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At least 73 records · Page 4Linked to original sources

PubMed2003

Somatic embryogenesis and plant regeneration from seeds of wild Dicentra spectabilis (L.) LEM.

Regeneration via somatic embryogenesis from callus was studied in Dicentra spectabilis. To obtain somatic embryogenic callus, we cultured D. spectabilis seeds on MS basal media supplemented with various concentrations of 2,4-dichlorophenoxyacetic acid (2,4-D). The highest percentage of embryogenic callus formation was observed on media containing 1.0 mg/l 2,4-D under dark conditions. Somatic embryogenesis was studied by transferring the callus onto MS basal medium containing different concentrations (0.0, 0.1, 0.5, 1.0, 2.0 mg/l) of KIN (kinetin) and/or BAP. Somatic embryogenesis on MS basal media with 1.0 mg/l of KIN was excellent under light conditions. Somatic embryos were rooted by transferring them to half-strength MS basal media containing 2 g/l Phytagel. About 64.2% of the somatic embryos converted to rooted plantlets, 4% showed secondary embryogenesis and 31.8% did not develop and died. Rooted plantlets showed a 46% survival rate when acclimatized ex vitro.

2,4-Dichlorophenoxyacetic Acid↗

PubMed2003

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.

Adenine↗

PubMed2005

High-frequency plant regeneration through cyclic secondary somatic embryogenesis in black pepper (Piper nigrum L.).

A high-frequency plantlet regeneration protocol was developed for black pepper (Piper nigrum L.) through cyclic secondary somatic embryogenesis. Secondary embryos formed from the radicular end of the primary somatic embryos which were originally derived from micropylar tissues of germinating seeds on growth regulator-free SH medium in the absence of light. The process of secondary embryogenesis continued in a cyclic manner from the root pole of newly formed embryos resulting in clumps of somatic embryos. Strength of the medium and sucrose concentration influenced the process of secondary embryogenesis and fresh weight of somatic embryo clumps. Full-strength SH medium supplemented with 1.5% sucrose produced significantly higher fresh weight and numbers of secondary somatic embryos while 3.0 and 4.5% sucrose in the medium favored further development of proliferated embryos into plantlets. Ontogeny of secondary embryos was established by histological analysis. Secondary embryogenic potential was influenced by the developmental stage of the explanted somatic embryo and stages up to "torpedo" were more suitable. A single-flask system was standardized for proliferation, maturation, germination and conversion of secondary somatic embryos in suspension cultures. The system of cyclic secondary somatic embryogenesis in black pepper described here represents a permanent source of embryogenic material that can be used for genetic manipulations of this crop species.

Germination↗

PubMed2005

Mitogen-activated protein kinases are developmentally regulated during stress-induced microspore embryogenesis in Brassica napus L.

Plant mitogen-activated protein kinase (MAPK) cascades are involved in extracellular stress signalling pathways, leading to different cellular responses. Stress-induced microspore embryogenesis involves the internalization of an extracellular stress signal, generating a number of cellular responses where MAPK cascades might be involved. These responses include a change of the developmental programme, the entry into an early proliferative stage and, subsequently, into differentiation stages during haploid embryogenesis. In this work we studied the expression during microspore embryogenesis of several kinases, to assess their putative role in these events. The known Brassica napus MAP kinase kinase kinases (MAP3Ks BnMAP3Kalpha1, BnMAP3Kbeta1 and BnMAP3Kepsilon, the BnBSKtheta kinase and B. napus extracellular signal-regulated kinase (ERK) homologues were analysed by electron microscope (EM) in situ hybridization, immuno-gold labelling, immunofluorescence and western blotting. The differential in situ expression of these kinases suggests a role for them during embryogenesis. Two different expression patterns were observed, indicating a different regulation. BnMAP3Kalpha1, BnMAP3Kepsilon, and the ERKs showed a pattern consistent with a role mainly in proliferative events. Conversely, BnMAP3Kbeta1 and BnBSKtheta, presented a pattern that suggested an involvement in differentiation stages. In addition, ERK homologues migrate to the nucleus immediately after induction, being found in a phosphorylated state in a larger amount.

Blotting, Western↗

PubMed2002

Similarity of expression patterns of knotted1 and ZmLEC1 during somatic and zygotic embryogenesis in maize ( Zea mays L.).

Expression of knotted1 ( kn1) and ZmLEC1, a maize homologue of the Arabidopsis LEAFY COTYLEDON1 ( LEC1) was studied using in situ hybridization during in vitro somatic embryogenesis of maize ( Zea mays L.) genotype Hi-II. Expression of kn1 was initially detected in a small group of cells (5-10) in the somatic embryo proper at the globular stage, in a specific region where the shoot meristem is initiating at the scutellar stage, and specifically in the shoot meristem at the coleoptilar stage. Expression of ZmLEC1 was strongly detected in the entire somatic embryo proper at the globular stage, gradually less in the differentiating scutellum at the scutellar and coleoptilar stages. The results of analyses show that the expression pattern of kn1 during in vitro somatic embryogenesis of maize is similar to that of kn1 observed during zygotic embryo development in maize. The expression pattern of ZmLEC1 in maize during in vitro development is similar to that of LEC1 in Arabidopsis during zygotic embryo development. These observations indicate that in vitro somatic embryogenesis likely proceeds through similar developmental pathways as zygotic embryo development, after somatic cells acquire competence to form embryos. In addition, based on the ZmLEC1 expression pattern, we suggest that expression of ZmLEC1 can be used as a reliable molecular marker for detecting early-stage in vitro somatic embryogenesis in maize.

Arabidopsis Proteins↗

PubMed2004

Expression of photosynthesis-related genes and their regulation by light during somatic embryogenesis in Daucus carota.

To clarify the spatial and temporal pattern of gene expression for photosynthesis-associated proteins during somatic embryogenesis in Daucus carota L., the localization of mRNAs for three genes, rbcL, Lhcb and por, was examined in dark-grown and light-irradiated somatic embryos by in situ hybridization. The three mRNAs were expressed in common in the mesophyll precursor cells of light-irradiated embryos at the late torpedo and plantlet stages, but characteristic expression patterns of each photosynthesis-related gene were also observed. Expression of rbcL mRNA first occurred throughout the embryo but gradually became localized in the mesophyll precursor cells and cortex during early embryogenesis. Localization of Lhcb mRNA in the mesophyll precursor cells and shoot apical meristem became clear in the early torpedo stage. Expression of Lhcb mRNA was not affected by light during early embryogenesis, but could be induced by light in the torpedo stage, suggesting that light-inducible expression of Lhcb mRNA arises within the torpedo stage. At the late torpedo stage, clear localization of por mRNA started in mesophyll precursor cells of the cotyledon in light-irradiated embryos. Greening potency of the embryo also appeared first at this stage. Therefore, greening and initial differentiation of photosynthetic tissues during somatic embryogenesis seem to be associated with coordinated expression of mRNA for rbcL, Lhcb and por in late torpedo-shaped embryos.

Darkness↗

PubMed2005

Possible involvement of abscisic acid in the induction of secondary somatic embryogenesis on seed-coat-derived carrot somatic embryos.

When seed coats (pericarps) were picked from 14-day-old carrot (Daucus carota) seedlings and cultured on agar plates, embryogenic cell clusters were produced very rapidly at a high frequency on the open side edge. Embryo induction progressed without auxin treatment; indeed treatment caused the formation of non-embryogenic callus. The embryogenic tissues (primary embryos) developed normally until the torpedo stage; however, after this a number of secondary somatic embryos were produced in the hypocotyl and root regions. "Tertiary" embryos were formed on some of the secondary embryos, but many developed into normal plantlets. The primary embryos contained significantly higher levels of abscisic acid (ABA) than the hypocotyl-derived normal and seed-coat-derived secondary embryos. Fluridone inhibited the induction of secondary embryogenesis, while exogenously supplied ABA induced not only "tertiary" embryogenesis on the seed-coat-derived secondary embryos, but also secondary embryos on the hypocotyl-derived normal somatic embryos. These results indicate that ABA is one of the important endogenous factors for the induction of secondary embryogenesis on carrot somatic embryos. Higher levels of indole-3-acetic acid (IAA) in primary embryos also suggest the presence of some concerted effect of ABA and IAA on the induction of secondary embryogenesis in primary embryos.

Abscisic Acid↗

PubMed2000

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

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

Asteraceae↗

PubMed2006

The relationship between induction of embryogenesis and chromosome doubling in microspore cultures.

The objective of this paper is to review the relationship between induction of microspore embryogenesis and chromosome doubling. It has been augmented with relative data on chromosome doubling by nuclear fusion. Some of the treatments used for induction of embryogenesis may also lead to doubling of the chromosome number, either through nuclear fusion or endomitosis. High frequencies of spontaneous chromosome doubling in cereal species appear to be induced by treatments that block cell wall formation during the first cell divisions, resulting in coenocytic cells in which the nuclei are able to fuse. The use of mannitol as a pretreatment for induction of embryogenesis in barley, wheat, and maize microspore cultures provides examples of nuclear fusion. The use of antimicrotubule agents for embryo induction via treatments during the first few hours of microspore culture has also resulted in high frequencies of chromosome doubling. Factors such as the doubling agent concentration, temperature during treatment, and duration of treatment may be critical for individual species. Actin filament as well as microtubule assembly studies related to new cell wall formation provide further evidence at the molecular level for the relationship between microspore embryogenesis and chromosome doubling.

Cell Nucleus↗

PubMed1985

An analysis of dopa decarboxylase expression during embryogenesis in Drosophila melanogaster.

Dopa decarboxylase (DDC) activity appears near the end of embryogenesis in Drosophila. High titers of 20-OH-ecdysone on the other hand are found at midembryogenesis. Several explanations for this lag were investigated, since this hormone has been shown to induce a rapid increase in DDC activity at pupariation (G. P. Kraminsky, W. C. Clark, M. A. Estelle, R. D. Gietz, B. A. Sage, J. D. O'Connor, and R. B. Hodgetts, 1980, Proc. Natl. Acad. Sci. USA 77, 4175-4179). Using immunological and genetical criteria, it was shown that the same structural gene encodes DDC in embryos, mature larvae, and young adults. This rules out the existence of a distinct embryonic DDC gene unresponsive to 20-OH-ecdysone. Second, no evidence was found to support the hypothesis that a delay in the translation of DDC transcripts, produced in response to the elevated titer of 20-OH-ecdysone at midembryogenesis, caused the lag. Northern analysis of the RNA molecules homologous to cloned genomic sequences revealed that DDC transcripts were present at two different times during embryogenesis. A transcript was found in both ovaries and 0- to 2-hr embryos. However, this species disappeared by 4 hr and DDC transcript levels remained low until late in embryogenesis, when a significant increase occurred. This increase was presumably responsible for the appearance of the enzyme at this time. The northern blotting revealed nine DDC transcript species were present during embryogenesis and hybridization to intron-specific probes indicated that five of these contained at least part of one (or both) of the two introns. Three putative mature mRNA species were identified by their small size, relative abundance, apparent lack of intron sequence, and their presence on polysomes. The two mature species found during the late stages were postulated to differ in the length of their poly(A)+ tails. The third mature species was found only in ovaries and very young embryos and may well be of maternal origin. Data are examined in light of the possibility that this species is derived from a precursor initiated at a novel promotor.

Animals↗

PubMed1995

GTP-binding proteins and early embryogenesis in Xenopus.

During early embryogenesis the specification of body axes and the determination of cell subtypes proceeds through cell interactions and movements which involve the decoding of various signals in a spatial and temporal manner. An increasingly abundant literature has revealed the participation of growth factors and their receptors in the induction and regionalization of the mesoderm. The question therefore arises as to whether other signal transducing systems are expressed and play a role in early embryogenesis. In this mini review we describe the main developmental events occurring during early embryogenesis in Xenopus and the signalling pathways that are potentially involved; we then summarize the major properties of heterotrimeric GTP-binding proteins; finally, we present results suggesting that heterotrimeric GTP-binding proteins are expressed during early embryogenesis and discuss their potential function.

Animals↗

PubMed2006

The utilization of lipovitellin during blue crab (Callinectes sapidus) embryogenesis.

Embryos of the blue crab Callinectes sapidus develop in egg sacs carried on the abdomen of the female. They develop over a period of 10-13 days at 28 degrees C and are nutritionally dependent on yolk until they emerge from the egg sacs as free-swimming zoeae. The principal component of blue crab yolk is lipovitellin (LpII), a water-soluble lipoprotein composed of approximately equal amounts of lipid and protein. We followed changes in the concentration of apoproteins of LpII during embryogenesis by ELISA and Western blots, using monoclonal antibodies against two LpII apoprotein associated peptides identified as Protein A (107 kDa) and Protein B (75 kDa). During embryogenesis there was a decrease in Protein B but an increase in two smaller peptides (52 and 35 kDa) that reacted with the Protein B antibody. Utilization of LpII during embryogenesis was also followed morphologically by immunohistochemistry. Utilization of LpII was slow in early embryonic stages, followed by rapid utilization in late embryonic stages, such that only traces of LpII were present at the end of embryogenesis. The cells of the developing hepatopancreas appear to play an important role in the utilization of LpII.

Animals↗

PubMed2000

Abscisic acid induced somatic embryogenesis in immature embryo explants of coconut (Cocos nucifera L.).

Somatic embryogenesis in coconut (Cocos nucifera L.) is generally induced by gradual reduction of auxin concentration in the culture medium and incorporation of cytokinins. Although plant regeneration through somatic embryogenesis is possible, the protocol is yet to be perfected. In this study, nodular callus was obtained from 7-9 months old immature zygotic embryos of coconut on a medium containing 24 µM 2,4-dichlorophenoxy acetic acid (2,4-D). As a novel approach, abscisic acid (ABA) at a concentration of 2.5-7.5 µM was incorporated into the culture medium for 3-7 weeks to induce somatic embryogenesis. Alternately, callus was subcultured at 5 weekly intervals on media containing gradually reducing concentrations of 2,4-D to induce somatic embryogenesis. Incorporation of ABA enhanced the production of somatic embryos. Application of 2.5-5 µM ABA for 5 weeks was found to be effective. A large number of somatic embryos developed on media containing ABA formed normal shoots and complete plants as compared to those produced in the media with low levels of 2,4-D.

Journal Article↗

PubMed1999

Expression of the genes for alpha-type and beta-type calcitonin gene-related peptide during rat embryogenesis.

Throughout rat embryogenesis we analysed the expression patterns of the three mature transcripts generated from the two calcitonin gene-related peptide genes: calcitonin, alpha-calcitonin gene-related peptide, and beta-calcitonin gene-related peptide messenger RNAs. In addition, we examined in parallel the distribution of calcitonin gene-related peptide and calcitonin immunoreactivity. Of the three transcripts, beta-calcitonin gene-related peptide messenger RNA was first detected in sensory ganglia on embryonic day 14, and by embryonic day 15 was seen to a lesser degree in motor neurons and autonomic ganglia. Starting at embryonic day 16, however, the highest levels of beta-calcitonin gene-related peptide messenger RNA were found in motor neurons rather than sensory ganglia. Alpha-calcitonin gene-related peptide messenger RNA was first detected on embryonic day 16 in both sensory ganglia and motor neurons, but at lower levels than beta-calcitonin gene-related peptide, particularly in the motor neurons of the spinal cord. By embryonic day 20, transcripts for alpha- and beta-calcitonin gene-related peptide were expressed in distinct brain regions. High levels of alpha-calcitonin gene-related peptide messenger RNA were detected in hypoglossal, facial, and parabrachial nuclei, and moderate levels in the trigeminal motor and ambiguus nuclei. By contrast, beta-calcitonin gene-related peptide messenger RNA was detected at low levels in hypoglossal, ambiguus, facial, and parabrachial nuclei, and at high levels in the trigeminal nucleus. In the oculomotor-trochlear nucleus, beta-calcitonin gene-related peptide messenger RNA was the sole isotype expressed. Low levels of messenger RNA for both calcitonin gene-related peptide transcripts were appreciated in the inferior olive. Outside the nervous system, alpha-calcitonin gene-related peptide messenger RNA was weakly expressed in the thyroid gland and beta-calcitonin gene-related peptide messenger RNA in the thymus. Throughout embryogenesis, calcitonin gene-related peptide immunoreactivity usually followed the expression of either alpha- or beta-calcitonin gene-related peptide messenger RNA. Calcitonin messenger RNA and protein were detected only in the thyroid gland from embryonic day 18 onward. This work shows that of the three mature transcripts produced by the two calcitonin gene-related peptide genes, beta-calcitonin gene-related peptide messenger RNA is the predominant transcript produced early in rat embryogenesis. However, by perinatal stages alpha-calcitonin gene-related peptide shows the highest expression in the brain and spinal cord. In autonomic ganglia, beta-calcitonin gene-related peptide is either the sole or the predominant transcript. Unlike the chick embryo in which calcitonin messenger RNA is expressed early in the CNS, in rat it was only expressed outside the nervous system in the thyroid gland during the last days of embryogenesis.

Animals↗

PubMed2006

Relationship between ploidy variation of citrus calli and competence for somatic embryogenesis.

This study focuses on the relationship between the genetic variation of calli and the competence for somatic embryogenesis in citrus. The DNA content of 35 citrus calli of different genotypes was measured three times by flow cytometry during a period of four years. The results showed that 71.4 % of the genotypes had a progressive increase of varied cells, while those of Page tangelo, Shamouti sweet orange, Russ navel orange and Cleopatra decreased; significant difference in the variation degree (percentages) existed among genotypes. Studies carried out on the induction of somatic embryogenesis revealed that 9 out of the 35 genotypes had still kept the competence of somatic embryogenesis, and the rest 26 had lost the competence. Correlation analysis indicated that there was no significant relationship between the variation degree and the embryogenesis competence r = -0.10 (P < 0.01), neither for the relationship between the subculture duration and the regeneration capacity.

Aneuploidy↗

PubMed2003

Hsp70 and Hsp90 change their expression and subcellular localization after microspore embryogenesis induction in Brassica napus L.

A stress treatment of 32 degrees C for at least 8h was able to change the gametophytic program of the microspore, switching it to embryogenesis in Brassica napus, an interesting model for studying this process in vitro. After induction, some microspores started symmetric divisions and became haploid embryos after a few days, whereas other microspores, not sensitive to induction, followed their original gametophytic development. In this work the distribution and ultrastructural localization of two heat-shock proteins (Hsp70 and Hsp90) throughout key stages before and after embryogenesis induction were studied. Both Hsp proteins are rapidly induced, localizing in the nucleus and the cytoplasm. Immunogold labeling showed changes in the distribution patterns of these proteins, these changes being assessed by a quantitative analysis. Inside the nucleus, Hsp70 was found in association with RNP structures in the interchromatin region and in the nucleolus, whereas nuclear Hsp90 was mostly found in the interchromatin region. For Hsp70, the accumulation after the inductive treatment was accompanied by a reversible translocation from the cytoplasm to the nucleus, in both induced (embryogenic) and noninduced (gametophytic) microspores. However, the translocation was higher in embryogenic microspores, suggesting a possible additional role for Hsp70 in the switch to embryogenesis. In contrast, Hsp90 increase was similar in all microspores, occurring faster than for Hsp70 and suggesting a more specific role for Hsp90 in the stress response. Hsp70 and Hsp90 colocalized in clusters in the cytoplasm and the nucleus, but not in the nucleolus. Results indicated that stress proteins are involved in the process of microspore embryogenesis induction. The differential appearance and distribution of the two proteins and their association at specific stages have been determined between the two systems coexisting in the same culture: embryogenic development (induced cells) and development of gametes (noninduced cells).

Antibodies, Monoclonal↗

PubMed1999

Synchronization of somatic embryogenesis at high frequency using carrot suspension cultures: model systems and application in plant development.

Materials and methods for the high frequency induction and synchronous somatic embryogenesis from cultured cells of higher plants are described, using carrot suspension cultures as a model system of higher plants. The following four synchronous systems of somatic embryogenesis, which were established in our laboratories, are reported: (1) Somatic embryogenesis from single cells. a) Small spherical single cells, obtained from suspension cultures in the presence of 2,4-D, zeatin and mannitol by sieving, density gradient centrifugation in Percoll solutions and manual picking up, form embryogenic cell clusters, which differentiate to embryos at high frequency, when embryogenic cell clusters are transferred to a medium lacking 2,4-D. b) Explants of hypocotyls of regenerated plantlets from somatic embryos were cultured after treatment with 2,4-D for 12-24 h, and then transferred into a fresh medium lacking 2,4-D. Single cells are released from hypocotyl explants and differentiated into embryos at high frequency. In this system, a large number of single cells and embryogenic cells can be collected. (2) Somatic embryogenesis from embryogenic cell clusters, which are obtained from suspension cultures by sieving, density gradient centrifugation in Ficoll solutions, and subsequent centrifugation at a low speed, differentiate synchronously to globular embryos at high frequency. Plantlets are formed from globular embryos. (3) Embryogenic cell clusters obtained according to the procedure described in (2) are cultured at cell densities of 2x10(3) cell clusters ml(-1). Globular embryos differentiate to torpedo-shaped embryos and subsequently to plantlets at high frequency when they are cultured at densities below 150 globular embryos ml(-1).

Daucus carota↗

PubMed2001

4-Hydroxybenzyl alcohol accumulates in suspension-cell cultures and inhibits somatic embryogenesis in carrot.

Somatic embryogenesis in carrot (Daucus carota L.) is strongly inhibited by certain factors that accumulate in culture medium of high-density cultures of embryogenic cells. We previously identified 4-hydroxybenzyl alcohol (4HBA) as one of the inhibitory factors. In this study, we analyzed the accumulation pattern of 4HBA in the cultures of carrot suspension cells. When somatic embryogenesis was induced by culturing embryogenic cells in phytohormone-free Murashige and Skoog medium at various initial cell densities, 4HBA accumulated in the culture medium. The concentration of 4HBA in high cell density cultures was higher than in low cell density cultures. The accumulation of 4HBA in high cell density cultures was rapid during the early days of culture. This rapid accumulation of 4HBA in high cell density cultures might result in the strong inhibition of somatic embryogenesis. The production of 4HBA decreased as the somatic embryos developed. In addition, embryogenic cells released larger amount of 4HBA into the culture medium compared with non-embryogenic cells. These results suggest that the production of 4HBA is both related to embryogenic competence and developmentally regulated during somatic embryogenesis.

Journal Article↗