propagation and enhanced metabolite production of active metabolites through biotechnological approaches has gained attention in recent years.ResultsPresent study reveals that 6-benzyladenine (BA) either alone or in combination with 1-naphthalene acetic acid (NAA) supplemented in Murashige and Skoog (MS) medium at different concentrations produced good quality callus from leaf explants of C. asiatica. The calli produced on different plant growth regulators at different concentrations were mostly embryogenic and green. Highest shoot regeneration efficiency; 10 shoots per callus explant, from non-embryogenic callus was observed on 4.42 μM BA with 5.37 μM NAA. Best rooting response was observed at 5.37 and 10.74 μM NAA with 20 average number of roots per explant. Calli and regenerated plants extracts inhibited bacterial growth with mean zone of inhibition 9-13 mm diameter when tested against six bacterial strains using agar well diffusion method. Agar tube dilution method for antifungal assay showed 3.2-76% growth inhibition of Mucor species, Aspergillus fumigatus and Fusarium moliniformes.ConclusionsThe present investigation reveals that non-embryogenic callus can be turned into embryos and plantlets if cultured on appropriate medium. Furthermore, callus from leaf explant of C. asiatica can be a good source for production of antimicrobial compounds through bioreactor.

Characterization of metabolic differences between embryogenic and non-embryogenic cells in forest trees

Somatic embryogenesis (SE) in forest trees is considered as one powerful approach in cloning elite genotypes. However, SE is difficult to achieve in tissue or cell beyond the mature embryo phase in trees and its physiological process involved are also poorly understood. Recently metabolic profiling studies have been contributed to understand the mechanism involved in SE process of plant [1-3]. In addition, there are also a number of reports indicating that polyamines (PAs) play a crucial role in SE process [4,5]. As part of an ongoing study of the transition of somatic cells to an embryogenic state in adult trees, embryogenic (EC) and non-embryogenic cells (NEC) in various forest species were investigated for its metabolic compositions including PAs. A comparison of metabolic compositions of NEC and EC using gas chromatography/mass spectrometry (GC/MS) identified around 50 compounds, partly displaying significant changes in metabolite levels, e.g., highly elevated levels of xanthosine and methyloxazole in EC compared to NEC of broadleaves and conifer species (Fig.​(Fig.1).1). Changes in the polyamine content were also analyzed in both cell types. Free polyamine contents varied according to the species and cell types, the highest levels occurring in the NEC on proliferation medium, when putrescine and spermidine were most abundant. However, the putrescine/spermine+spermidine (put/spm+spd) ratio was higher in EC of yellow poplar and hybrid pine. Analysis of PAs in both cell types indicated that total polyamine concentration was always higher in NEC than in EC, and spermine was present in only minute quantities and showed only a small change. From this analysis, we have identified numerous compounds involved with embryogenic state, and could characterize its differences between broadleaves and conifer trees. Figure 1 Non-embryogenic (NEC; A, C) and embryogenic cells (EC; B, D) of yellow poplar and hybrid pine ‘rigida x taeda’ respectively, and its metabolic compositions (NEC: a, c and EC: b, d).

Extracellular matrix as the early structural marker for Centella asiatica embryogenic tissues

Embryogenic and non-embryogenic calli were induced from the Centella asiatica leaf explants on Murashige and Skoog medium supplemented with kinetin and 2,4-dichlorophenophenoxyacetic acid. The extracellular matrix (ECM) layer was seen on the surface of embryogenic cells but not on the non-embryogenic cells. The ECM formed bridges with net-like material between the embryogenic cells. This network like structure was believed to play an important role in plant morphogenesis and can serve as an early structural marker of embryogenic competence in Centella asiatica calli culture.

Developmental localization and the role of hydroxyproline rich glycoproteins during somatic embryogenesis of banana (Musaspp. AAA)

BackgroundHydroxyproline rich glycoproteins (HRGPs) are implicated to have a role in many aspects of plant growth and development but there is limited knowledge about their localization and function during somatic embryogenesis of higher plants. In this study, the localization and function of hydroxyproline rich glycoproteins in embryogenic cells (ECs) and somatic embryos of banana were investigated by using immunobloting and immunocytochemistry with monoclonal JIM11 and JIM20 antibodies as well as by treatment with 3,4-dehydro-L-proline (3,4-DHP, an inhibitor of extensin biosynthesis), and by immunomodulation with the JIM11 antibody.ResultsImmunofluorescence labelling of JIM11 and JIM20 hydroxyproline rich glycoprotein epitopes was relatively weak in non-embryogenic cells (NECs), mainly on the edge of small cell aggregates. On the other hand, hydroxyproline rich glycoprotein epitopes were found to be enriched in early embryogenic cells as well as in various developmental stages of somatic embryos. Embryogenic cells (ECs), proembryos and globular embryos showed strong labelling of hydroxyproline rich glycoprotein epitopes, especially in their cell walls and outer surface layer, so-called extracellular matrix (ECM). This hydroxyproline rich glycoprotein signal at embryo surfaces decreased and/or fully disappeared during later developmental stages (e.g. pear-shaped and cotyledonary stages) of embryos. In these later developmental embryogenic stages, however, new prominent hydroxyproline rich glycoprotein labelling appeared in tri-cellular junctions among parenchymatic cells inside these embryos. Overall immunofluorescence labelling of late stage embryos with JIM20 antibody was weaker than that of JIM11. Western blot analysis supported the above immunolocalization data. The treatment with 3,4-DHP inhibited the development of embryogenic cells and decreased the rate of embryo germination. Embryo-like structures, which developed after 3,4-DHP treatment showed aberrant non-compact epidermis with discontinuous ECM at the outer surface as well as much less immunolabelling with the JIM11 antibody. This treatment also decreased the plant regeneration capacity in embryogenic banana cultures. Finally, immunomodulation of surface hydroxyproline rich glycoproteins by co-culture of embryos with the JIM11 antibody resulted in a much lower germination capacity of these embryos.ConclusionsThese results suggest that hydroxyproline rich glycoproteins play an important developmental role, especially in the process of regeneration and germination of embryos during plant regeneration via somatic embryogenesis. Proper content and localization of hydroxyproline rich glycoproteins seem to be essential for the formation and regeneration of banana somatic embryos.

Competence versus Recalcitrance for in Vitro Regeneration

Plant regeneration from tissue cultures of many species has been reported so far, but various groups, families and genera are still regarded as recalcitrant, and tools susceptible to distinguish as early in culture as possible the regeneration-competent cells and tissues from those that will never regenerate would be of interest, whatever the regeneration pathway. We have examined various cytological parameters to identify those that might serve as early indicators of cell competence to undergo somatic embryogenesis and/or organogenesis. Water potential of the culture medium declined preceding the onset of embryogenesis while intracellular osmolarity and cell surface increased concomitantly in embryogenic cells. In addition, cellulose accumulated in the walls of non-embryogenic cells while cell walls became thinner with onset of embryogenesis, and diminished further as embryos matured. In parallel experiments, the cytogenetic, proteomic and molecular processes governing the competence for embryogenesis and/or organogenesis from plant cells were studied with Arabidopsis thaliana, and more recently also with Medicago truncatula and pea, both by flow cytometry, immuno-cytohistology, mono and bidimensional electrophoresis and comparing insertion mutants and RNAis with their respective wild types. The implications of these results when applying biotechnology approaches for grain legume breeding, and in terms of plant regeneration competence in general are discussed.

Large impact of the apoplast on somatic embryogenesis in Cyclamen persicum offers possibilities for improved developmental control in vitro.

BackgroundClonal propagation is highly desired especially for valuable horticultural crops. The method with the potentially highest multiplication rate is regeneration via somatic embryogenesis. However, this mode of propagation is often hampered by the occurrence of developmental aberrations and non-embryogenic callus. Therefore, the developmental process of somatic embryogenesis was analysed in the ornamental crop Cyclamen persicum by expression profiling, comparing different developmental stages of embryogenic cell cultures, zygotic vs. somatic embryos and embryogenic vs. non-embryogenic cell cultures.ResultsThe analysis was based on a cDNA microarray representing 1,216 transcripts and was exemplarily validated by realtime PCR. For this purpose relative transcript abundances of homologues of a putative receptor kinase, two different glutathione S-transferases (GST), a xyloglucan endotransglycosylase (XET) and a peroxidase (POX) were quantitatively measured by realtime PCR for three different comparisons. In total, 417 genes were found to be differentially expressed. Gene Ontology annotation revealed that transcripts coding for enzymes that are active in the extracellular compartment (apoplast) were significantly overrepresented in several comparisons. The expression profiling results are underpinned by thorough histological analyses of somatic and zygotic embryos.ConclusionsThe putative underlying physiological processes are discussed and hypotheses on improvement of the protocol for in vitro somatic embryogenesis in Cyclamen persicum are deduced. A set of physiological markers is proposed for efficient molecular control of the process of somatic embryogenesis in C. persicum. The general suitability of expression profiling for the development and improvement of micropropagation methods is discussed.

A novel regeneration system for a wild passion fruit species (Passiflora cincinnata Mast.) based on somatic embryogenesis from mature zygotic embryos

The objective of the present work was to induce somatic embryogenesis from zygotic embryos of Passiflora cincinnata Masters. Zygotic embryos formed calli on media with different concentrations of 2,4-dichlorophenoxyacetic acid (2,4-D) and 4.5 μM benzyladenine (BA) after 30 days of in vitro culture. A concentration of 18.1 μM 2,4-D resulted in the largest number of somatic embryos. Embryogenic calli were yellowish and friable, forming whitish proembryogenic masses. Morphologically, embryogenic cells were small and had large nuclei and dense cytoplasm, whereas non-embryogenic cells were elongated, with small nuclei and less dense cytoplasm. Calli cultured under white light on basal Murashige and Skoog’s medium with activated charcoal produced embryos in all developmental stages. There were differences among the treatments, with some leading to the production of calli with embryos and some only to callus formation. Some abnormalities were associated with somatic embryos, including fused axes, fused cotyledons and polycotyledonary embryos. Production of secondary somatic embryos occurred in the first cycle of primary embryo development. Secondary embryos differentiated from the surface of the protodermal layer of primary embryos with intense cell proliferation, successive mitotic divisions in the initial phase of embryoid development, and a vascular system formed with no connection to the parental tissue. This secondary embryogenic system of P. cincinnata is characterized by intense proliferation and maintenance of embryogenic competence after successive subcultures. This reproducible protocol opens new prospects for massive propagation and is an alternative to the current organogenesis-based transformation protocol.

Cell morphometry and osmolarity as early indicators of the onset of embryogenesis from cell suspension cultures of grain legumes and model systems

Whatever the in vitro regeneration pathway, it would be of interest to be able to distinguish regeneration-competent from non-regenerating cells and tissues, and as early in culture as possible, as this would allow a dramatic improvement of biotechnology breeding, particularly for the so-called recalcitrant species. With this aim, we examined a range of genotypes of pea (Pisum sativum) and grass pea (Lathyrus sativus), and of the two model species Medicago truncatula, another legume, and Arabidopsis thaliana. This was done by comparing cell suspension cultures of different ages (young [<6 transfers-old]vs. mature [>6 transfers-old]), densities (dense [>107 cells/ml] or sparse [<106 cells/ml]) and regeneration abilities (non-embryogenic vs. embryogenic), in order to identify early indicators of competence for somatic embryogenesis. All such cell suspensions were subcultured every 14 days and several parameters were assessed every 3–4 days during each 14-day cycle. These included the time course pH and osmolarity of the culture medium, the internal osmolarity of cells, the cell surface and the cell wall thickness (by examining cellulose accumulation in Calcofluor White-stained cells under UV light). As cells underwent embryogenesis they enlarged. Cellulose accumulated in the walls of non-embryogenic cells, but walls became thinner with the onset of embryogenesis, and diminished further as embryos matured. Although medium osmolarity decreased at the onset of embryogenesis, this was never observed for non-embryogenic cell suspensions. Conversely, there was a concomitant increase in intracellular osmolarity for embryogenic cells. Medium pH (analysed with the model species only) was not significantly correlated with regeneration competence of cells. For all genotypes and species, the kinetics of cell wall thickness and cell surface, and that of medium and cell osmolarity were reliable early indicators of the competence of cells to undergo somatic embryogenesis. The implication of these results for biotechnological breeding of grain legumes and for plant regeneration competence in general are discussed.

Embryo-specific Proteins in Cyclamen persicum Analyzed with 2-D DIGE

Somatic embryogenesis can be used to produce artificial seeds of Cyclamen persicum, one of the most important ornamental plants for the European market, both as a potted plant in northern Europe and a bedding plant in the cool winters in southern Europe. The aim of this study was to obtain new insights into the molecular biology of somatic embryogenesis, which in turn can be useful for the improvement of tissue culture methodology. Total proteins were characterized from two isogenic cell lines of Cyclamen persicum, one that was embryogenic and one that never has shown any embryogenic capacity. The extracted proteins were separated by two-dimensional differential gel electrophoresis (2-D DIGE) and selected proteins were treated using the ETTAN Dalt Spot Handling Workstation. Protein identification was performed using MALDI-TOF-MS. More than 1200 Cyclamen proteins were detected; 943 proteins were common to both lines. The different protein patterns of the embryogenic and non-embryogenic cell lines were obvious: One hundred eight proteins were more abundant in the embryogenic cells, and 97 proteins in the non-embryogenic cells. Among the differentially expressed proteins, 128 were identified. MALDI-TOF-MS analysis enabled 27 spots to be proposed as candidates for embryo-specific proteins, as they were unique to the embryogenic cell line. The proteins identified are involved in a variety of cellular processes, including cell proliferation, protein processing, signal transduction, stress response, metabolism, and energy state, but the majority are involved in protein processing and metabolism. The main functions of the putative embryo-specific proteins have been discussed in proportion to their role in the somatic embryogenesis process.

Analysis of Cytological Activities in Sugar Beet Calluses

The present study focus on the results of cytological studies of embryogenic (E) and Non-Embryogenic (NE) calluses as the main tissues for cytological studies and the cells of primary root meristem derived from seedling or in vitro cloned plant, as reference tissues. The cellular parameters, such as Mitotic Index (MI), cell-doubling time (Cdt), cell and nuclear area and DNA C value of the different tissues, which may be involved with cell activity, have quantitatively been compared. Embryogenic callus of sugar beet, containing meristematic centers unlike the non-embryogenic one, the cytological behavior of their cells was very close to typical meristematic tissue i.e,, primary root of seedling. In non-embryogenic cells, increased in the rate of cytoplasm/nuclear (C/N) area, endoreduplication and reduction of mitotic index followed by increasing of cell doubling times were observed. © 2008 Asian Network for Scientific Information.

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.

Identification of genes expressed during early somatic embryogenesis in Pinus radiata

Analysis of cDNA-AFLPs was used to study gene expression underlying the early embryogenic process in the gymnosperm Pinus radiata. Somatic embryogenesis in this species was used as a model as it resulted in the generation of a large number of embryos at defined stages of development. The gene expression patterns of three embryogenic stages were compared with non-embryogenic cells. Fifty transcript-derived fragments (TDFs) that are upregulated and 32 TDFs that are down-regulated in the embryogenic stages were selected, sequenced and their homologies sought in the databases. Expression of a selected subset of differentially expressed genes was confirmed by RT-PCR and their levels of expression were quantified. Of the 50 up-regulated TDFs, 16 are homologous to genes encoding either known or putative proteins in higher plants, 19 are homologous to conifer ESTs and 15 did not show significant matches. Of the down-regulated TDFs, 8 are homologous to genes encoding either known or putative proteins, 20 are homologous to conifer ESTs and 4 of them did not show significant matches in DNA or protein sequence database. The known up-regulated genes were similar to genes involved in cellular metabolism and in the stress response and the known down-regulated genes were similar to genes involved in proteolysis, cell wall modification and signaling pathways. Their putative individual function is briefly reviewed based on published information, and the potential roles of these genes in embryo development are discussed.

Conifer WOX-related homeodomain transcription factors, developmental consideration and expression dynamic of WOX2 during Picea abies somatic embryogenesis

In angiosperms, the WOX family of transcription factors has important functions in meristem regulation and in control of the partitioning of developing embryos into functional domains. In this study, a putative WOX2 homologous gene was isolated from Picea abies, and its expression pattern during somatic embryo development was followed using real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR). We used strategies of both absolute and relative quantification of gene expression, and benefits and disadvantages of the two methods are presented and discussed. During embryogenesis, PaWOX2 expression was highest at the earliest stages of development, but low levels were also detected in seedling tissues. No PaWOX2 expression was detected in a non-embryogenic cell culture, indicating that PaWOX2 plays a fundamental role during early somatic embryo development, and can be used as a possible marker for embryogenic potential. Additional results show that conifers, like angiosperms, contain a large number of WOX-related genes, many of them expressed during embryo development. In phylogenetic analysis based on the deduced homeodomain of retrieved pine and spruce EST sequences, no conifer WUS homolog was found. Neither did we find any homeodomain to cluster with WOX5. Interestingly, a clade including only conifer sequences derived from various tissues was resolved as sister to a Physcomitrella WOX-like gene, suggestive of the early origin of this gene family. Our results thus provide basic information for further studies of the evolution of this gene family and of their function in relation to meristem dynamics and specification of stem cells in gymnosperms.

Arginine and ornithine decarboxylases in embryogenic and non-embryogenic carrot cell suspensions

The in vivo activities of arginine and ornithine decarboxylases, key enzymes in the biosynthesis of putrescine and thus polyamines, were measured in three different cell lines of carrot (Daucus carota) during growth and somatic embryogenesis. The activities of these two enzymes differed in the different cell lines in the presence of various levels of auxin (2,4 dichlorophenoxy acetic acid), but was highest during periods of active cell division. During somatic embryo development, the activities of both enzymes were highest during globular stage formation. Thus, both enzymes were found to be active during growth and somatic embryogenesis and could contribute to polyamine biosynthesis.

When microspores decide to become embryos — cellular and molecular changesThis review is one of a selection of papers published in the Special Issue on Plant Cell Biology.

Cultured microspores can be induced to develop into fully functional haploid embryos instead of mature pollen. The ability of these cells to change their development in response to environmental stimuli is an exceptional example of totipotency in plants. Discovering the triggers of embryo development in microspore cultures could lead to a greater understanding of the early stages of embryogenesis in plants and might be used to increase the range of crop plants to which microspore culture can be applied for the production of double haploid homozygous lines. The information might also help to define the general characteristics of pluripotent cells in any organism. In this review, the changes that occur in cellular organization and gene expression in early-stage microspore cultures of several species are discussed. Responding cells in these cultures enlarge, their nuclei are repositioned to their cell centres, and their cytoplasms become filled with fragmented vacuoles. We used flow cytometry to track cellular changes in canola ( Brassica napus L.) microspore cultures as well as microarray analyses and real-time PCR to compare gene expression in embryogenic and nonembryogenic cells. A model for embryogenic cell activation in plants that involves alkalinization, Ca2+ signaling, and changes in GTPase activity that lead to significant changes in gene expression is discussed.